Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Micronucleus assay: OECD 487, GLP, TK6 cells, 34.38 – 1100.00 µg/ml (with and without S9), three experiments. No evidence of genotoxicity.

 

HPRT: OECD 476, GLP, CHO, 68.8 – 1100.00 µg/ml (with and without S9), four experiments. No evidence of genotoxicity.

 

Ames: Equivalent to 471, GLP, S. typhimurium TA100, TA 98, TA1535, TA1537, and TA1538, 100-10,000 µg/plate. No evidence of genotoxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jul - Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
Jul 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 640/2012; B.49
Version / remarks:
JUl 2012
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: BASF SE Ludwigshafen, Germany
- Batch No.of test material: 83294456P0
- Date of production: 10 Dec 2016
- Purity: 99.9%
- Physical state, appearance: liquid, colorless, clear


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability was guaranteed until 08 Nov 2018
- Homogeneity: Homogeneity was guaranteed on account of the high purity and was ensured by mixing before preparation

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with the vehicle to achieve the required stock solution. The test substance preparation was shaken thoroughly.
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Cytokinesis block (if used):
CytB
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction obtained from phenobarbital and beta-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
1st experiment: 34.38, 68.75, 137.50, 275.00, 550.00, and 1100.00 µg/ml (with and without S9)
2nd experiment: 137.50, 275.00, 550.00, and 1100.00 µg/ml (with S9)
3rd experiment: 200.00, 400.00, 550.00, 800.00, and 1100.00 µg/ml (with and without S9)

A pretest was performed using 1100 µg/ml as top concentration. No cytotoxicity about or below 55% +/- 5% of control was observed.
Vehicle / solvent:
- Vehicle used: RPMI 1640 (culture medium)
- Justification for choice of solvent: The test substance has a good solubility in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle control is negative control
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 h (with and without S9), Experiment II: 4 h (with S9), Experiment III: 4 h (with S9) and 24 h (without S9)
- Recovery time: Experiment I and II: 20 h (with and without S9), Experiment III: 40 h (with S9)
- Harvest time: Experiment I and II: 24 h (with and without S9), Experiment III: 24 h (without S9) and 44 h (with S9)

SPINDLE INHIBITOR (cytogenetic assays): CytB (final concentration: 3 µg/ml; stock: 0.6 mg/ml in DMSO)

STAIN (for cytogenetic assays): 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/ml)

NUMBER OF REPLICATIONS: Two

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Slide preparation was carried out based on the method described by Fenech (1993). Single cell suspensions were prepared from each test group by resuspending. Then, the cell number per flask of each cell suspension was determined using a cell counter. Subsequently, 5x10^4 cells per slide were centrifuged at 600 rpm for 7 minutes onto labeled slides using a Cytospin centrifuge. At least two slides per flask were prepared. In the case of strongly reduced cell numbers no slides were prepared. After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes.
Before scoring, the slides were stained with a mixture of DAPI and propidium iodide in Fluoroshield™ at a concentration of 0.25 μg/mL each. By the use of the combination of both fluorescence dyes it can be differentiated between DNA (DAPI; excitation: 350 nm, emission: 460 nm) and cytoplasm (PI; excitation: 488 nm, emission: 590 nm).

NUMBER OF CELLS EVALUATED: As a rule, at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not specified

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The analysis of micronuclei was carried out following the criteria of Countryman and Heddle (1976):
− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only binucleated cells were scored.

DETERMINATION OF CYTOTOXICITY
- Method: reduced cell number, Cytokinesis-block proliferation index (CBPI)

OTHER EXAMINATIONS:
- pH value, osmolality, solubility
Evaluation criteria:
Assessment criteria

A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data

A test substance is considered to be clearly negative if the following criterion is met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data
Statistics:
The statistical evaluation of the data was carried out using an appropriate statistical analysis.
The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE).
If the results of this test were statistically significant compared with the respective vehicle control (p ≤ 0.05), labels (S) have been printed in the tables.
Furthermore, a statistical trend test (SAS; Proc Reg) was performed to assess a possible dose-related increase of micronucleated cells. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the micronuclei rate of each test group and the independent variable was the concentration. The trend was performed one-sided (increased dose-related trend) with a significande level of 0.05.
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced by test substance
- Effects of osmolality: not influenced by test substance
- Water solubility: yes
- Precipitation: no
- Definition of acceptable cells for analysis: Before use each batch was checked for mycoplasma contamination, karyotype stability, and growth characteris

