Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity: negative with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or WP uvrA (RCC, 2004)

Chromosome aberration: negative with or without metabolic activation in Chinese hamster V79 cells (RCC, 2004).

Mammalian mutagenicity study: read-across from 1,3,5-trimethyl-1,1,3,5,5-pentaphenyltrisiloxane, negative with and without metabolic activation in Mouse lymphoma L5178Y cells (Charles River, 2016) (OECD TG 490).

The studies were conducted according to an appropriate OECD test guideline, and in compliance with GLP.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 May 2004 to 12 August 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro chromosome aberration test
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: Not indicated by the sponsor
- Solubility and stability of the test substance in the solvent/vehicle: stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in ethanol. The final concentration of ethanol in the culture medium was 0.5% (v/v)
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing
- Suitability of cells: recommended in guideline
- Cell cycle length, doubling time or proliferation index: 12 hours
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: stored in liquid nitrogen
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: not specified
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I, 4 hours exposure without metabolic activation: 0.025, 0.050, 0.100, 0.150, 0.200, 0.300 µL/mL
Experiment II, 18 hours exposure without metabolic activation: 0.006, 0.013, 0.025, 0.050, 0.100, 0.200 µL/mL
Experiment II, 24 hours exposure without metabolic activation: 0.013, 0.025, 0.050, 0.100 µL/mL
Experiment I, 4 hours exposure with metabolic activation: 0.003, 0.006, 0.013, 0.025, 0.050, 0.100 µL/mL
Experiment II, 4 hours exposure with metabolic activation: 0.013, 0.025, 0.050, 0.100, 0.200 and 0.400 µL/mL
Repetition of Experiment II, 4 hours exposure with metabolic activation, due to missing test item precipitation: 0.040, 0.080, 0.160, 0.310, 0.630, 1.250, 2.500, 5.000 µL/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 40000 cells/slide

METABOLIC ACTIVATION: 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. Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl, 33 mM KCI, 5 mM glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

DURATION
- Preincubation period: 48 hours
- Exposure duration: Experiment I, 4 hours with and without metabolic activation; Experiment II, 18 and 28 hours without metabolic activation; Experiment II, 4 hours with metabolic activation
- Expression time (cells in growth medium): Experiment I, 14 hours with and without metabolic activation; Experiment II, 14 or 28 hours with metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): Experiment I 18 hours with and without metabolic activation, Experiment II, 18 or 28 hours with and without metabolic activation.

SPINDLE INHIBITOR (cytogenetic assays): colcemid for 2.5 hours

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Per experiment two slides per group were prepared.

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not applicable

Rationale for test conditions:
The highest concentration used in the cytogenetic experiments was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible. 5 µL/mL of CBI were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 0.04 and 5 µL/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test item after 4 hours treatment was observed at 0.16 µL/mL and above in the absence of S9 mix and at 0.04µL/mL in the presence of S9 mix.

Since no clear toxicity was observed in the pre-test on toxicity, the test item was tested up to a concentration exhibiting clear test item precipitation as recommended in the OECD Guideline 473. Therefore, 0.3µL/mL was chosen as top treatment concentration in the absence of S9 mix and 0.1 µL/mL in the presence of S9 mix.

Dose selection of experiment Il was also based on toxicity data and the occurrence of precipitation. In the range finding experiment clearly reduced cell numbers were observed after 24 hours exposure with 0.04 to 0.63 µL/mL. Therefore, 0.2 µL/mL and 0.1 µL/mL were chosen as top treatment concentrations for continuous treatment (18 hrs and 28 hours, respectively) in the absence of S9 mix. In the presence of S9 mix 0.4 µL/mL were chosen as top treatment concentration with respect to the results obtained in experiment l. Due to missing test item precipitation, this experimental part was repeated with a top test item concentration of 5 µL/mL.

