Tetravinylsilane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro genetic tests are available for the submission test.

Ames test:

The mutagenicity of the test material was investigated in an Ames test (pre-incubation method) similar to OECD 471. The test item was not mutagenic under the conditions of this Ames test.

In vitro chromosome aberration study:

The ability of test item to induce chromosomal aberrations was investigated by using Chinese hamster lung fibroblasts (CHL/IU cell), based on OECD 473. It was concluded that test item did not induce chromosomal aberrations under the present test conditions.

In Vitro Mammalian Cell Gene Mutation Test:

The study was performed to evaluate the test item for its ability to cause gene mutations in vitro cultured mammalian cells (L5178Y) in compatible with OECD 490. The test item is non-mutagenic under the conditions of this study.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2015-07-28 to 2015-09-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Japan MHLW Testing Methods for New Substance

Version / remarks:

March 29 2011, Revised April 2 2012

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

Duplicate cultures were used for the test material, whereas triplicate plates are specified by OECD 471. This deviation does not affect the integrity of the study given the clear negative responses seen in three separate assays.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Lot No.: 150715
Purity: 99.1%

Target gene:

Reversion to histidine / tryptophan independence

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 / 5,6-benzoflavone-induced male SD rat liver S9 fraction
Composition of S9 mix:
S9 mix was prepared just before use. One milliliter of S9 mix consisted of 8 μmol MgCl2, 33 μmol KCl, 5 μmol Glucose-6-phophate, 4 μmol NADPH, 4 μmol NADH, 100 μmol sodium-phosphate buffer (pH 7.4) and 0.1 mL of S9.

- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix

Test concentrations with justification for top dose:

Test concentrations were based on the results of a preliminary cytotoxicity assay performed at concentrations of up to 5000 µg/plate and were limited by toxicity. There was no evidence of precipitation.
In the main assay, the highest concentrations (-S9) were 19.5 µg/plate (TA100, TA1535, TA98, TA1537) and 313 µg/plate (WP2uvrA); (+S9) 78.1 µg/plate (TA1535) and 313 µg/plate (TA100, WP2uvrA, TA98, TA1537). Cytotoxicity (reduced numbers of revertant colonies and/or reduced background lawn) was seen at the highest concentrations used for each strain in the absence and presence of cytotoxicity.

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: AF-2 (TA100, WP2uvrA, TA98; -S9); ICR-191 (TA1537, -S9). 2-aminoanthracene (all strains; +S9)

Remarks:

Activity of the S9 batch had also been demonstrated using 2-AA, B(a)P and dimethylanthracene

Details on test system and experimental conditions:

The study was performed using the pre-incubation method using duplicate plates (triplicate plates were used for the solvent controls). Cultures were exposed to the test material or positive controls with or without S9 mix for 20 minutes at 37°C with shaking. Following the addition of soft agar, revertant colonies were counted after incubation for 48 hours.

- Confirmation of Sterility:
The highest concentration of the test item solution (0.05 mL) and S9 mix (0.5 mL) were individually mixed with 2 mL of the soft agar and were poured onto each minimal glucose agar plate in order to examine bacterial contamination. Bacterial contamination was judged with those plates after incubation at 37±0.5°C for 48 hours.

- Colony counting:
For the plates at which the bacterial growth inhibition was observed, the number of colonies was counted manually, and for the other plates with a colony analyzer (CA-11D, SYSTEM SCIENCE). Square correction and miscounting correction were conducted when counting with the colony analyzer.

Rationale for test conditions:

A preliminary range-finding assay was performed using concentrations of up to 5000 µg/plate in the absence and presence of S9. A second range-finding assay using lower concentrations was performed due to the level of toxicity seen in the initial assay. There was no evidence of precipitation at any concentration

Evaluation criteria:

A positive response was concluded where the number of revertant colonies in any strain exceeded twice that seen in the relevant control, and where the response was concentration-related and/or reproducible.

