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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative

In vitro chromosome aberration test (OECD TG 473): negative

In vitro Gene mutation in mammalian cells (OECD TG 490): negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 April 2019 - 23 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
bacteria
Species / strain / cell type:
E. coli WP2 uvr A
Remarks:
bacteria
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight)
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP, and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The solution was filter-sterelized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 mL S9-mix in 3mL top molten agar with 0.1 mL of a dilution of the test item in vehicle and 0.1 mL of fresh bacterial culture.
- quality controls of S9: Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.
Test concentrations with justification for top dose:
Experiment 1:
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate.
The highest concentration of SHR 1396 used in the subsequent mutation assays was 5000 μg/plate or the level at which the test item exhibited limited solubility.
Main study: TA1535, TA1537 and TA98 (without and with S9-mix): 52, 164, 512, 1600 and 5000 µg/plate

Experiment 2, TA1535, TA1537, TA100 and WP2uvrA:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2A, TA98:
Without S9 mix: 26, 82, 256, 800, 2500 µg/plate
With S9 mix: 26, 82, 256, 800, 2500, 5000 µg/plate
Vehicle / solvent:
- Solvent used for test item: Ethanol
- Justification for choice of solvent: A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in ethanol at concentrations of 100 mg/mL and below.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Saline
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9: 5 µg/plate in saline for TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
without S9: 2.5 µg/plate in DMSO for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9: 15 µg/plate in DMSO for TA1537 (pre-incubation assay)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9: 10 µg/plate in DMSO for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 650 µg/plate in DMSO for TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9: 10 µg/plate in DMSO for WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9: 2.5 µg/plate in DMSO for TA1535, TA 1537; 1 µg/plate in DMSO for TA98 and TA100 (direct plate assay); 5 µg/plate in DMSO for TA100 (pre-incubation assay); 15 µg/plate in DMSO for WP2uvrA.
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

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

DOSE RANGE FINDING TEST
- Strains TA100 and WP2uvrA, with and without S9-mix.
- Concentrations: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate, tested in triplicate.

TREATMENT:
First experiment: Direct plate assay. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in ethanol and either 0.5 ml S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Second experiment: Pre-incubation assay. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C, either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays), 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.05 mL of a dilution of the test item in ethanol. After the pre-incubation period the solutions were added to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

ENVIRONMENTAL CONDITIONS
- Incubation at 37.0 ± 1.0°C (actual range 35.4 - 38.1°C).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER:
- The presence of precipitation of the test compound on the plates was determined.



Evaluation criteria:
ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

INTERPRETATION
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No statistical analysis was performed.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only in the second experiment, with and without S9 mix
Vehicle controls validity:
valid
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:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
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
Remarks:
only in the second experiment, without S9 mix
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRECIPITATE
In the dose rage finding, precipitation of SHR 1396 on the plates was not observed at the end of the incubation period in any tester strain.
In the first experiment, precipitation of SHR 1396 on the plates was observed at 1600 and 5000 μg/plate at the end of the incubation period.
In the second experiment, precipitation of SHR 1396 on the plates was observed at the concentrations of 1600, 2500 and/or 5000 μg/plate at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES
In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

TOXICITY
In the first experiment, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In strain TA1537 (absence and presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reduction are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.
In the second experiment cytotoxicity was observed in tester strains TA100 in the absence and presence of S9-mix, at 52 μg/plate and upwards and 512 μg/plate and upwards respectively, and TA1537 in the absence of S9-mix at the highest concentration of 5000 μg/plate.