HISTORICAL CONTROL DATA (with ranges)
- Positive historical control data: 2.0 - 6.5% micronucleated cells
- Negative (solvent/vehicle) historical control data: 0.1 - 1.3% micronucleated cells

Table 1: Analysis of micronuclei - 1st Experiment;

4 hours exposure, 24 hours harvest time, without S9 mix

 

 

Test groups

 

Culture

 

No. of Cells

 

Cells containing Micronuclei

 

n

n

%

 

Negative control*

 

A

 

1000

 

9

 

0.9

 

 

B

1000

12

1.2

 

Test substance

 

Total

2000

21

1.1

275.0

µg/mL

A

1000

11

1.1

 

 

B

1000

11

1.1

 

 

Total

2000

22

1.1

550.0

µg/mL

A

1000

12

1.2

 

 

B

1000

11

1.1

 

 

Total

2000

23

1.2

1100.0

µg/mL

A

1000

11

1.1

 

 

B

1000

6

0.6

 

 

Total

2000

17

0.9

MMC 0.125

µg/mLS

A

1000

49

4.9

 

 

B

1000

29

2.9

 

 

Total

2000

78

3.9

 

* RPMI 1640

SFrequency statistically significant higher than corresponding control values

 

 Table 2: Analysis of micronuclei – 2nd Experiment;

4 hours exposure, 24 hours harvest time, with S9 mix

 

 

Test groups

 

Culture

 

No. of Cells

 

Cells containing Micronuclei

 

n

n

%

 

 

A

 

1000

 

13

 

1.3

Negative control*

B

1000

9

0.9

 

Test substance

 

Total

2000

22

1.1

275.00

µg/mL

A

1000

12

1.2

 

 

B

1000

9

0.9

 

 

Total

2000

21

1.1

550.00

µg/mL

A

1000

10

1.0

 

 

B

1000

9

0.9

 

 

Total

2000

19

1.0

1100.00

µg/mL

A

1000

9

0.9

 

 

B

1000

12

1.2

 

 

Total

2000

21

1.1

CPP 0.5

µg/mL

A

1000

21

2.1

 

 

B

1000

12

1.2

 

 

Total

2000

33

1.7

CPP 1.0

µg/mLS

A

1000

38

3.8

 

 

B

1000

41

4.1

 

 

Total

2000

79

4.0

 

* RPMI 1640

SFrequency statistically significant higher than corresponding control values

Table 3: Analysis of micronuclei – 3rd Experiment;

24 hours exposure, 24 hours harvest time, without S9 mix

 

 

Test groups

 

Culture

 

No. of Cells

 

Cells containing Micronuclei

 

n

n

%

 

Negative control*

 

A

 

1000

 

10

 

1.0

 

 

B

1000

7

0.7

 

Test substance

 

Total

2000

17

0.9

550.0

µg/mL

A

1000

10

1.0

 

 

B

1000

8

0.8

 

 

Total

2000

18

0.9

800.0

µg/mL

A

1000

7

0.7

 

 

B

1000

8

0.8

 

 

Total

2000

15

0.8

1100.0

µg/mL

A

1000

5

0.5

 

 

B

1000

7

0.7

 

 

Total

2000

12

0.6

MMC 0.015

µg/mL

A

1000

11

1.1

 

 

B

1000

13

1.3

 

 

Total

2000

24

1.2

MMC 0.030

µg/mLS

A

1000

21

2.1

 

 

B

1000

22

2.2

 

 

Total

2000

43

2.2

 

* RPMI 1640

 

SFrequency statistically significant higher than corresponding control values

 

 

 Table 4: Analysis of micronuclei – 3rd Experiment;

4 hours exposure, 44 hours harvest time, with S9 mix

 

 

Test groups

Culture

No. of Cells n

Cells containing Micronuclei n %

 

Negative control*

 

Test substance

550.0 µg/mL

 

800.0 µg/mL

 

1100.0 µg/mL

 

CPP 0.5 µg/mL

 

CPP 1.0 µg/mLS

 