Statistics:

Not required.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There was no evidence of precipitation at any concentration of the test material.
The bacterial growth inhibition was observed at 19.5 μg/plate for TA100, TA98 and TA1537 without S9 mix, at 9.77 μg/plate or more for TA1535 without S9 mix, at 156 μg/plate or more for WP2urA without S9 mix. The bacterial growth inhibition was observed at 156 μg/plate or more for TA100, TA98 and TA1537 with S9 mix, at 78.1 μg/plate for TA1535 with S9 mix, at 313 μg/plate for WP2uvrA with S9 mix.
Exposure to the test material did not induce any increase in the number of revertant colonies at any concentration either in the absence or presence of metabolic activation.

Results of Range-finding Study 1

µg/plate	TA100		TA1535		WP2uvrA		TA98		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	107	98	12	10	30	29	24	31	13	15
4.88	110	108	11	6	33	40	21	35	13	18
19.5	88*	108	6*	9	40	35	18*	37	9*	16
78.1	91*	88	9*	8*	35	25	14*	22	8*	12
313	100*	80*	5*	9*	33*	19*	16*	16*	10*	13*
1250	88*	87*	4*	4*	31*	26*	11*	20*	8*	12*
5000	0*	29*	0*	2*	26*	21*	0*	17*	3*	2*
AF-2	934				143		360
NaN3			267
ICR-191									532
2AA		1159		273		519		276		147

Mean numbers of revertant colonies

*growth inhibition observed

Results of Range-finding Study 2

µg/plate	TA100		TA1535		WP2uvrA		TA98		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	118	100	11	13	28	21	23	26	9	10
0.610	129		11				19		9
1.22	110		7				22		10
2.44	107		10	14			23		9
4.88	103		12	7			26		14
9.77	84	112	12*	9	36	29	17	24	9	11
19.5	76*	114	6*	14	43	30	17*	29	12*	7
39.1		103		12	22	27		35		9
78.1		89*		5*	27	28		25		13
156		86*			24*	35		19*		11
313		78*			23*	26*		18*		10
AF-2	1358				145		377
NaN3			295
ICR-191									639
2AA		1023		294		396		318		176

Mean numbers of revertant colonies

*growth inhibition observed

 

Results of Main Study

µg/plate	TA100		TA1535		WP2uvrA		TA98		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	98	109	11	9	34	36	20	24	14	12
0.610	112		6				27		15
1.22	109		11				18		12
2.44	94		9	12			23		10
4.88	106		13	8			21		7
9.77	106	97	9*	8	41	23	16	20	11	17
19.5	96*	103	9*	7	128	34	16*	17	7	14
39.1		104		8	25	46		27		9
78.1		114		8*	43	36		25		9
156		71*			23*	42		23*		12*
313		92*			26*	35*		23*		10*
AF-2	1113				173		461
NaN3			337
ICR-191									527
2AA		1075		325		397		329		184

Mean numbers of revertant colonies

*growth inhibition observed

Conclusions:

No evidence of mutagenicity was seen under the conditions of this study.

Executive summary:

The mutagenicity of the test material was investigated in an Ames test (pre-incubation method) similar to OECD 471. Duplicate cultures of strains S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2uvrA (triplicate cultures for the negative control) were exposed to concentrations of the test material (dissolved in acetone), negative (solvent) or positive controls in the absence or presence of an exogenous metabolic activation system. Test concentrations were based on the results of a preliminary cytotoxicity assay performed at concentrations of up to 5000 µg/plate and were limited by toxicity. There was no evidence of precipitation. In the main assay, the highest concentrations (-S9) were 19.5 µg/plate (TA100, TA1535, TA98, TA1537) and 313 µg/plate (WP2uvrA); (+S9) 78.1 µg/plate (TA1535) and 313 µg/plate (TA100, WP2uvrA, TA98, TA1537). Cytotoxicity (reduced numbers of revertant colonies and/or reduced background lawn) was seen at the highest concentrations used for each strain in the absence and presence of cytotoxicity. Exposure to the test material did not induce any biologically relevant increase in the number of revertant colonies of any strain at any concentration either in the absence or presence of metabolic activation. The laboratory's criteria for a positive response was not seen for any strain. Appropriate positive control substances confirmed the sensitivity of the assay and the metabolic activation system. It was concluded that the test item was not mutagenic under the conditions of this Ames test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2019-05-16 to 2019-08-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Batch No.: 707001
Purity: 98.6%