RESULTS
- Individual plate counts : see 'detail tables' in attachment
- Mean number of revertant colonies per plate and standard deviation : see 'summary tables' in attachment

HISTORICAL CONTROL DATA: see tables included in 'any other information on results including tables'

Historical control data solvent control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

3 – 29

3 – 27

3 – 20

3 – 23

8 - 61

8 – 60

61 – 176

60 - 176

10 – 61

9 - 68

Mean

10

11

6

6

16

22

112

108

27

33

SD

3

3

2

3

5

7

18

21

8

9

n

3303

3265

3232

3212

3251

3326

3336

3246

3021

2993

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Apr 2016 and Apr 2019

Historical control data of the positive control items

 

TA1535

TA1537

TA98

S9-mix

-

+

-

+

-

+

Range

128 – 1530

73 – 1481

58 – 1422

54 – 1239

365 – 1978

250 – 2018

Mean

919

256

802

328

1305

910

SD

172

122

362

154

236

355

n

3215

3122

2777

3187

3202

3216

 

 

TA100

WP2uvrA

S9-mix

-

+

-

+

Range

439 – 1993

408 - 2379

93 – 1999

109 - 1968

Mean

907

1308

1073

437

SD

167

386

537

158

n

3231

3179

2923

2987

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Apr 2016 and Apr 2019. 

Conclusions:
In an AMES test, performed according to OECD471 guideline and GLP principles, SHR 1396 was found not to be mutagenic with or without metabolic activation.
Executive summary:

An in vitro gene mutation (AMES) test was performed according to OECD 471 guideline and in accordance with GLP principles. All bacterial strains TA1535, TA1537, TA100, TA98 and WP2uvrA showed negative responses up to 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. Cytotoxicity was observed in tester strains TA100 in the absence and presence of S9 mix and in the tester strain TA 1537 in the absence of S9 mix. No cytotoxicity and/or precipitation of the test substance was observed in the other tester strains. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that SHR 1396 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 April 2019 - 15 July 2019
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:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Sex, age and number of blood donors:
Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2018): age 24 - 31; AGT: 12.6 - 13.5h
- Whether whole blood or separated lymphocytes were used:
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One,The Netherlands). Immediately after blood collection lymphocyte cultures were started.
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Culture medium consisted of RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 32 - 95%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 38.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Rat S9 homogenate was obtained from Trinova Biochem GmbH and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES.
The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: 1.8% (v/v).
Test concentrations with justification for top dose:
Dose range finding test:
- Without/with S9-mix, 3hr exposure; 24hr fixation: 7.8, 15.6, 31, 63, 125, 250 μg/mL
- Without S9-mix, 24hr expsoure; 24hr fixation: 15.6, 31, 63, 125, 250, 500 μg/mL
- Without S9-mix, 48hr exposure; 48hr fixation: 31, 63, 125, 250, 500, 1000 μg/mL
First cytogenetic test:
- Without and with S9-mix, 3hr exposure time, 24hr fixation time: 10, 100, 150, 200, 250, 300, 350, 400 μg/mL
Second cytogenetic test:
- Without S9-mix, 24hr exposure; 24hr fixation: 10, 50, 65, 80, 95, 110, 125 μg/mL
- Without S9-mix, 48hr exposure; 48hr fixation: 10, 30, 40, 50, 60 μg/mL
Repeat experiment cytogenic test:
- Without S9-mix, 48hr exposure; 48hr fixation: 10, 30, 40, 50, 60, 70, 80 μg/mL
Vehicle / solvent:
- Solvent used: Ethanol
- Justification for choice of solvent: solubility test was performed based on visual assessment. The test item formed a clear colorless solution in ethanol.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 hr
- Exposure duration/duration of treatment: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Harvest time after the end of treatment (sampling/recovery times): 24 and 48 hr

FOR CHROMOSOME ABERRATION
- Spindle inhibitor: colchicine (0.5 μg/mL medium)
- Methods of slide preparation and staining technique used including the stain used: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/etherand cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper.
- Number of cells spread and analysed per concentration: One hundred and fifty metaphase chromosome spreads per culture were examined in duplicate.
- Criteria for scoring chromosome aberrations: Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%).

ACCEPTABILITY CRITERIA
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analyzed by the Fisher’s exact test (one-sided, p < 0.05).
Evaluation criteria:
A test item is considered positive (clastogenic) in the chromosome aberration test if:
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 a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None 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) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
Statistics:
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used.
Key result
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: The highest concentration analyzed was selected based on the solubility of the test item in the culture medium (3 h exposure time) or on toxicity, inhibition of the mitotic index of about 50% or greater (24h and 48h exposure time).
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
At a concentration of 500 μg/mL SHR 1396 precipitated in the culture medium. Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays.
The highest concentration analyzed was selected based on the solubility of the test item in the culture medium (3 h exposure time) or on toxicity, inhibition of the mitotic index of about 50% or greater (24h and 48h exposure time).

