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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria: negative in a Salmonella typhimurium and Escherichia coli reverse mutation assay (OECD 471)


In vitro cytogenicity study in mammalian cells: negative in the in vitro micronucleus test in human lymphocytes (OECD 487)


In vitro gene mutation study in mammalian cells: No data for the test item are available. Read-across to CAS 91031-31-1: not mutagenic (OECD 476).  Furthermore, the read across substances propane-1,3-diol and octanoic acid were negative in the in vitro mammalian gene mutation study according to OECD Guideline No. 476 (data obtained from the respective ECHA disseminated REACH Dossiers).


 

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:
22 January 2020 to 16 April 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
please see "Principles of method if other than guideline"
Principles of method if other than guideline:
A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human lymphocytes, primary culture
Details on mammalian cell type (if applicable):
CELLS USED
- Suitability of cells: The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Sex, age and number of blood donors: Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a female donor (29 years old in Experiment I and 30 years old in Experiment III) and from a male donor (21 years old) for Experiment II.
- Mitogen used for lymphocytes: PHA

MEDIA USED
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5% CO2 in humidified air.
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- method of preparation of S9 mix: The S9 was prepared and stored according to the currently valid version of the ICCR-Roßdorf SOP for rat liver S9 preparation.
- concentration or volume of S9 mix and S9 in the final culture medium: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
The protein concentration of the S9 preparation used for this study was 30.2 mg/mL.
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
Test concentrations with justification for top dose:
Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterised by the percentages of reduction in the CBPI in comparison to the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.
This preliminary test was designated Experiment I, since the cultures fulfilled the acceptability criteria and appropriate concentrations could be selected for cytogenetic evaluation.
Vehicle / solvent:
- Solvent used: Ethanol, the final concentration of Ethanol in the culture medium was 0.5 %.
- Justification for choice of solvent: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: demecolcine (without metabolic activation)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours (pulse exposure), 20 hours (continuous exposure)
- Harvest time after the end of treatment: 36 hours (pulse exposure), 20 hours (continuous exposure)
- Identity of cytokinesis blocking substance: Cytochalasin B (4 μg/mL), duration and period of cell exposure: 20 hours
- Methods of slide preparation and staining technique: The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa, mounted after drying and covered with a coverslip.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.
- Criteria for scoring micronucleated cells: The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (COUNTRYMAN P.I. and HEDDLE J.A. (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index


Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.

Statistics:
Statistical significance was confirmed by the Chi Square Test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
A linear regression was performed using a validated test script of “R”, to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Key result
Species / strain:
lymphocytes: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In Experiment I in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration. The next higher tested concentration, however, which was separated by a factor smaller than requested by the guideline was not evaluable for cytogenetic damage, due to strong cytotoxic effects. In the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration which showed phase separation. In Experiment III in the absence of S9 mix, clear cytotoxicity (47.2% cytostasis) was observed at the highest evaluable concentration. In Experiment I in presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. In Experiment I the absence of S9 mix, statistically significant increases in micronucleated cells were observed after treatment with all evaluated concentrations. The value of 1.65% micronucleated cells after treatment with 653 μg/mL clearly exceeded the range of the historical control data (0.00 – 1.04% micronucleated cells) and dose dependency, tested by trend test, was observed.
In the confirmatory Experiment II in the absence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. The value 1.15% micronucleated cells after treatment with 299 μg/mL, however, exceeded the range of the 95% control limit of the historical control data (0.00 – 1.04% micronucleated cells), but was clearly within the min-max range (0.05 – 1.20% micronucleated cells). Neither a statistically significant increase nor dose dependency, tested by trend test, was observed. In Experiment III in the absence of S9 mix after continuous treatment, no relevant increases in the numbers of micronucleated cells and no dose dependency, tested by trend test, were observed after treatment with the test item.

Table 2 Summary of results







































































































































































 

Exp.



Preparation interval



Test item  concentration in µg/mL



Proliferation index CBPI



Cytostasis  in %*



Micronucleated cells in %**



95% Ctrl limit



Exposure period 4 h without S9 mix



I



40 h



Solvent control 1/#



1.46



 



0.55



0.00-1.04



Positive control 2



1.56



n.c.



10.25S



213#



1.50



n.c.



1.03S



373#



1.44



4.3



0.98S



653#



1.45



3.2



1.65S



Trend test: p-value 0.491 T



II



40 h



Solvent control 1



1.56



 



0.95



0.00-1.04



Positive control 3



1.51



10



9.50S



171



1.66



n.c.