A

 

1000

 

8

 

0.8

B

1000

12

1.2

Total

2000

20

1.0

 

A

 

1000

 

9

 

0.9

B

1000

24

2.4

Total

2000

33

1.7

A

1000

8

0.8

B

1000

7

0.7

Total

2000

15

0.8

A

1000

11

1.1

B

1000

16

1.6

Total

2000

27

1.4

A

1000

11

1.1

B

1000

13

1.3

Total

2000

24

1.2

A

1000

59

5.9

B

1000

42

4.2

Total

2000

101

5.1

 

* RPMI 1640

 

SFrequency statistically significant higher than corresponding control values

 

 

Conclusions:
Thus, under the experimental conditions chosen here, the conclusion is drawn that the test substance has not the potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in human TK6 cells in the absence and the presence of metabolic activation.
Executive summary:

The test substance was assessed for its potential to induce micronuclei in TK6 cells in vitro (clastogenic or aneugenic activity). Three independent experiments were carried out 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. The test groups printed in bold type were evaluated.

1st Experiment

4 hours exposure, 24 hours harvest time, without S9 mix

0; 34.38; 68.75; 137.50; 275.00; 550.00 and 1100.00 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix (not valid)

0; 34.38; 68.75; 137.50; 275.00; 550.00 and 1100.00 μg/mL

2nd Experiment

4 hours exposure, 24 hours harvest time, with S9 mix

0; 137.50; 275.00; 550.00 and 1100.00 μg/mL

3rd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix

0; 200.00; 400.00; 550.00; 800.00 and 1100.00 μg/mL

4 hours exposure, 44 hours harvest time, with S9 mix

0; 200.00; 400.00; 550.00; 800.00 and 1100.00 μg/mL

In the 1st Experiment cultures treated with the positive control in the presence of S9 mix did not show an increase in the micronucleus frequency. Thus, this experimental part is considered as invalid and was repeated in the 2nd Experiment. The results of the invalid part of the 1st Experiment will not be shown in this report. The raw data of this experimental part will be archived with the raw data of this study.

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.

The negative controls gave frequencies of micronucleated cells within our historical negative control data range for TK6 cells. Both positive control substances, mitomycin C (MMC) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei.

Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in human TK6 cells in the absence and the presence of metabolic activation.

On the basis of the results of the present study, the test substance did not cause any

biologically relevant increase in the number of cells containing micronuclei either without S9

mix or after adding a metabolizing system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan - Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Jul 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008; B.17
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht
Type of assay:
other: HPRT
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: BASF SE Ludwigshafen, Germany
- Batch No.of test material: 83294456P0
- Date of production: 10 Dec 2016
- Purity: 99.9%
- Physical state, appearance: liquid, colorless, clear


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability was guaranteed until 08 Nov 2018
- Homogeneity: Homogeneity was guaranteed on account of the high purity and was ensured by mixing before preparation

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with the vehicle to achieve the required stock solution. The test substance preparation was treated with ultrasonic waves thoroughly.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction obtained from phenobarbital and beta-naphthoflavone induced rats
Test concentrations with justification for top dose:
Experiment I: 68.8, 137.5, 275.0, 550.0, and 1100.0 µg/ml (with and without S9)
Experiment II: 87.5, 175.0, 350.0, 700.0, and 1100.0 µg/ml (with and without S9)
Experiment III: 87.5, 175.0, 350.0, 700.0, and 1100.0 µg/ml (with and without S9)
Experiment IV: 87.5, 175.0, 350.0, 700.0, and 1100.0 µg/ml (with S9)

Top concentration was established in a pretest. No cytotoxicity was observed as indicated by a reduced relative servival of about or below 20% of control.
Vehicle / solvent:
- Vehicle used: Ham's F12 (culute medium)
- Justification for choice of vehicle: The test substance has a good solubility in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Solvent control is negative control
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: 20*10^6

DURATION
- Preincubation period: 20-24 h
- Exposure duration: 4 h
- 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 of cells): 5-7 days