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Health Science Research Resources Bank, Japan Health Sciences Foundation
- Suitability of cells: recommended in the OECD method
- Normal cell cycle time (negative control):
- Modal number of chromosomes: 25 per cell
- Doubling time: About 15 hours
- Mycoplasma: Negative
- Spontaneous frequencies of cells with structural aberrations and the numerically aberrant cells: <5%

MEDIA USED
- Type and composition of media: L-Glutamine (final concentration: 0.292 g/L) and sodium hydrogen carbonate (final concentration: approximately 1.95 g/L) were added to Eagle’s minimum essential medium (Lot number 751810 and 754903, Nissui Pharmaceutical) and basal medium (MEM) was prepared. This medium was then supplemented with 10 vol% heat-inactivated NBCS (NBCS of Lot number 1774292 (Life Technologies) and NBCS of Lot number S14368S0750 (Bio West) were mixed in a ratio of 4:1).

CULTURE CONDITION
- Incubator: CO2 incubator (MCO-18AIC, SANYO Electric)
- Temperature: 37°C
- Humidity: Under humid condition
- CO2 concentration: 5%

SUBCULTURE
- Culture vessel: 90-mm diameter Petri dish (Nunc)
- Frequency of passage: Twice or three times a week
- Passage number of cells: 6 for cell growth inhibition test, 7-17 for chromosomal aberration test

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- S9 and S9 mix: from Ieda Trading Corporation, prepared just before use.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.2 mL of S9 mix

Test concentrations with justification for top dose:

- The first chromosomal aberration test:
The following doses were set to obtain the dose which RPD or RICC was 40% or more and 50% or less based on Cell Growth Inhibition Test.
Short-term treatment (S9-): 20.0, 40.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0 and 80.0 μg/mL
Short-term (S9+) and 24 hours continuous treatment (S9-): 20.0, 30.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0 and 70.0 μg/mL
- The second chromosomal aberration test:
In the first chromosomal aberration test for “- S9 mix”, the fluctuation of the cytotoxicity parameter was observed between 2 flasks in this dose. And for “+ S9 mix" and 24 hours continuous treatment, the doses adequate for the evaluation of the chromosomal aberration were not obtained, Therefore, the specimen preparation was not carried out and the second chromosomal aberration test was carried out.
Short-term treatment: S9-: 20.0, 40.0, 56.0, 58.0, 60.0, 62.0, 64.0, 66.0 and 68.0 μg/mL; S9+: 20.0, 40.0, 56.0, 58.0, 60.0, 62.0, 64.0, 66.0, 68.0, 70.0 and 74.0 μg/mL.
24 hours continuous treatment: 20.0, 40.0, 42.0, 44.0, 46.0, 48.0, 50.0 and 52.0 μg/mL
- The third chromosomal aberration test:
In the second chromosomal aberration test for “- S9 mix”, the doses which RPD or RICC was less than 50% or less were not obtained and the doses adequate for the evaluation of the chromosomal aberration were not obtained. Therefore, the specimen preparation was not carried out and the third chromosomal aberration test was carried out at the following doses:
Short-term treatment (S9-): 20.0, 40.0, 56.0, 58.0, 60.0, 62.0, 64.0, 66.0, 68.0, 70.0 and 74.0 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone

- Justification for choice of solvent/vehicle: The test substance was suspended in water at 13.6 mg/mL and not suspended in DMSO at 136 mg/mL, but soluble in acetone at 136 mg/mL. The test substance solution of 136 mg/mL prepared with acetone was considered to be stable from the facts that there were no change in color, exothermic reaction nor gas generation at room temperature and ice bath within 2 hours after preparation. Therefore, acetone was selected as a vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

