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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-01-15 to 2016-03-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2014
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Method

Species / strain
Species / strain / cell type:
other: Peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg) induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1: 1.7, 5.4, 17, 52, 164 µg/ml, with and without metabolic activation
Experiment 2: 5.4, 17 and 52 µg/ml, without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The solvent was chosen based on the results from the solubility test.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation, 48 hour exposure period
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation, 3 hour exposure period
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

ACTIVATION: To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. The added co-factores were: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES.

DURATION

- Exposure duration:
Experiment 1: 3 hours, with and without metabolic activation
Experiment 2: 24 and 48 hours, without metabolic activation
- Expression time (cells in growth medium):
Experiment 1: 20-22 hours

- Fixation time (start of exposure up to fixation or harvest of cells):
Experiment 1: 24 hours
Experiment 2: The cells were fixed immediately after exposure

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/ml medium) was added during the last 2.5 - 3 h of the culture period
STAIN (for cytogenetic assays): 5 % v/v Giemsa

NUMBER OF REPLICATIONS: duplicate cell cultures

NUMBER OF CELLS EVALUATED: 1000 cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A test item is considered positive (clastogenic) in the chromosome aberration test if all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with an Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
Statistics:
Fisher’s exact test and Cochran Armitage trend test

Results and discussion

Test results
Key result
Species / strain:
other: Peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation:
Experiment 1: At the 3 h exposure time a concentration of 164 µg/ml the test item precipitated in the culture medium
Experiemnt 2: ). At 24 and 48 hour exposure times a concentration of 52 µg/ml test item precipitated in the culture medium

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Polyploidy: The test item did not increase the number of polyploid cells.
- Endoreplication: The test item did not increase the number of endoreplicated chromosomes
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

Table 1: Mitotic index of human lymphocyte cultures treated with the test item in the first (experiement 1) cytogenetic assay

Test item concentration (µg/ml)

Number of metaphases

 

 

Absolute

Number of cells scored

Percentage of control

3 hour exposure, 24 hour fixation time, without metabolic activation

Control (DMSO)

96 - 110

1027 - 1011

100

17

82 - 89

1018 - 1010

83

52

86 - 95

1008 - 1011

88

164

72 - 93

1015 - 1002

80

MMC-C; 0.5 µg/ml

55 - 63

1020 - 1011

57

MMC-C; 0.75 µg/ml

45 - 43

1022 - 1007

43

3 hour exposure, 24 hour fixation time, with metabolic activation

Control

107 – 98

1007 – 1014

100

17

99 – 85

1009 – 1026

90

52

95 – 101

1016 – 1008

96

164

94 – 82

1002 – 1012

86

CP; 10 µg/ml

62 - 50

1014 - 1004

55

Table 2: Chromosome aberrations in human lymphocyte cultures treated with the test item in the absence of S9-mix in the first (experiment 1) cytogenetic assay (3 h exposure time, 24 h fixation time)

Concentration

DMSO

(1.0% v/v)

17

µg/ml

52

µg/ml

164

µg/ml

MMC-C

0.5µg/ml

Culture

A           B     A+B

A           B     A+B

A           B     A+B

A           B     A+B

A           B     A+B

Mitotic

Index (%)

          100

            83

            88

            80

            57

No. of

Cells scored

150    150     300

150    150     300

150    150     300

150    150     300

150    150     300

No. of

Cells with

aberrations

(+ gaps)a)

1

0

1

0

0

0

0

0

0

0

0

0

40

48

***)

88

 

No. of

Cells with

aberrations

(- gaps)

1

0

1

0

0

0

0

0

0

0

0

0

40

46

***)

86

 

total aberr

(+ gaps)

1

0

 

0

0

 

0

0

 

0

0

 

51

48

 

total aberr

(- gaps)

1

0

 

0

0

 

0

0

 

0

0

 

51

46

 

Table 3: Chromosome aberrations in human lymphocyte cultures treated with the test item in the presence of S9-mix in the first (experiment 1) cytogenetic assay (3 h exposure time, 24 h fixation time)

Conc                                                 

DMSO

(1.0% v/v)

17

µg/ml

52

µg/ml

164

µg/ml

CP

10µg/ml

Culture

A           B     A+B

A           BA+B

A           BA+B

A           BA+B

A           B A+B

Mitotic

Index (%)

          100

            90

            96

            86

            55

No. of

Cells scored

150    150     300

150    150300

150    150300

150    150300

150    150 300

No. of

Cells with

aberrations

(+ gaps)a)