0.60



299



1.47



15.7



1.15



522PS



1.46



17.8



0.40



Trend test: p-value 0.491



Exposure period 20 h without S9 mix



III



40 h



Solvent control 1#



1.74



 



0.43



0.00-0.86



Positive control 4



1.70



6.6



3.60S



135#



1.71



5.2



0.65



237#



1.66



11.6



0.40



356#



1.39



47.2



0.68



Trend test: p-value 0.522



Exposure period 4 h with S9 mix



I



40 h



Solvent control 1



1.49



 



0.65



0.00-1.03



Positive control 5



1.37



26



3.40S



213



1.51



n.c.



0.40



373



1.50



n.c.



0.80



653PS



1.46



5.9



0.65



Trend test: p-value 0.754



* For the positive control groups and the test item treatment groups the values are related to the solvent controls


** The number of micronucleated cells was determined in a sample of 2000 binucleated cells


# The number of micronucleated cells was determined in a sample of 4000 binucleated cells


PS Phase separation occurred at the end of treatment


S The number of micronucleated cells is statistically significantly higher than corresponding control values


T Trend analysis via linear regression is significant (p ˂ 0.05)


n.c. Not calculated as the CBPI is equal or higher than the solvent control value


solvent control 1 Ethanol 0.5 % (v/v)


positive control 2: MMC 1.0 μg/mL


positive control 3 MMC 0.8 μg/mL


positive control 4 Demecolcine 50 ng/mL


positive control 5 CPA 15.0 μg/mL

Conclusions:
The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Executive summary:

The test item dissolved in ethanol was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. In each experimental group, two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.


The highest applied concentration in this study (2000 μg/mL of the test item) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. In Experiment I in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration. The next higher tested concentration, however, which was separated by a factor smaller than requested by the guideline was not evaluable for cytogenetic damage, due to strong cytotoxic effects. In the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration which showed phase separation. In Experiment III in the absence of S9 mix, clear cytotoxicity (47.2% cytostasis) was observed at the highest evaluable concentration.


In Experiment I in presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. In Experiment I in the absence of S9 mix, statistically significant increases in micronucleated cells were observed after treatment in all evaluated concentrations. The value of 1.65% micronucleated cells after treatment with 653 μg/mL clearly exceeded the range of the historical control data (0.00 – 1.04% micronucleated cells) and dose dependency, tested by trend test, was observed. In the confirmatory Experiment II in the absence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. The value of 1.15% micronucleated cells after treatment with 299 μg/mL, however, exceeded the range of the 95% control limit of the historical control data (0.00 – 1.04% micronucleated cells), but was clearly within the min-max range (0.05 – 1.20% micronucleated cells). Neither a statistically significant increase nor dose dependency, tested by trend test, was observed.


Taken together, none of the findings in Experiment I were confirmed in the confirmatory experiment and these can therefore be regarded as biologically irrelevant.


In Experiment III in the absence of S9 mix after continuous treatment, no relevant increases in the numbers of micronucleated cells and no dose dependency, tested by trend test, were observed after treatment with the test item.


Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June - August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 21, 1997
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 2008
Qualifier:
according to guideline
Guideline:
other: The recommendations of the 'International Workshop on Genotoxicity Tests Workgroup' (the IWGT), published in the literature (Clive et ai., 1995, Moore et al, 1999. 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- method of preparation of S9 mix: Rat liver microsomal enzymes were routinely prepared from adut maie Wistar rats, which were obtained from Charles River, Sulzfeld, Germany. The rats were orally dosed at three consecutive days with a suspension of phenobarbital (Bufa B.V., Usselstein, The Nethedands; 80 mg/kg body weight) and 13-naphthoflavone (Sigma Aldrich Chemie; 100 mg/kg body weight) in com oit (Roth, Karlsruhe, Germany) food access denied 3 to 4 hours befor dosing. One day afler the final exposure (24 h), the rats were sedated and then killed. The rats received a limited quantity of food during the night before sacrification. Livers of the rats were removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4, Merck) containing 0.1 mM Na2-EDTA (Merck). Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer wfth a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supematant (S9-fraction) was transferred into sterile ampules, which were stored in liquid nitfagen (-196°C) for a maximum of 1 year.
Test concentrations with justification for top dose:
First test: 0.1. 0.3. 1, 3. 10, 33, 100 and 333 µg/mL (absence and presence of 8% (v/v) S9-mix)
Second test: 3, 10, 33, 100, 125, 140, 175, 200, 225, 250 and 275 µg/mL (without S9-mix)
0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/mL (with 12 % (v/v) S9-mix)
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h (presence of S9-mix), 3 and 24 hours (absence of S9-mix)
- Expression time (cells in growth medium): 2 days