Evaluation criteria:
A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (9) (p < 0.05).
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: tested up to cytotoxic and/or precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Evaporation from medium: not examined
- Water solubility: not examined
- Precipitation:

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment, precipitation of the test item in culture medium was observed after 4 hours treatment with 0.16 µL/mL and above in the absence of S9 mix and with 0.04 µL/mL and above in the presence of S9 mix. In addition, after 24 hours continuous treatment precipitation occurred with 0.04 µL/mL and above in the absence of S9 mix. No relevant influence of the test item on the pH value was observed (solvent control: pH 7.3 versus pH 7.3 at 5 µL/mL). Due to technical problems at measuring the osmolarity of the highest concentration with the oily phase on the top the osmolarity was measured at 2.5 µL/mL (371 mOsm versus 425 mOsm in the solvent control). No relevant influence on the osmolarity was observed.


HISTORICAL CONTROL DATA
- Positive historical control data: within the ranges for positive control data
- Negative (solvent/vehicle) historical control data: within the ranges for negative control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: In the cytogenetic experiments, toxic effects indicated by reduced cell numbers of below 50 % of control were observed in experiment Il after 18 hours continuous treatment with O. 100 µL/mL in the absence of S9 mix. In all experimental parts of this study with 4 hours treatment with and without S9 mix no clear toxic effects indicated by reduced mitotic indices or cell numbers were observed up to the highest tested test item concentration.

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.8 - 2.3 %) as compared to the rates of the solvent controls (1.1 - 1.9 %).

See attachment for results table.

Conclusions:
The test substance has been tested for ability to cause chromosome aberrations in Chinese hamster ovary cells according to OECD TG 473 and under GLP (RCC, 2004). No increase in the number of cells with aberrations was observed either with or without metabolic activation in Chinese hamster V79 cells. Appropriate solvent, positive and negative controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of this study.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 June 2004 to 05 July 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: Not indicated by the sponsor
- Solubility and stability of the test substance in the solvent/vehicle: stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in ethanol. The final concentration of ethanol in the culture medium was 0.5% (v/v)
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
33, 100, 333, 1000, 2500 and 5000µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility
properties and its relative non-toxicity to the bacteria.
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding (if applicable):

ACTIVATION: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgC12, 33 mM KCI , 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.


DURATION
- Preincubation period: In the pre-incubation assay 100 PL test solution, 500µl S9 mix I S9 mix substitution buffer and 100 PL bacterial suspension were mixed in a test tube and shaken at 370 C for 60 minutes. After pre-incubation 2.0 mL overlay agar (450 C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 370 C in the dark.
- Exposure duration: 60 minutes
- Expression time (cells in growth medium): 48 hours

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: not specified

DETERMINATION OF CYTOTOXICITY: cloning efficiency; relative total growth


Rationale for test conditions:
In the pre-experiment the concentration range of the test item was 3 — 5000 µg/plate. The pre-experiment is reported as part of experiment I since no toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.
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:
No statistical evaluation was performed.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not specified
- Effects of osmolality: not specified
- Evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: not specified

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.


HISTORICAL CONTROL DATA
- Positive historical control data: within ranges of historical positive control data
- Negative (solvent/vehicle) historical control data: within ranges of historical negative control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

See attachment for results table

Conclusions:
The test substance has been tested in a valid bacterial reverse mutation assay, according to the OECD TG 471 (1989), and under GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or WP uvrA (RCC, 2004).
No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to limit concentration. Appropriate negative, positive and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-06-07 to 2016-08-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0006760363
- Expiration date of the lot/batch: 8th November 2016
- Purity test date: 8th October 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: dimethyl sulfoxide
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: stable in dimethyl sulfoxide