0

1

1

0

0

0

0

0

0

0

0

0

36

30

***)

66

 

No. of

Cells with

aberrations

(- gaps)

0

1

1

0

0

0

0

0

0

0

0

0

36

30

***)

66

 

total aberr

(+ gaps)

0

1

 

0

0

 

0

0

 

0

0

 

42

37

 

total aberr

(- gaps)

0

1

 

0

0

 

0

0

 

0

0

 

42

37

 

 

Table 4: Mitotic index of human lymphocyte cultures treated with the test item in the second (experiement 2) cytogenetic assay

Test item concentration (µg/ml)

Number of metaphases

 

 

Absolute

Number of cells scored

Percentage of control

24 hour exposure, 24 hour fixation time, without metabolic activation

Control

69 - 78

1021 - 1013

100

5.4

68 - 69

1013 - 1016

93

17

76 - 66

1033 -1002

97

52

73 - 69

1030 - 1018

97

MMC-C; 0.2 µg/ml

39 - 45

1029 - 1005

57

MMC-C; 0.3 µg/ml

18 - 23

1017 - 1010

28

48 hour exposure, 48 hour fixation time, without metabolic activation

Control

44 - 44

1014 - 1013

100

5.4

47 – 43

1013 - 1021

102

17

37 - 37

1003 - 1004

84

52

38 - 37

1013 - 1006

85

MMC-C; 0.1 µg/ml

34 - 36

1020 – 1016

80

MMC-C; 0.15 µg/ml

29 - 31

1003 - 1021

68

Table 5: Chromosome aberrations in human lymphocyte cultures treated with the test item in the absence of S9-mix in the second (experiment 2) cytogenetic assay (24 h exposure time, 24 h fixation time)

Conc

DMSO

(1.0% v/v)

5.4

µg/ml

17

µg/ml

52

µg/ml

MMC-C

0.2µg/ml

Culture

A           B  A+B

A           B  A+B

A           B  A+B

A           B  A+B

A           B    A+B

Mitotic

Index (%)

          100

            93

            97

            97

            57

No. of

Cells scored

150    150   300

150    150   300

150    150   300

150    150   300

150    150     300

No. of

Cells with

aberrations

(+ gaps)a)

0

1

1

1

1

2

2

0

2

1

0

1

53

43

***)

96

 

No. of

Cells with

aberrations

(- gaps)

0

1

1

1

1

2

2

0

2

1

0

1

52

42

***)

94

 

total aberr

(+ gaps)

0

1

 

1

1

 

2

0

 

1

0

 

60

56

 

total aberr

(- gaps)

0

1

 

1

1

 

2

0

 

1

0

 

59

53

 

Table 6: Chromosome aberrations in human lymphocyte cultures treated with the test item in the absence of S9-mix in the second (experiement 2) cytogenetic assay (48 h exposure time, 48 h fixation time)

Conc

DMSO

(1.0% v/v)

5.4

µg/ml

17

µg/ml

52

µg/ml

MMC-C

0.1µg/ml

Culture

A           B     A+B

A           B     A+B

A           B     A+B

A           B     A+B

A           B     A+B

Mitotic

Index (%)

          100

          102

            84

            85

            80

No. of

Cells scored

150    150     300

150    150     300

150    150     300

150    150     300

150    150     300

No. of

Cells with

aberrations

(+ gaps)a)

0

1

1

0

0

0

0

1

1

0

0

0

42

53

***)

95

 

No. of

Cells with

aberrations

(- gaps)

0

1

1

0

0

0

0

0

0

0

0

0

42

52

***)

94

 

total aberr

(+ gaps)

0

1

 

0

0

 

0

1

 

0

0

 

51

66

 

total aberr

(- gaps)

0

1

 

0

0

 

0

0

 

0

0

 

51

65

 

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001

Applicant's summary and conclusion

Conclusions:
1,3,5-Trimethyl-1,1,3,5,5-pentaphenyltrisiloxane has been tested in a valid study for ability to induce chromosome aberrations in Peripheral human lymphocytes according to OECD TG 473 (2014), and in compliance with GLP. No statistically significant or biologically relevant increase in the number of cells with chromosome aberrations was observed either with or without metabolic activation when tested up to precipitating concentrations. Appropriate positive and negative controls were used and gave the expected results. It is concluded that the test item is negative for the induction of chromosome aberrations under the conditions of the study.