In the second and the third chromosomal aberration test, distilled water was set as a negative control of the positive control substance.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: Cell growth inhibition test 1.5E+04 cells/mL, 5E+03 for the first Chromosomal aberration test, 1.5 E+04 for the second and third Chromosomal aberration test.
- Volume of cell suspension: 5 mL
- Pre-culture period: 2 or 3 days
- Treatment:
(1) For the short-term treatment without S9 mix (- S9 mix), the medium was removed from a pre-culture, and the cells were treated in well-mixed medium made of 3 mL of the fresh medium and 30 μL of the test substance solutions or the vehicle.
(2) For the short-term treatment with S9 mix ( + S9 mix), the medium was removed from a pre-culture, and the cells were treated in well-mixed medium made of 2.8 mL of fresh medium, 30 μL of the test substance solutions or the vehicle, and 0.2 mL of S9 mix.
(3) For “- S9 mix" and “+ S9mix”, after treatment for 6 hours, the medium was removed, and the cells were rinsed 3 times with 2 mL of Dulbecco’s physiological phosphate buffered solution without Ca2+ and Mg2+. Cells were then cultured for another 18 hours in 5 mL of fresh medium.
(4) For the 24 hours continuous treatment, the medium was removed from a pre-culture, and the cells were treated for 24 hours with well-mixed medium made of 5 mL of fresh medium and 50 μL of the test substance solutions or the vehicle.


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Specimen preparation:
(1) Leftover cells were collected by a centrifugation at 1000 rpm for 5 minutes and were treated hypotonically with 3 mL of 0.075 mol/L KCI at 37 °C for 15 minutes. After the hypotonic treatment, the cells were pre-fixed once with approximately 0.3 mL of a fixative solution (methanol : acetic acid = 3 : 1 ), and were completely fixed twice with 3 mL of fixative solutions. Then, the cell suspension was prepared with appropriate amount of a fixative solution, the suspension was dropped onto a glass slide. One specimen per dose was prepared.
(2) A specimen was dried and stained for about 15 minutes with 2 vol% Giemsa solution made with 1/15 mol/L phosphate buffer solution (pH 6.8).
- Dose for observation: The specimens were observed in the third chromosomal aberration test for “- S9 mix” and “+ S9 mix" and the second chromosomal aberration test for 24 hours continuous treatment. All specimens of the negative and the positive controls were observed.
- Structural aberration:
Three hundred metaphase cells per dose (75 cells per specimen) containing 25±2 chromosomes were observed.
- Numerical aberration:
The numbers of polyploid cells with triploid or more (38 or more chromosomes) and endoreduplicated cells among 300 metaphase cells per dose (75 cells per specimen) were recorded.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative population doubling (RPD); relative increase in cell count (RICC)
- Any supplementary information relevant to cytotoxicity:
Fifty microliters of a 10 μg/mL demecolcine solution was added to each flask at 2 hours before the end of the culture.
At the end of the culture, cells were suspended with 2 mL of 0.25 w/v% trypsin. After 200 μL of the cell suspension was diluted with 10 mL of Cell Pack (Sysmex), the number of the cells was measured using a Particle counter/analyzer (CDA-1000, Sysmex) and the cell growth rate compared with a negative control (Relative Cell Count, RCC), RPD and RICC were calculated.



- OTHER:

Evaluation criteria:

Regarding the frequencies of cells with chromosomal aberrations, the test substance was judged to be negative if a) or b) was satisfied:
a) all results are inside the distribution of the historical data of the negative control group,
b) outside the distribution of the historical data of the negative control group, but none of the doses of the test substance exhibits a statistically significant increase compared with the concurrent negative control.
Regarding the frequencies of cells with chromosomal aberrations, the test substance was judged to be positive if cases of c) to e) or f) were fulfilled:
c) outside the distribution of the historical data of the negative control group,
d) there is a statistically significant increase compared with the concurrent negative control,
e) the increase of the frequencies of cells with chromosomal aberrations is dose-related,
f) both chromosomal aberration test and confirmation test are fulfilled in cases of c) and d).

Statistics:

Statistical analysis was performed in order to evaluate the frequencies of cells with structural aberrations and numerically aberrant cells. Significance level of these tests was set at 1 % and 5% for both sides.
Fisher’s exact test was performed in order to compare in the negative control group with positive control group.
Fisher’s exact test was performed in order to compare in the negative control group with test substance group when the frequencies of cells with chromosomal aberrations in each test substance group were outside the distribution of the historical data of the negative control group. When the test substance induced a statistically significant increase in the Fisher’s exact test, the dose-response relation was tested by the Cochran-Armitage trend test.