SELECTION AGENT: fluorothymidine

Second mutagenicity test: To test the mutagen potential of the test item, a second mutation experinent was performed in the absence of S9-mix with a 24 hour treatment period and in the presence of 12% (v1v) S9-mix with a 3 hour incubation.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The mutation assay was considered acceptable if following criteria were met:
a) The absolute cloning efficiency of the solvent controls (CEday2) is, between 65 and 120%. An acceptable number of surviving cells 10^6 could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent controi is ≥ 50 x 10^-6 and ≤ 170 x 10^-6.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 x 10^-6 and for CP not below 700 x 10^-6.
Statistics:
The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126. A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more then MF(contols) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative resut can be made after an additional confirmation study. A test substance is considered negative (not mutagenic) in the mutation assay if: a) None of the tested concentrations reaches a mutation frequency (MF) of controls + 126. b) The resuits are conflrmed in an indepen repeated test.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
First mutagenicity test: No severe toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item cultures were comparable to the numbers of small and large colonies of the solvent controls.
Second mutagenicity test: In the absence of S9-mix, the dose levels of 200 to 275 µg/ml were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were: 3, 10, 33, 100, 125, 140 and 175 µg/ml. In the absence of S9-mix, the relative total growth of the highest test substance concentration 175 µg/mL was reduced by 91% compared solvent control. The relative total growth at 140 µg/mL was reduced by 80% compared to the total growth of the solvent controls
In the presence of S9-mix, no toxicity was observed up to and including the highest tested dose level. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Table 1: Experiment 1: Cytotoxic and mutagenic response of C-SAT 100024 in the mouse lymphoma L5178Y test system

 

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

Mutation frequency x 10-5

total

( small

large )

Without metabolic activation

3 hours treatment

SC1

100

70

100

100

55

( 35

20 )

SC2

101

67

( 48

18 )

0.1

97

101

118

114

56

( 29

25 )

0.3

92

101

118

108

52

( 28

23 )

1

102

118

138

140

66

( 48

16 )

3

102

91

107

109

63

( 46

15 )

10

97

95

111

108

66

( 32

33 )

33

92

97

113

104

76

( 46

28 )

100(1)

90

91

107

97

81

( 32

46 )

333(1)

64

102

120

77

64

( 48

14 )

MMS

64

60

70

45

685

( 521

118 )

With 8% (v/v) metabolic activation

3 hours treatment

SC1

100

80

100

100

70

( 37

31 )

SC2

102

63

( 35

26 )

0.1

93

108

119

111

74

( 47

25 )

0.3

97

89

97

94

71

( 45

24 )

1

95

108

119

113

64

( 42

20 )

3

100

98

107

107

78

( 53

23 )

10

96

95

104

100

87

( 54

29 )

33

81

108

119

96

55

( 32

22 )

100(1)

83

98

107

89

83

( 60

21 )

333(1)

89

94

103

92

63

( 23

39 )

CP

48

60

66

32

1074

( 829

144 )

 

Note: all calculations were made without rounding off
RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate; CP = Cyclophosphamide
(1) = C-SAT 100024 precipitated in the exposure medium

 

Table 2: Experiment 2: Cytotoxic and mutagenic response of C-SAT 100024 in the mouse lymphoma L5178Y test system

 

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

Mutation frequency x 10-5

total

( small

large )

Without metabolic activation

24 hours treatment

SC1

100

120

100

100

50

( 23

25 )

SC2

110

51

( 29

20 )

3

79

135

117

92

58

( 42

14 )

10

91

137

119

109

51

( 39

11 )

33

74

133

115

85

71

( 32

35 )

100(1)

31

110

96

30

100

( 35

60 )

125(1)

30

118

103

31

70

( 31

36 )

140(1)

20

118

103

20

103

( 51

47 )

175(1)

10

102

89

9

134

( 53

72 )

MMS

84

102

89

75

853

( 456

230 )