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database
The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

- Genotoxicity results
SHR 1396 did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments (see attached background material for results of each seperate experiment).
No effects of SHR 1396 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

TEST-SPECIFIC CONFOUNDING FACTORS
In general at a concentration of 250 μg/mL or higher SHR 1396 precipitated in the culture medium in the experiments.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- See any other information on results incl. tables for historical control data.

Historical Control Data for in vitro Chromosome Aberration Studies of the Solvent Control

 

 

 

3 hours exposure time

24 hours exposure time

48 hours exposure time

Gaps included

Gaps excluded

Gaps included

Gaps excluded

Gaps included

Gaps excluded

+ S9-mix

- S9-mix

+ S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

Mean number of aberrant cells per 100 cells

0.40

0.50

0.31

0.42

0.44

0.37

0.87

0.69

SD

0.53

0.73

0.45

0.68

0.65

0.63

2.44

2.28

n

130

130

130

130

126

126

122

122

Upper control limit

(95% control limits)

1.68

2.32

1.39

1.95

1.92

1.71

3.92

3.13

Lower control limit

(95% control limits)

-0.89

-1.32

-0.77

-1.10

-1.03

-0.96

-2.17

-1.74

SD = Standard deviation n = Number of observations

Distribution historical negative control data from experiments performed between September 2015 and September 2018.

Historical Control Data for in vitro Chromosome Aberration Studies of the Positive Control Substances 

 

 

 

3 hours exposure time

24 hours exposure time

48 hours exposure time

Gaps included

Gaps excluded

Gaps included

Gaps excluded

Gaps included

Gaps excluded

+ S9-mix

- S9-mix

+ S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

Mean number of aberrant cells per 100 cells

21.63

24.16

20.78

23.65

26.76

26.09

33.65

32.92

SD

10.45

12.83

10.26

13.04

14.43

14.41

15.28

15.26

n

128

130

128

130

124

124

120

120

Upper control limit

(95% control limits)

39.56

44.96

38.16

44.70

51.64

50.92

63.20

62.08

Lower control limit

(95% control limits)

3.70

3.35

3.39

2.60

1.89

1.25

4.10

3.76

SD = Standard deviation n = Number of observations

Distribution historical positive control data from experiments performed between September 2015 and September 2018.

           

Historical Control Data for Numerical Aberrations for in vitro Chromosome Aberration Studies of the Solvent Control

 

 

 

3 hours exposure time

24 hours exposure time

48 hours exposure time

Poly

Endo

Poly

Endo

Poly

Endo

+ S9-mix

- S9-mix

+ S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

Mean number numerical

aberrations per

100 cells

0.06

0.02

0

0.01

0.05

0.02

0.03

0.01

SD

0.24

0.12

0

0.08

0.24

0.12

0.14

0.12

n

130

130

130

130

126

126

122

122

Upper control limit

(95% control limits)

0.30

0.13

0

0.04

0.25

0.11

0.16

0.07

Lower control limit

(95% control limits)

-0.19

-0.09

0

-0.02

-0.15

-0.06

-0.10

-0.05

Endo = endoreduplication

D = Standard deviation n = Number of observations Poly = polyploidy

Distribution historical negative control data from experiments performed between September 2015 and September 2018.