Species / strain:

Chinese hamster lung (CHL/IU)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Cell Growth Inhibition Test
In all treatment methods, the precipitation of the test substance was observed at 170 μg/mL or more at the start of the treatment. In “+ S9 mix", the precipitation of the test substance was observed at 170 μg/mL or more at the end of the treatment.
At the start of the treatment, the color of the medium was changed to red at 1360 μg/mL in all treatment methods.
The pH at 1360 μg/mL was within range (6.6 - 7.8), thus the pH did not affect the evaluation of the chromosomal aberration.
The corrosion of the culture flask was not observed in all treatment methods.
- Chromosomal Aberration Test
-Precipitation of the test substance, the color change of medium and the corrosion of the culture flask: not observed for all treatment methods.


STUDY RESULTS
- Concurrent vehicle negative and positive control data
In each treatment method, the frequency of cells with chromosomal aberrations in the negative control group and the frequency of cells with structural aberration in the positive control group were within the range of the historical data. The frequency of cells with structural aberrations in the positive control group showed a statistically significant increase compared with the negative control group. Cell proliferation criteria in the negative control met the criteria in the testing facility.

Chromosome aberration test (CA) in mammalian cells:
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps : Gaps were defined as an achromatic region smaller than the width of one chromatid. (1) Chromatid break (2) Chromatid exchange (3) Chromosome break (4) Chromosome exchange (dicentric, ring and translocation) (5) Fragmentation
- Genotoxicity results
- Frequency of cells with structural aberrations:
Short-term treatment (-S9mix): The frequencies were within the range of the historical data of the negative control.
Short-term treatment (+S9mix): At 98.0 μg/mL, the frequency was not within the range of the historical data of the negative control. But a statistically significant increase was not observed.
24 hours continuous treatment: The frequencies were not within the range of the historical data of the negative control. But a statistically significant increase was not observed.
-Frequency of numerically aberrant cell:
Short-term treatment (-S9mix): The frequencies were within the range of the historical data of the negative control.
Short-term treatment (+S9mix): The frequencies were within the range of the historical data of the negative control.
24 hours continuous treatment: The frequencies were within the range of the historical data of the negative control.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
Please see Appendix 1 in attached backgroud material.

Conclusions:

It was concluded that the test item did not induce chromosomal aberrations under the present test conditions.

Executive summary:

The ability of test item to induce chromosomal aberrations was investigated by using Chinese hamster lung fibroblasts (CHL/IU cell), based on OECD 473.

In the chromosomal aberration test, the observation doses for evaluation were selected to be 20.0, 58.0 and 68.0 μg/mL, and 20.0, 86.0 and 98.0 μg/mL in the shot-term treatments without and with S9 mix, respectively, and 20.0, 46.0 and 50.0 μg/mL in the 24 hours continuous treatment.

The frequencies of cells with structural aberrations at all observation doses of the test substance in the short-term treatment without S9 mix was within the range of the historical data of the negative control. The frequency of cells with structural aberrations at 98.0 μg/mL in the short-term treatment with S9 mix was not within the range of the historical data of the negative control. However, there was no statically significant increase in the frequency of cells with structural aberrations in this dose as compared to the concurrent negative control. The frequencies of cells with structural aberrations at all observation doses of the test substance in 24 hours continuous treatment were not within the range of the historical data of the negative control. However, there were no statistically significant increase in the frequencies of cells with structural aberrations in these doses as compared to the concurrent negative control. Therefore, structural aberration was judged to be negative.

The frequencies of numerically aberrant cells at all observation doses of the test substance in all treatment methods were within the range of the historical data of the negative control. Therefore, numerical aberration was judged to be negative.