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- pH, osmolality, solubility, cell morphology
Evaluation criteria:
Assessment 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 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
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 (1979). 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.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Without S9, pH was not influenced. With S9, there was a slight shift in the pH values towards an acid pH value in the highest applied concentrations. However, the increased pH values did not influence the results on cytotoxicity and genotoxicity of this study.
- Effects of osmolality: not influenced by test substance
- Water solubility: yes
- Precipitation: no
- Definition of acceptable cells for analysis:
- Cell morphology: After 4 h treatment, neither with nor without S9, cell morphology and attachment of cells was not adversely influenced in any test group.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: 42.47-419.90 (without S9), 21.52-270.48 (with S9)
- Negative (solvent/vehicle) historical control data: 0.00-6.49 (without S9), 0.00-7.43 (with S9)

Table 1: Mutant frequency and viability - 1stExperiment 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

Negative control

115

146

65.3

100.0

2

1

0.75

1.15

68.8

n.c.1

n.c.1

137.5

120

150

67.5

103.4

5

7

3.00

4.44

275.0

164

154

79.5

121.8

0

0

0.00

0.00

550.0

154

149

75.8

116.1

7

5

3.00

3.96

1100.0

154

172

81.5

124.9

1

0

0.25

0.31

EMS 400.0

141

127

67.0

102.7

245

254

124.75

186.19S

SMutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)

 

Table 2: 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

Negative control

113

114

56.8

100.0

4

5

2.25

3.96

68.8

n.c.1

n.c.1

137.5

114

129

60.8

107.0

7

8

3.75

6.17

275.0

126

105

57.8

101.8

1

3

1.00

1.73

550.0

100

102

50.5

89.0

0

5

1.25

2.48

1100.0

159

121

70.0

123.3

3

4

1.75

2.50

DMBA 1.25

75

91

41.5

73.1

120

117

59.25

142.77S

S Mutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)

 Table 3: Mutant frequency and viability – 3rdExperiment 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

Negative control

163

154

79.3

100.0

3

8

2.75

3.47

87.5

n.c.1

n.c.1

175.0

151

135

71.5

90.2

8

11

4.75

6.64

350.0

131

137

67.0

84.5

7

4

2.75

4.10

700.0

127

152

69.8

88.0

7

3

2.50

3.58

1100.0

135

133

67.0

84.5

5

4

2.25

3.36

EMS 400.0

94

98

48.0

60.6

136

182

79.50

165.63S

SMutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)

 Table 4: Mutant frequency and viability – 4thExperiment 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

Negative control

134

154

72.0

100.0

3

3

1.50

2.08

87.5

n.c.1

n.c.1

175.0

165

149

78.5

109.0

1

4

1.25

1.59

350.0

145

179

81.0

112.5

3

3

1.50

1.85

700.0

147

165

78.0

108.3

0

1

0.25

0.32

1100.0

134

151

71.3

99.0

5

7

3.00

4.21

DMBA 1.25

118

91

52.3

72.6

227

211

109.50

209.57S

SMutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)

 

 

 

Conclusions:
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.
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. Four independent experiments were carried out, with and/or 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 top concentration was determined to be the limit concentration of approx. 10 mM (1100 μg/mL). In the main experiments the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

without S9 mix

0; 68.8; 137.5; 275.0; 550.0; 1100.0 μg/mL

with S9 mix

0; 68.8; 137.5; 275.0; 550.0; 1100.0 μg/mL

2nd Experiment (not valid; data not shown)

without S9 mix

0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL

with S9 mix

0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL

3rd Experiment

without S9 mix

0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL

with S9 mix (not valid; data not shown)

0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL

4th Experiment

with S9 mix

0; 87.5; 175.0; 350.0; 700.0; 1100.0 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 negative 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 all experiments, in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.