With 12% (v/v) metabolic activation

3 hours treatment

SC1

100

113

100

100

67

( 44

21 )

SC2

105

77

( 50

24 )

0.1

99

110

101

100

73

( 49

21 )

0.3

103

123

113

117

72

( 47

23 )

1

111

104

95

105

82

( 52

27 )

3

117

97

89

104

94

( 62

29 )

10

120

115

105

126

87

( 57

26 )

33

111

107

98

108

85

( 59

23 )

100(1)

102

131

120

123

80

( 51

26 )

333(1)

94

97

89

83

92

( 64

24 )

CP

91

70

64

59

979

( 621

221 )

 

Note: all calculations were made without rounding off
RSG = Relative Suspension Growth; CE = Cloninq Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate; CP = Cyclophosphamide
(1) = C-SAT 100024 precipitated in the exposure medium

Conclusions:
The evaluation of the mutagenic activity of the test item in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat), no mutagenicity was detected.
Executive summary:

The effects of the test item on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells was assessed. The test was performed in two independent experiments in the absence and presence of S9 -mix (rat liver S9-mix induced by a combination of phenobarbital and & β-naphthoflavone). The study procedures described in this report were based on the OECD guideline 476. The test item was suspended in dimethyl sulfoxide (DMSO). In the first experiment, the test item was tested up to concentrations of 333 µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. However the test item precipitated already in the exposure medium at 100 mg/mL. In the second experiment, the test item was tested up to a concentrations of 333 µg/mL in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours and 3 hours for incubation in absence and presence of S9-mix, respectively. The test item was tested up to cytotoxic level of 91% in the absence of S9--mix. No toxicity was observed in the presence of S9-mix. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical controls and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 11- and 17-fold for MMS in the absence of S9-mix, and by 16- and 14-fold for CIP in the presence of S9-mix. It was therefore concluded that the test conditions, both in absence and presence of S9-mix were appropriate and that the metabolic activation system (S9-mix) worked properly. In absence of S9-mix, the test item did not induce a significant increase in mutation frequency in the first experiment. The result was confirmed in an independently repeated experiment with modification in duration of treatment time. In presence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independently repeated experiment with modifications in the concentration of the S9 for metabolic activation. It is concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to chapter 13.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-16 to 2017-04-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium: histidine (his)
Escherichia coli: tryptophan (trp)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: phenobarbital/β-naphthoflavone induced rat liver
- method of preparation of S9 mix: 10 % v/v S9 in the S9 mix; S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4
- volume of S9 mix and S9 in the final culture medium: 500 μL S9 mix, S9 protein concentration of 35.4 mg/mL in the pre-experiment / experiment I and 32.3 mg/mL in experiment II
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
Test concentrations with justification for top dose:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
5000 μg/plate were chosen as maximal concentration based on pre-experiment and guideline.
Vehicle / solvent:
- Solvent used: DMSO

- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD): without metabolic activation for TA 1537, TA 98 2-aminoanthracene (2-AA): with metabolic activation for TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10^8-10^9 cells/mL, 100 μL used
- Test substance added in agar (plate incorporation) and preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 60 min
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition, reduction in the number of spontaneous revertants
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
Following was recorded: individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with S9 mix: starting at 2500 μg/plate without S9 mix: starting at 1000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with S9 mix: starting at 1000 μg/plate without S9 mix: starting at 2500 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with S9 mix: starting at 2500 μg/plate without S9 mix: starting at 333 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with S9 mix: starting at 2500 μg/plate without S9 mix: starting at 1000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 mix: starting at 2500 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Experiment I (Plate Incorporation)

















































































































































































































































Dose (µg/plate)Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
 TA1535TA1537TA98TA100WP2 uvrA
  
 Results without S9
DMSO8 ± 211 ± 130 ± 4199 ± 2045 ± 7
Untreated10 ± 410 ± 531 ± 4210 ± 1048 ± 6
38 ± 38 ± 326 ± 0216 ± 1251 ± 3
1012 ± 28 ± 323 ± 4217 ± 1648 ± 5
3311 ± 18 ± 330 ± 4193 ± 1441 ± 13
1009 ± 011 ± 230 ± 3207 ± 1252 ± 3
33310 ± 112 ± 529 ± 4154 ± 239 ± 6
10009 ± 112 ± 522 ± 452 ± 619 ± 2
25008 ± 2 PM12 ± 2 PM8 ± 2 PM6 ± 1 PM5 ± 1 PM
50001 ± 1 PM5 ± 2 PM1 ± 2 PM1 ± 1 PM1 ± 1 PM
NaN3 (10)1194 ± 227  2097 ± 74 
4-NOPD (10)  448 ± 39  
4-NOPD (50) 89 ± 6   
MMS (2.0 µL)    904 ± 24
  