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no specific handling conditions required
- Preliminary purification step (if any): no correction was made for the purity/composition of the test item
Target gene:
thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, USA
- Suitability of cells: recommended test system in international guidelines
- Cell cycle length, doubling time or proliferation index: no data
- Sex, age and number of blood donors if applicable: no data
- Whether whole blood or separated lymphocytes were used if applicable: no data
- Number of passages if applicable: no data
- Methods for maintenance in cell culture if applicable: no data
- Modal number of chromosomes: no data
- Normal (negative control) cell cycle time: no data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight) induced rat liver S9
Test concentrations with justification for top dose:
Dose range finding test: 1.7, 5.4, 17, 52, 164, 512 μg/ml (with and without S9 mix)
Experiment I: 0.056, 0.18, 0.55, 1.7, 5.4, 17, 52 and 164 μg/ml (with and without S9 mix)
Experiment II: 0.018, 0.056, 0.18, 0.55, 1.7, 5.4, 17, 52 and 164 μg/ml (without S9 mix)
Doses were selected based on cytotoxicity results from dose-range finding test
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: based on solubility in dimethyl sulfoxide observed during a solubility test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 9.6 x 10⁵ cells/ concentration

ACTIVATION: S9 mix was prepared immediately before use and kept on ice. S9-mix components contains per ml physiological saline: 1.63 mg MgCl2 H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES. The final concentration of the S9-fraction in the exposure medium was 4%.

DURATION
- Exposure duration:
Dose-range finding test: 3-hour treatment with and without metabolic activation
Experiment I: 3-hour treatment, with and without metabolic activation
Experiment II: 24-hour treatment, without metabolic activation
- Expression time (cells in growth medium): the remaining cells were cultured for 2 days after the treatment period.
- Selection time (if incubation with a selection agent): 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): selective medium (TFT-selection agent)

NUMBER OF REPLICATIONS: duplicate plates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: the plates of the TFT-selection were stained for 2 hours by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well.

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
- Any supplementary information relevant to cytotoxicity: suspension growth

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no

Rationale for test conditions:
The test concentrations in experiment I and II were selected based on cytotoxicity data from a dose-range finding study.
Evaluation criteria:
A test item is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency (MF) of more than MF(controls) + 126 in a dose-dependent manner. Any observed increase should be biologically relevant and will be compared with the historical control data range.
Statistics:
A Cochran Armitage trend test (p < 0.05) was performed to test whether there was a significant trend in the induction of mutations.
Key result
Species / strain:
mouse lymphoma L5178Y cells
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
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: the test item was insoluble in exposure medium and ethanol
- Water solubility: no data
- Precipitation: when mixing with exposure medium the test item precipitated at the concentration of 16.4 mg/ml and higher.
- Definition of acceptable cells for analysis: no data


RANGE-FINDING/SCREENING STUDIES: No toxicity in the suspension growth was observed up to and including the highest test item concentration of 512 μg/ml compared to the suspension growth of the solvent control both in the absence and presence of S9-mix. No toxicity in the relative suspension growth was observed up to the test item concentrations of 512 μg/ml compared to the solvent control.


HISTORICAL CONTROL DATA
- Positive historical control data: within the ranges of historical control data
- Negative (solvent/vehicle) historical control data: within the ranges of historical control data. One of the solvent/negative control cultures in experiment II was just above the 95% control limits of the historical control data range. The observed mutation frequency, however, was within the acceptability criteria of this assay.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No cytotoxicity was observed in experiment I and II.
- Other observations when applicable: In experiment I the number of small and large colonies in the test item treated cultures were comparable to the number of small and large colonies of the solvent controls. In experiment II the test item precipitated in the exposure medium at concentration of 52 µg/ml after 24-hour treatment.
Remarks on result:
other: No mutagenic potential
Remarks:
Experiment I and II

Table 1: Experiment I, 3-hour exposure without metabolic activation

Test Group

Concentrations

µg/ml

RCE %

RTG %

RSG %

MF

(mutants/106cells)

GEF exceeded

Statistical Significant Increase

SC1

0

100

100

100

79

-

-

SC2

0

100

100

100

124

-

-

1

0.056

102

101

99

93

-

-

2

0.18

104

92

89

97

-

-

3

0.55

111

102

92

106

-

-

4

1.7

134

126

94

95

-

-

5

5.4

105

103

98

51

-

-

6

17

111

100

90

64

-

-

7

52

104

96

93

76

-

-

8

164*

116

104

90

94

-

-

MMS

15 

63

39

62

632

+

+

Table 2: Experiment I, 3-hour exposure with metabolic activation

Test Group

Concentrations

µg/ml

RCE %

RTG %

RSG %

MF

(mutants/106cells)