Conclusions:
A chromosome aberration study with SHR 1396 was performed according to OECD TG 473 and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that SHR 1396 is not clastogenic in human lymphocytes.
Executive summary:

A chromosome aberration study with SHR 1396 was performed according to OECD TG 473 and in accordance with GLP principles, in cultured peripheral human lymphocytes. In the first cytogenetic assay, SHR 1396 was tested up to and including precipitating levels (250 μg/mL) for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-mix. In the second cytogenetic assay, SHR 1396 was tested up to 110 μg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 70 μg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. Reliable solvent and positive controls were included in both experiments. Both in the absence and presence of S9-mix SHR 1396 did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments. No effects of SHR 1396 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that SHR 1396 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations. Based on the results it can be concluded that SHR 1396 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 September 2019 - 19 November 2019
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:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Methods for maintenance in cell culture: stock cultures of the cells were stored in the freezer (- 150°C).
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:

Basic medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium:
For 3 hour exposure: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/mL trifluorothymidine (TFT).
Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).

Environmental conditions:
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 49 - 97%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 - 37.5 °C).
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal enzymes (S9 homogenate) was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES. The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: The concentration of the S9-fraction in the exposure medium was 4% (v/v).
Test concentrations with justification for top dose:
- Dose-range finding test, with S9 (3h) and without S9 (3h & 24h): 6.3, 12.5, 25, 50 and 120 μg/mL
- Experiment 1, with S9 (3h): 0.5, 1, 2, 5, 10, 25, 50, 60, 70, 80, 90, 100, 110 and 120 μg/mL
- Experiment 1, without S9 (3h): 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30 and 35 μg/mL
- Experiment 2 without S9 (24h): 0.6, 1.3, 2.5, 5, 10, 20, 40, 45, 50, 55 and 60 μg/mL
- Repeat experiment 2, without S9 (24h): 0.6, 1.3, 2.5, 5, 10, 20, 25, 30, 35, 40, 45 and 50 μg/mL

Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 120 μg/mL exposure medium.
Vehicle / solvent:
- Vehicle used: Ethanol
- Justification for choice of vehicle: A solubility test was performed based on visual assessment. The test item formed a clear colourles solution in ethanol.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures: 1 per test concentration; positive control: 1; solvent control: 2
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): below 1 x 10^6 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Short term treatment: 3 hours (with and without S9-mix)
Prolonged treatment: 24 hours (without S9-mix)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selective agent): 11 or 12 days
- Method used: microwell plates
- Selective agent is used: 5 μg/mL trifluorothymidine (TFT)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium. For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFTselection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
- Criteria for small (slow growing) and large (fast growing) colonies:
The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (see 'Any other information on materials and methods incl. tables' for details on calculations)
Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWGT 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 item is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more than the mutation frequency in the controls + 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 item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of the mutation frequency in the controls + 126.
ACCEPTABILITY CRITERIA:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6.
At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).
Statistics:
Not performed
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
SHR 1396 precipitated in the exposure medium at concentrations of 120 μg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- In the 3-hour exposure period, In the absence of S9-mix, the relative suspension growth was 11% at the test item concentration of 25 μg/mL compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at test item concentrations of 50 μg/mL and above. In the presence of S9-mix, no toxicity in the relative suspension growth was observed up to test item concentrations of 50 μg/mL compared to the solvent control. Hardly any cell survival was observed at the test item concentrations of 120 μg/mL.
- In the 24-hour exposure period (without S9-mix), The relative suspension growth was 15% at the test item concentration of 50 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test item concentrations of 120 μg/mL.

STUDY RESULTS (result tables are reported in the attached background material).
- Concurrent vehicle negative and positive control data:
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Gene mutation tests in mammalian cells:
The dose levels selected to measure mutation frequencies at the TK-locus in experiment 1 were:
Without S9-mix: 0.1, 0.5, 1, 2, 5, 10, 15 and 20 μg/mL exposure medium.
With S9-mix: 0.5, 1, 2, 5, 10, 25, 50 and 60 μg/mL exposure medium.

The dose levels selected to measure mutation frequencies at the TK-locus in experiment 2 were:
Without S9-mix: 1.3, 10, 20, 25, 30, 35, 40 and 45 μg/mL exposure medium.

- Results from cytotoxicity measurements:
The RTG at the highest concentration was 35% and 13% compared to the total growth of the solvent controls without and with metabolic activation respectively in experiment 1
The RTG at the highest concentration was 14% compared to the total growth of the solvent controls in experiment 2

- Genotoxicity results:
No biologically relevant 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.