 Results with S9
DMSO13 ± 317 ± 345 ± 8200 ± 1449 ± 8
Untreated16 ± 418 ± 552 ± 5200 ± 1459 ± 7
314 ± 316 ± 642 ± 12186 ± 866 ± 8
1010 ± 118 ± 641 ± 6183 ± 354 ± 5
3311 ± 211 ± 139 ± 7200 ± 1955 ± 4
10013 ± 511 ± 144 ± 1176 ± 1856 ± 14
33311 ± 415 ± 643 ± 13199 ± 758 ± 5
100015 ± 415 ± 131 ± 4167 ± 540 ± 2
250013 ± 3 P9 ± 3 PM22 ± 6 P143 ± 10 P32 ± 5 P
500014 ± 2 P5 ± 1 PM16 ± 3 PM65 ± 17 P8 ± 2 PM
2-AA (2.5)47 ± 20232 ± 203601 ± 3933992 ± 163 
2-AA (10.0)    428 ± 25

 


Table 2: Experiment II (Pre-Incubation)

















































































































































































































































Dose (µg/plate)Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
 TA1535TA1537TA98TA100WP2 uvrA
  
 Results without S9
DMSO13 ± 512 ± 327 ± 1198 ± 544 ± 6
Untreated10 ± 411 ± 427 ± 9196 ± 746 ± 8
3n.a.n.a.25 ± 6156 ± 744 ± 7
1010 ± 311 ± 528 ± 5141 ± 1049 ± 10
3314 ± 210 ± 227 ± 8152 ± 144 ± 4
1009 ± 315 ± 325 ± 5111 ± 1144 ± 2
33312 ± 29 ± 3 R27 ± 9 R62 ± 4 RM29 ± 3 R
100012 ± 2 R5 ± 2 MR15 ± 6 MR3 ± 0 RM8 ± 1 MR
25004 ± 1 MR1 ± 1 MR1 ± 1 RM0 ± 0 R0 ± 0 R
50000 ± 0 RP0 ± 0 RP0 ± 0 RP0 ± 0 RP0 ± 0 RP
NaN3 (10)1338 ± 69  1848 ± 59 
4-NOPD (10)  312 ± 29  
4-NOPD (50) 83 ± 4   
MMS (2.0 µL)    927 ± 37
  
 Results with S9
DMSO12 ± 514 ± 437 ± 5195 ± 749 ± 7
Untreated11 ± 214 ± 336 ± 7187 ± 762 ± 13
3n.a.n.a.47 ± 5122 ± 2059 ± 9
1011 ± 217 ± 435 ± 9130 ± 558 ± 16
3311 ± 216 ± 135 ± 8132 ± 1046 ± 6
10014 ± 311 ± 535 ± 5135 ± 2055 ± 6
33310 ± 120 ± 335 ± 4127 ± 1447 ± 3
100012 ± 414 ± 410 ± 1 MR130 ± 242 ± 8
25008 ± 3 R2 ± 1 MR6 ± 0 MR69 ± 24 R20 ± 5 R
50009 ± 1 RMP4 ± 2 MRP0 ± 0 RP2 ± 1 RP10 ± 2 MRP
2-AA (2.5)380 ± 11108 ± 194058 ± 3373801 ± 112 
2-AA (10.0)    337 ± 12

R = Reduced background growth
M = Manual count
P = Precipitate

Conclusions:
The test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

A study according to OECD 471 was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.


The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:


Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate


Experiment II:


Strains TA 1535 and 1537: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate


The remaining strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate


The test item precipitated in the overlay agar in the test tubes only in experiment II at 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 2500 to 5000 μg/plate and in experiment II at 5000 μg/plate. The undissolved particles had no influence on the data recording.


The plates incubated with the test item showed reduced background growth only in experiment II in all strains used with and without S9 mix.


Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in all strains used.


No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.


Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.


In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.


Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay


A study according to OECD 471 was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:


Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate


Experiment II: Strains TA 1535 and 1537: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate; the remaining strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate


The test item precipitated in the overlay agar in the test tubes only in experiment II at 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 2500 to 5000 μg/plate and in experiment II at 5000 μg/plate. The undissolved particles had no influence on the data recording. The plates incubated with the test item showed reduced background growth only in experiment II in all strains used with and without S9 mix. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in all strains used. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.


 


In vitro cytogenicity study in mammalian cells


The test item dissolved in ethanol was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. In each experimental group, two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage. In Experiment I in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration. The next higher tested concentration, however, which was separated by a factor smaller than requested by the guideline was not evaluable for cytogenetic damage, due to strong cytotoxic effects. In the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration which showed phase separation. In Experiment III in the absence of S9 mix, clear cytotoxicity (47.2% cytostasis) was observed at the highest evaluable concentration. In Experiment I in presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. In Experiment I in the absence of S9 mix, statistically significant increases in micronucleated cells were observed after treatment in all evaluated concentrations. The value of 1.65% micronucleated cells after treatment with 653 μg/mL clearly exceeded the range of the historical control data (0.00 – 1.04% micronucleated cells) and dose dependency, tested by trend test, was observed. In the confirmatory Experiment II in the absence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. The value of 1.15% micronucleated cells after treatment with 299 μg/mL, however, exceeded the range of the 95% control limit of the historical control data (0.00 – 1.04% micronucleated cells), but was clearly within the min-max range (0.05 – 1.20% micronucleated cells). Neither a statistically significant increase nor dose dependency, tested by trend test, was observed. Taken together, none of the findings in Experiment I were confirmed in the confirmatory experiment and these can therefore be regarded as biologically irrelevant. In Experiment III in the absence of S9 mix after continuous treatment, no relevant increases in the numbers of micronucleated cells and no dose dependency, tested by trend test, were observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. As a conclusion, the test item was not mutagenic in this assay.


 


In vitro gene mutation study in mammalian cells


No data on the test item itself are available. Therefore read-across was applied.


CAS 91031-31-1 (Fatty acids, C16-18, esters with ethylene glycol):


The effects of the source substance on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells was assessed. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and & β-naphthoflavone). The study procedures described in this report were based on the OECD guideline 476. The substance was suspended in dimethyl sulfoxide (DMSO). In the first experiment, the test item was tested up to concentrations of 333 µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. However the test item precipitated already in the exposure medium at 100 mg/mL. In the second experiment, the test item was tested up to a concentration of 333 µg/mL in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours and 3 hours for incubation in absence and presence of S9-mix, respectively. The test item was tested up to cytotoxic level of 91% in the absence of S9-mix. No toxicity was observed in the presence of S9-mix. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical controls and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 11- and 17-fold for MMS in the absence of S9-mix, and by 16- and 14-fold for CIP in the presence of S9-mix. It was therefore concluded that the test conditions, both in absence and presence of S9-mix were appropriate and that the metabolic activation system (S9-mix) worked properly. In absence of S9-mix, the test item did not induce a significant increase in mutation frequency in the first experiment. The result was confirmed in an independently repeated experiment with modification in duration of treatment time. In presence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independently repeated experiment with modifications in the concentration of the S9 for metabolic activation. It is concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.


CAS 504-63-2 (propane-1,3-diol) and CAS 124-07-2 (octanoic acid) are also suitable for read across because compared to the target substance they degrade to common products as the target substance is a reaction product of these source substances. For comparison, the following data was obtained from the ECHA disseminated REACH-Dossiers of these two substances:


The potential of 1,3-propanediol to induce gene mutations in the HPRT locus in V79 cells of the Chinese hamster was investigated according to OECD Guideline No. 476. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. The following concentrations were tested: 0, 250, 1000, 2500, and 5000 µg/mL. Up to the highest concentration tested, no relevant increases in mutant colony numbers was obtained in the two independent experiments. Therefore, it was concluded that the test substance did not induce gene mutations at the HPRT locus in V79 cells and is considered to be non-mutagenic in this HPRT assay.


Octanoic acid was itself assessed by read-across (category approach). The test item was decanoic acid, which is a structural anlogue to ocatnoic acid only differing in the carbon chain length. The assay according to OECD Guideline 476 was conducted with mouse lymphoma L5178Y cells. Decanoic acid is considered to be non-mutagenic in this assay.

Justification for classification or non-classification

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


Based on available data on genetic mutation, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.