GEF exceeded

Statistical Significant Increase

SC1

0

100

100

100

91

-

-

SC2

0

100

100

100

117

-

-

1

0.056

100

90

90

105

-

-

2

0.18

86

75

87

116

-

-

3

0.55

104

97

93

100

-

-

4

1.7

97

83

86

136

-

-

5

5.4

110

82

75

81

-

-

6

17

110

96

87

85

-

-

7

52

94

82

87

97

-

-

8

164*

118

103

87

92

-

-

CP

 7.5

25

7

27

1938

+

+

Table 3: Experiment II, 24 -hour treatment without metabolic activation

Test Group

Concentrations

µg/ml

RCE %

RTG %

RSG %

MF

(mutants/106cells)

GEF exceeded

Statistical Significant Increase

SC1

0

100

100

100

118

-

-

SC2

0

100

100

100

133

-

-

1

0.018

117

109

93

72

-

-

2

0.056

104

95

91

122

-

-

3

0.18

104

93

89

102

-

-

4

0.55

117

111

95

70

-

-

5

1.7

106

107

101

108

-

-

6

5.4

124

120

97

81

-

-

7

17

114

109

96

85

-

-

8

52*

99

87

88

108

-

-

MMS

 15

88

65

74

583

+

+

* the test item precipitated in the exposure medium

RSG: Relative suspension growth

RCE: Relative cloning efficiency

RTG: Relative total growth

SC: solvent control

MMS: methylmethanesulfonate

CP: cyclophosphamine

Conclusions:
1,3,5-Trimethyl-1,1,3,5,5-pentaphenyltrisiloxane has been tested for mutagenicity in mouse lymphoma L5178Y cells, according to OECD 490 and in compliance with GLP. No test substance induced increase in the number of mutations was observed when tested up to precipitating concentration. Appropriate solvent and positive controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The submission substance has been tested in a valid bacterial reverse mutation assay, according to the OECD TG 471 (1989), and under GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or WP uvrA (RCC, 2004).

No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to limit concentration. Appropriate negative, positive and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

The submission substance has been tested for ability to cause chromosome aberrations in Chinese hamster ovary cells according to OECD TG 473 and under GLP (RCC, 2004). No increase in the number of cells with aberrations was observed either with or without metabolic activation in Chinese hamster V79 cells. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of this study.

No mutagenicity data in mammalian cells was available for the submission substance, therefore data were read-across from a structural analogue. 1,3,5-Trimethyl-1,1,3,5,5-pentaphenyltrisiloxane has been tested for mutagenicity in mouse lymphoma L5178Y cells, according to OECD 490 and in compliance with GLP (Charles River, 2016). No test substance induced increase in the number of mutations was observed when tested up to precipitating concentration. Appropriate solvent and positive controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study.

Read-across justification

There are no available measured mammalian mutagenicity or cytogenicity data for Reaction Mass of 3,3-diphenylhexamethyltrisiloxane; 3,3,5,5-tetraphenylhexamethyltetrasiloxane and 3-trimethylsiloxy-3,5,5-triphenylhexamethyltetrasiloxane (CAS 352230-22-9) for mutagenicity in mammalian cells. Therefore, the Annex requirements are fulfilled by data on structurally analogous substances. The analogue approach for fulfilling this endpoint by read-across from one source substance, 1,3,5-trimethyl-1,1,3,5,5-pentaphenyltrisiloxane (CAS 3390-61-2), according to the Read-across Assessment Framework (RAAF) is described.

Read-across is proposed in accordance with RAAF Scenario 2: “This scenario covers the analogue approach for which the read-across hypothesis is based on different compounds which have the same type of effect(s). For the REACH information requirement under consideration, the effects obtained in a study conducted with one source substance are used to predict the effects that would be observed in a study with the target substance if it were to be conducted. The same type of effect(s) or absence of effect is predicted. The predicted strength of the effects may be similar or based on a worst-case.”