Consequently, it was concluded that test item did not induce chromosomal aberrations under the present test conditions.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2019-06-26 to 2019-08-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Batch No.: 707001
Purity: 98.5%

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: ATCC®CRL-9518
- Suitability of cells: sensitive cell of this test and is accredited by OECD Guideline
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages: 9 and 10
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
RPMI1640 (RPMI in short), and three types of 0%, 10% and 20% Donor Equine Serum RPMI medium (RPMI 0, RPMI 10 and RPMI 20) were prepared by adding horse serum (0% or 10 or 20%), 10000U/mL Penicillin-Streptomycin (1%) and Sodium pyruvate (2%).
RPMI0 was used during cells treatment (3-hour treatment, 3h), RPMI10 was used during cells daily culture and cells treatment (24-hour treatment group, 24h), and RPMI20 was used during cells cloning culture in 96-well plate.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : in-house, prepared from the livers of the rats induced with Aroclor1254
- method of preparation of S9 mix: 10% S9 mix was prepared according to the recipe below table in any other information on materials and methods immediately before being used and placed in the mixture of water and ice. The remaining S9 mix was discarded.
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

THE FIRST TEST:
According to the preliminary test and OECD Guidelines for Testing of Chemicals, No.490( 2016), L5178Y were exposed at six doses 2000, 800, 320, 128, 51.2 and 20.5 μg/mL.
THE SECOND TEST: 80, 40, 25, 20, 10 and 5 μg/mL media for 3hours with metabolic activation, and 40, 25, 20, 10, 5 and 2.5 μg/mL medium for 3hours and 24 hours without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

- Justification for choice of solvent/vehicle: basing on the results of test item solubility (the test item could be dissolved in ethanol at the concentration of 400mg/mL), ethanol was used as solvent.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 3E+05 cells/mL
- The test solutions or control solutions were added to the corresponding mixture. All the cell cultures were incubated at 35.2~37.3℃in the humidified atmosphere of 5.0% CO2. The mixtures in the treatment conditions of exposure for 3h with and without S9 were shaking treated for near 3 hours, and the mixtures in the treatment conditions of exposure for 24h without S9 were stationary treated for near 23 hours.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3h with metabolic activation and for 24h and 3h without metabolic activation
- Harvest time after the end of treatment (sampling/recovery times): After the treatment, for the cells in the treatment conditions of exposure for 3h with and without S9, the cell cultures were centrifuged at 1000rpm/min for 5 min, discarded the supernatant, and the cells were washed with Hank,s balanced salt solution (HBSS) for twice, resuspended in 20mL RPMI10 and cultured to make the cells mutant phenotypic expression.
For the cells in the treatment condition of exposure for 24h without S9, after being resuspended in 20mL RPMI10 following the same conductions as above, the cells were counted, adjusted the density to 3E+05 cells/mL (The cells less than 3E+05 cells/mL were incubated without adjustment) andcontinued to be cultured to make the cells mutant phenotypic expression.


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2days
- Selection of mutant cells: After expression period, PE plates and TFT plates were prepared for cell viability and TFT resistance.
After expression period, cells were suspended in media with and without the selective agent triflurothymidine (TFT)for the determination of thenumber of mutants (selection plates =TFT plates) and for cloning/planting efficiency (viability plates = PE plates), respectively.
Recording the number of cells on the day 2, the cells were adjusted to 1E+04 cells/mL in the volume of 50mL as cell culture B. Then part of cell culture B was taken out and diluted as the table below to 25mL of 8 cells/mL as cell culture D. The selective agent, trifluorothymidine (TFT), was added in the remaining cells of the culture B, and the final concentration of TFT in cell culture was 3 μg/mL.
For each cell culture, two 96-microwell plates wereprepared with cell culture B as TFT plates, and one 96-microwell plate wasprepared with cell culture D as PE plate. For all plates, 0.2mL of the cell culture was added into each well.
- Cell colonies observation:
All plates above were incubated for 12 days. After the incubation, cell colonies in the plates were observed and counted.
For PE plate, empty wells and total wells were counted respectively.
For TFT plate, wells with viable colony and total wells were counted respectively. In addition, for TFT plates of the highest concentrations, untreated controls, solvent controls and positive controls, large colony and small colony were counted respectively. The small colony and large colony are defined as below:
1) Large colony:
Size: the diameter was not less than a quarter of diameter of the well; Morphology: thin and dispersive
2) Small colony:
Size: the diameter was less than a quarter of diameter of the well; Morphology: compact and dense massive structure