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 all experiments performed independently of each other.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline with acceptable restrictions (Purity unknown, missing E.coli/TA 102 strain to detect crosslinking and oxidising agents; therefore, reliability score "1" was not adopted from OECD SIDS)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
E. coli/ TA102 strain is missing (former OECD guideline 472)
Principles of method if other than guideline:
according to Ames B.N. et al., Mutat. Res., 31. 347-364, (1975)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his-
Species / strain / cell type:
other: S. typhimurium TA100, TA 98, TA1535, TA1537, and TA1538
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254-induced rat liver S-9 mix
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333, 10000 ug/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: not specified
Details on test system and experimental conditions:
In the absence of S-9 mix, 100 ul of tester strain and 50 ul of vehicle, positive control, or test article were added to 2.5 ml selective top agar. When S-9 mix was required, 0.5 ml S-9, 100 ul tester strain, and 50 ul of vehicle, positive control, or test article were added to 2.0 ml selective top agar. After votexing, the agar mixture was overlaid onto the surface of plates containing minimal bottom agar. Plates were allowed to solidify, then inverted and incubated for 48 hr at 37 degrees C. Plates not counted immediately after 48 hr were stored at 4 degrees C.
Test concentrations of propyl acetate (0, 100, 333, 1000, 3333, and 10,000 ug/plate) were prepared using dimethylsulfoxide (DMSO) as the solvent; a maximum of 0.5 ml solvent was added to each plate. Each dose was tested in triplicate without activation, and with 10% rat liver S-9. Concurrent positive and solvent controls were run with each trial.
Evaluation criteria:
A material was considered mutagenic if it produced a doubling in the mean reverants per plate in at least one tester strain. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.
Species / strain:
other: S. typhimurium TA100, TA 98, TA1535, TA1537, and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The test substance was non-mutagenic in the Salmonella typhimurium plate incorporation assay.
Executive summary:

The bacterival reverse mutation assay (Ames) was conducted to investigate the genotoxic potential of the test substance. Ames was performed according former OECD guideline 472 and did not include E.Coli strain or TA 102. Five strains of S. typhimurium (TA 100, TA 98, TA 1535, TA 1537, and TA 1538) were treated with 100, 333, 1000, 3333, or 10000 ug/plate both with and without a metabolic activation system (Arochlor 1254-induced rat liver S9-mix). Adequate controls were included. No evidence of a genotoxic potential was observed for any strain tested. Cytotoxicity was not reported but the test substance was tested up to limit concentrations. As a result, the test substance was non-mutagenic in the Salmonella typhimurium plate incorporation assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro

Bacteria

Propyl acetate was negative in a guideline conform Ames test (Microbiological Associates 1989 and OECD SIDS).

The bacterival reverse mutation assay (Ames) was conducted to investigate the genotoxic potential of the test substance. Ames was performed according former OECD guideline 472 and did not include E.Coli strain or TA 102. Five strains of S. typhimurium (TA 100, TA 98, TA 1535, TA 1537, and TA 1538) were treated with 100, 333, 1000, 3333, or 10000 ug/plate both with and without a metabolic activation system (Arochlor 1254-induced rat liver S9-mix). Adequate controls were included. No evidence of a genotoxic potential was observed for any strain tested. Cytotoxicity was not reported but the test substance was tested up to limit concentrations. As a result, the test substance was non-mutagenic in the Salmonella typhimurium plate incorporation assay.

Chromosome aberration

The test substance was assessed for its potential to induce micronuclei in TK6 cells in vitro (clastogenic or aneugenic activity). Three independent experiments were carried out 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. The test groups printed in bold type were evaluated.

1st Experiment

4 hours exposure, 24 hours harvest time, without S9 mix

0; 34.38; 68.75; 137.50;275.00; 550.00and1100.00 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix (not valid)

0; 34.38; 68.75; 137.50;275.00; 550.00and1100.00 μg/mL

2nd Experiment

4 hours exposure, 24 hours harvest time, with S9 mix

0; 137.50;275.00; 550.00and1100.00 μg/mL

3rd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix

0; 200.00; 400.00;550.00; 800.00and1100.00 μg/mL

4 hours exposure, 44 hours harvest time, with S9 mix

0; 200.00; 400.00;550.00; 800.00and1100.00 μg/mL

In the 1st Experiment cultures treated with the positive control in the presence of S9 mix did not show an increase in the micronucleus frequency. Thus, this experimental part is considered as invalid and was repeated in the 2nd Experiment. The results of the invalid part of the 1st Experiment will not be shown in this report. The raw data of this experimental part will be archived with the raw data of this study. A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.

The negative controls gave frequencies of micronucleated cells within our historical negative control data range for TK6 cells. Both positive control substances, mitomycin C (MMC) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei.

Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in human TK6 cells in the absence and the presence of metabolic activation.