The read-across justification is presented (Table 5.7.2) according to RAAF scenario 2 assessment elements (AE) as outlined in Table B1 of the RAAF:

Table 5.7.2. RAAF scenario 2 assessment elements (AE) as given in Appendix B (Table B1) of the RAAF

AE A.1

Characterisation of source substance

AE A.2

Link of structural similarity and differences with the proposed Prediction

AE A.3

Reliability and adequacy of the source study

AE 2.1

Compounds the test organism is exposed to

AE 2.2

Common underlying mechanism, qualitative aspects

AE 2.3

Common underlying mechanism, quantitative aspects

AE 2.4

Exposure to other compounds than to those linked to the prediction

AE 2.5

Occurrence of other effects than covered by the hypothesis and Justification

AE A.4

Bias that influences the prediction

1. AE A.1 Identity and characterisation of the source substance

The source substance, 1,3,5-trimethyl-1,1,3,5,5-pentaphenyltrisiloxane (CAS 3390-61-2), is a linear trisiloxane with phenyl and methyl groups bound to silicon. The substance is highly insoluble in water.  Therefore, hydrolysis half-lives and hydrolysis products are not relevant for this endpoint as hydrolysis is expected to be very slow.

The source substance has a log Kow of 9 at 20°C (QSAR), vapour pressure of 1.3E-08 Pa at 25°C (QSAR) and water solubility of 9.0E-11 mg/L at 20°C (QSAR).

2. AE A.2 Link of structural similarities and differences with the proposed prediction

Reaction Mass of 3,3-diphenylhexamethyltrisiloxane and 3,3,5,5-tetraphenylhexamethyltetrasiloxane is a multi-constituent substance containing two main constituents 3,3-diphenylhexamethyltrisiloxane (55-70%) and 3,3,5,5-tetraphenylhexamethyltetrasiloxane (15-25%).

The read-across substance, 1,3,5-trimethyl-1,1,3,5,5-pentaphenyltrisiloxane (CAS 3390-61-2), is structurally similar to the main constituent of the submission substance because they are both linear trisiloxanes with phenyl and methyl groups bound to silicon. The difference is that read-across substance has 5 phenyl groups and 3 methyl groups and the main constituent of the submission substance has 2 phenyl groups and 6 methyl groups. The second constituent of the submission substance is also a structural analogue of the read-across substance but has a longer siloxane chain (4 silicon atoms instead of 3, with 4 phenyl groups and 6 methyl groups).

Both the source and the target substances have consistent physicochemical properties. They have very high log Kow, very low water solubility, very low vapour pressure and slow hydrolysis. Hydrolysis products are different but considering the long hydrolysis half-lives and high log Kow the test organisms would be exposed to the parent substances.

Table 5.7.3. Physicochemical properties of the target and source substances

Property

Target substance

Source substance

Substance name

Reaction Mass of 3,3-diphenylhexamethyltrisiloxane; 3,3,5,5-tetraphenylhexamethyltetrasiloxane; 3-trimethylsiloxy-3,5,5-triphenylhexamethyltetrasiloxane

1,3,5-Trimethyl-1,1,3,5,5-pentaphenyltrisiloxane

CAS number

352230-22-9

3390-61-2

Hydrolysis half-life at pH 7

>200 h for Constituent 1

>630 h for Constituent 2

>200 h

log Kow value

9.0 at 20°C (QSAR) for Constituent 1 and Constituent 2

9 at 20°C (QSAR)

Vapour pressure

0.068 Pa at 25°C (whole substance) (OECD 104) 

3.6E-03 Pa at 25°C (QSAR, Constituent 1)

1.3E-07 Pa at 25°C (QSAR, Constituent 2)

1.3E-08 Pa at 25°C (QSAR)

Water solubility

 <0.5234 mg/L at 20°C (whole substance) (OECD 105) 