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)

Evaluation criteria:

1) When IMF(s) in one or several doses are more thanGEF* (GEF= 126E-06), and the increase is concentration- related and/or replicated, the result is evaluated as positive.
2) The result is evaluated as negative if none of the above criteria is met.
3) In case the criteria above are not met at the same time, the repeated test should be performed using modified experimental conditions, and the biological relevance of the result should be considered. If the result is still not clear, the result is conclude to be equivocal.
*GEF = Global Evaluation Factor, established for the MLA making use of the version of microtiter plates as average background mutation frequency of negative controls.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- The first test:
There was precipitate of test item in the cultures only at 2000μg/mL at the beginning and end of the treatment in each condition. In the second test, no test item precipitate was observed in the cell cultures at all designed doses in each treatment condition.
The results of the cells count showed that obvious cytotoxicity was observed at 51.2 μg/mL and above dose levelsfor 3 hours and 24 hours without metabolic activation, it was forecasted that RTG＜10%, but RTG＞10% at 51.2 μg/mL for 3 hours with metabolic activation.According to the above cytotoxicity, the test could not continue. Subsequently, the dose was redesigned based on the results of cytotoxicity in the first test and retested.
- The second test:
Cytotoxicity: the RTG of the cells exposed for 3h in the absence of S9 at the doses of 40, 25, 20, 10, 5 and 2.5 μg/mL were 17%, 53%, 69%, 94%, 86% and 103%; the RTG of the cells exposed for 3h in the presence of S9 at the doses of 80, 40, 25, 20, 10 and 5μg/mL were 18%, 42%, 67%, 76%, 104% and101%; the RTG of the cells exposed for 24h in the absence of S9 at the doses of 40, 25, 20, 10, 5 and 2.5 μg/mL were 1%, 20%, 59%, 71%, 92% and 79 %.
The results of mutant frequency: the IMF at all doses in each treatment condition was less than GEF( GEF=126E-6).

Conclusions:

The test item is non-mutagenic to in vitro cultured L5178Y mouse lymphoma cells under the conditions of this study.

Executive summary:

The study was performed to evaluate the test item for its ability to cause gene mutations in vitro cultured mammalian cells (L5178Y), after being exposed for 3 hours with and without metabolic activation respectively, and exposed for 24 hours without metabolic activation in compatible with OECD 490.

In the first test, L5178Y were exposed at six exposure concentrations including 2000, 800, 320, 128, 51.2 and 20.5 μg/mL media in three treatment conditions above. In the second test, L5178Y were also exposed at six exposure concentrations including 40, 25, 20, 10, 5 and 2.5 μg/mL media for 3 hours and 24 hours without metabolic activation and 80, 40, 25, 20, 10 and 5 μg/mL media for 3 hours with metabolic activation. At the same time, the concurrent untreated controls, solvent (Ethanol) controls and positive controls were included in each condition in each test. The dose volume of each dose group and solvent control group was 5 μg/mL medium, in duplicate.

Cytotoxicity: the RTG of the cells exposed for 3h in the absence of S9 at the doses of 40, 25, 20, 10, 5 and 2.5 μg/mL were 17%, 53%, 69%, 94%, 86% and 103%; the RTG of the cells exposed for 3h in the presence of S9 at the doses of 80, 40, 25, 20, 10 and 5 μg/mL were 18%, 42%, 67%, 76%, 104% and 101%; the RTG of the cells exposed for 24h in the absence of S9 at the doses of 40, 25, 20, 10, 5 and 2.5 μg/mL were 1%, 20%, 59%, 71%, 92% and 79 %.

The results of mutant frequency: the IMF at all doses in each treatment condition was less than GEF( GEF=126E-6).

The test item is non-mutagenic to in vitro cultured L5178Y mouse lymphoma cells under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Data:

Ames test, OECD 471: negative

In vitro chromosome aberration study, OECD 473: negative

In Vitro Mammalian Cell Gene Mutation Test, OECD 490: negative

 

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, the test substance should not be classified as germ cell mutagens.