HISTORICAL CONTROL DATA
- See any other information on results incl. tables for historical control data.

Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay 

 

 

 

Mutation frequency per 106survivors

-S9 Mix

+S9 mix

3 hour treatment

24 hour treatment

3 hour treatment

Mean

100

100

102

SD

31

31

30

n

133

123

137

Lower Control Limit (95% Control Limits)

40

38

44

Upper Control Limit (95% Control Limits)

160

162

160

SD = Standard deviation n = Number of observations

Distribution historical negative control data from experiments performed between September 2016 and September 2019. 

  Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay 

 

 

 

Mutation frequency per 106survivors

-S9 Mix

+S9 mix

3 hour treatment

24 hour treatment

3 hour treatment

Mean

817

722

1461

SD

304

216

885

n

133

123

137

Lower Control Limit (95% Control Limits)

221

298

-273

Upper Control Limit (95% Control Limits)

1412

1147

3196

SD = Standard deviation n = Number of observations

Distribution historical positive control data from experiments performed between September 2016 and September 2019. 

Conclusions:
In a gene mutation test in mammalian cells performed according to OECD TG 490 and in accordance with GLP principles it is conlcluded that SHR 1396 is not mutagenic in the TK mutation test system.
Executive summary:

A gene mutation test in mammalian cells was performed according to OECD TG 490 and in accordance with GLP principles, to assess the potential of SHR 1396 to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. In the first experiment, the test item was tested up to and including concentrations of 35 and 120 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. The highest doses that were analyzed gave a cell survival of 35% (20 μg/mL) and 13% (60 μg/mL) in the absence and presence of S9-mix, respectively. In the second experiment, the test item was tested up to a concentration of 60 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The highest concentration analyzed gave a cell survival of 14% (45 μg/mL). Reliable negative and positive controls were included in both experiments. In the absence and presence of S9-mix, SHR 1396 did not induce a biologically relevant increase in the mutation frequency. In conclusion, SHR 1396 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test:

An in vitro gene mutation (AMES) test was performed according to OECD 471 guideline and in accordance with GLP principles. All bacterial strains TA1535, TA1537, TA100, TA98 and WP2uvrA showed negative responses up to 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. Cytotoxicity was observed in tester strains TA100 in the absence and presence of S9 mix and in the tester strain TA 1537 in the absence of S9 mix. No cytotoxicity and/or precipitation of the test substance was observed in the other tester strains. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that SHR 1396 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.

Chromosome aberration test:

A chromosome aberration study with SHR 1396 was performed according to OECD TG 473 and in accordance with GLP principles, in cultured peripheral human lymphocytes. In the first cytogenetic assay, SHR 1396 was tested up to and including precipitating levels (250 μg/mL) for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-mix. In the second cytogenetic assay, SHR 1396 was tested up to 110 μg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 70 μg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. Reliable solvent and positive controls were included in both experiments. Both in the absence and presence of S9-mix SHR 1396 did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments. No effects of SHR 1396 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that SHR 1396 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations. Based on the results it can be concluded that SHR 1396 is not clastogenic in human lymphocytes.

Mouse lymphoma test:

A gene mutation test in mammalian cells was performed according to OECD TG 490 and in accordance with GLP principles, to assess the potential of SHR 1396 to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. In the first experiment, the test item was tested up to and including concentrations of 35 and 120 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours.The highest doses that were analyzed gave a cell survival of 35% (20μg/mL) and 13% (60μg/mL) in the absence and presence of S9-mix, respectively. In the second experiment, the test item was tested up to a concentration of 60 μg/mL in the absence of S9-mix. The incubation time was 24 hours.The highest concentration analyzed gave a cell survival of 14% (45μg/mL). Reliable negative and positive controls were included in both experiments. In the absence and presence of S9-mix, SHR 1396 did not induce a biologically relevant increase in the mutation frequency. In conclusion, SHR 1396 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Justification for classification or non-classification

Based on the results of the Ames test, the in vitro chromosome aberration test and the mouse lymphoma test, the substance does not have to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 and its amendments.