Constituent 1: 2.8E-05 mg/L at 20°C (QSAR)

Constituent 2: 1.5E-10 mg/L at 20°C (QSAR)

9.0E-11 mg/L at 20°C (QSAR)

3. AE A.3 Reliability and adequacy of the source study

1,3,5-Trimethyl-1,1,3,5,5-pentaphenyltrisiloxane has been tested for mutagenicity in Mouse lymphoma L5178Y cells, according to OECD 490 and in compliance with GLP (Charles River, 2016). No test substance induced increase in the number of mutations was observed when tested up to precipitating concentration. Appropriate solvent and positive controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study.

4. AE A.4 Bias that influences the prediction

Data on the source substance 1,3,5-trimethyl-1,1,3,5,5-pentaphenyltrisiloxane (CAS 3390-61-2) were read-across to the registered (target) substance Reaction Mass of 3,3-diphenylhexamethyltrisiloxane and 3,3,5,5-tetraphenylhexamethyltetrasiloxane (CAS 352230-22-9). The source substance and the target substance have similar chemical structure and physicochemical properties. Both hydrolyse very slowly, and it is considered that the test organism would be exposed to the parent substances. Their toxicological properties are expected to be similar, with similar genotoxic effects. 1,3,5-Trimethyl-1,1,3,5,5-pentaphenyltrisiloxane (CAS 3390-61-2) is the closest structural analogue with available data.

5. AE A.2.1 Compounds the test organism is exposed to

The source substance as well as the target substance hydrolyse very slowly in contact with water. Therefore, their hydrolysis products are not considered to be relevant as the test organism would mainly be exposed to the parent substances. The source and target substance have consistent toxicological properties and none of the substances has been identified to be of any toxicological concern.

6. AE A.2.2 and A.2.3 Common underlying mechanism, qualitative and quantitative aspects

No toxicity data are available for the target substance Reaction Mass of 3,3-diphenylhexamethyltrisiloxane and 3,3,5,5-tetraphenylhexamethyltetrasiloxane, therefore data are read-across from the structurally analogous substance 1,3,5-trimethyl-1,1,3,5,5-pentaphenyltrisiloxane (CAS 3390-61-2). Both substances have consistent physicochemical properties. The have very high log Kow, very low water solubility, very low vapour pressure and slow hydrolysis. Hydrolysis products are different but considering the long hydrolysis half-lives and high log Kow the test organisms would be exposed to the parent substances. The hydrolysis products are all di- and trisilanols with phenyl and methyl substituents.

7. AE 2.4 Exposure to other compounds than to those linked to the prediction

Neither the target substance, Reaction Mass of 3,3-diphenylhexamethyltrisiloxane and 3,3,5,5-tetraphenylhexamethyltetrasiloxane, nor the source substance, 1,3,5-trimethyl-1,1,3,5,5-pentaphenyltrisiloxane, have impurities of toxicological concern.

The test/source substance in the study, 1,3,5-trimethyl-1,1,3,5,5-pentaphenyltrisiloxane has an analytical purity of 99.7%.

The target substance, Reaction Mass of 3,3-diphenylhexamethyltrisiloxane and 3,3,5,5-tetraphenylhexamethyltetrasiloxane (CAS 352230-22-9) is a multi-constituent substance containing two main constituents, 3,3-diphenylhexamethyltrisiloxane present at concentration of 55-70% and 3,3,5,5-tetraphenylhexamethyltetrasiloxane at concentration of 15-25%. The impurities are linear and branched siloxanes with 4-7 silicon atoms and phenyl/methyl substituents. These are structural analogues of the main constituents and share the same physicochemical properties (very high log Kow, very low water solubility, very low vapour pressure, slow hydrolysis).

8. AE 2.5 Occurrence of Other Effects than Covered by the Hypothesis and Justification

Not relevant

Justification for classification or non-classification

Based on the available data for the submission substance, no classification for genetic toxicity is required according to Regulation (EC) No 1272/2008.