On the basis of the results of the present study, the test substance did not cause any

biologically relevant increase in the number of cells containing micronuclei either without S9

mix or after adding a metabolizing system.

Gene mutation

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. Four independent experiments were carried out, with and/or 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 top concentration was determined to be the limit concentration of approx. 10 mM (1100 μg/mL). In the main experiments the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

without S9 mix

0; 68.8;137.5; 275.0; 550.0; 1100.0 μg/mL

with S9 mix

0; 68.8;137.5; 275.0; 550.0; 1100.0 μg/mL

2nd Experiment (not valid; data not shown)

without S9 mix

0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL

with S9 mix

0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL

3rd Experiment

without S9 mix

0; 87.5;175.0; 350.0; 700.0; 1100.0 μg/mL

with S9 mix (not valid; data not shown)

0; 87.5; 175.0; 350.0; 700.0; 1100.0 μg/mL

4th Experiment

with S9 mix

0; 87.5;175.0; 350.0; 700.0; 1100.0 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 negative 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 all experiments, in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.

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 all experiments performed independently of each other.

Mitotic aneuploidy assays in the absence of metabolic activation with the yeast, Saccharomyces cerevisiae, provided negative and ambiguous results for propyl acetate (Zimmermann et al. 1985, Zimmermann et al. 1988, Zimmermann et al. 1989; reliability score 3).

Analogous substances:

Butyl acetate

Butyl acetate (maximum concentration: 2 mg/mL) was negative for chromosomal aberrations in an in vitro test using Chinese hamster lung cells without metabolic activation (Ishidate et al. 1984).

Propan-1-ol

In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA 1535, TA 100, TA 1537, TA 98) and E.coli (E. coli WP2 uvrA) were exposed to propan-1 -ol using the standard plate test (SPT) and the preincubation test (PIT) in the presence and the absence of mammalian metabolic activation. The tests were performed using five different concentrations up to 5000µg/plate. There was no evidence of induced mutant colonies over background. The study was performed under GLP conditions and satisfies the requirements of the OECD guideline 471 (BASF SE, 2009).

In an OECD guideline conform in vitro mammalian cell gene mutation assay (HPRT test, OECD 476) Chinese hamster ovary (CHO) cells were exposed to propan-1-ol at 4 different concentrations of up to 600 µg/ml (10 mM) in the presence and absence of mammalian metabolic activation. Under the experimental conditions of this study, the test item propan-1-ol was not mutagenic in the HPRT locus assay (BASF SE, 2010).

 

In a mammalian cell cytogenetics assay (chromosome aberration), V79 cultures were exposed to propan-1-ol (99.8% pure, vehicle: Minimum essential medium) at concentrations between 75 and 600 µg/ml (with 2 fold increment in the concentration) with and without S9 mix. The cells were exposed for 4h and 18 hours. Harvesting was performed for each time point 18 and 28 hours after the start of incubation. Tested up to the limit concentration of 10 mM (600 µg/ml), no cytotoxicity was observed in V79 cells. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background (BASF AG, 2003). The study was performed according GLP standards and also satisfies the requirement for Test Guideline (in vitro mammalian cytogenetics (chromosome aberration) OECD 473 (chinese hamster lungs fibroblast, V79).

 

Propan-1 -ol (maximum dose: 6000 µg/mL) was negative in vitro SCE assays conducted in Chinese hamster lung cells with and without metabolic activation (Van der Hude et al. 1987).

 

In vivo

no data available

Read across justification to propan-1-ol and n-butyl acetate for filling data gaps of n-propyl acetate:

As indicated by toxicokinetic studies (see chapter on toxicokinetics, metabolism and distribution), n-propyl acetate is rapidly hydrolyzed to propan-1-ol and acetate (acetic acid). Available data on propan-1-ol is therefore suitable for filling data gaps of n-propyl acetate. N-propyl acetate and n-butyl acetate differ structurally by only one –CH2 group and both substances have a similar toxicological profile. The available data for n-butyl acetate is therefore suitable for filling the data gaps of n-propyl acetate due to structural similarities. For a detailed justification of read-across, please refer to IUCLID section 13.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation 1272/2008. Based on the criteria laid down in Regulation (EC) No. 1272/2008, as amended for the second time in Directive EC 286/2011, classification as a mutagen is not warranted.