Reaction mass of 2- (palmitoylamino)ethyl acrylate and 2-(stearoylamino)ethyl acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the test item was not considered to be mutagenic in S. typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA in the presence and absence of phenobarbital & 5,6-benzoflavone-induced rat liver S9. (OECD 471/GLP).

 

In vitro cytogenicity (chromosome aberration) study in mammalian cells: the test item did not induce any statistically significant increases in the frequency of structural or numerical aberrations in the presence or absence phenobarbital/5,6-benzo flavone-induced rat liver S9 metabolic activation in Chinese hamster lung fibroblasts (CHL/IU) cells (OECD 473/GLP).

 

Gene mutation (mammalian cell gene mutation assay): there was no evidence of induced mutant colonies over background in mouse lymphoma L5178Y cells exposed to the test item in the presence and absence of phenobarbital and 5,6-benzoflavone -induced rat liver S9 metabolic activation (OECD 490/GLP)

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 12, 2021-May 31, 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No vehicle justification or historical control data

Qualifier:

according to guideline

Guideline:

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

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:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Provided by Sponsor, Lot number: 91112Y
- Purity.: 92.4%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: (with no photoreactivity， hydrolyzability or effect of air) ,Room temperature(acceptable range 10.0-30.0℃)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stability

Target gene:

TK +/- 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 (IVGT) (hereafter referred to as “L5178Y”); Japanese Collection of Research Bioresources (JCRB) Cell Bank,
Laboratory of Cell Cultures, National Institutes of Biomedical Innovation, Health and Nutrition

For cell lines:
- Absence of Mycoplasma contamination: Free (confirmed on Aug. 3, 2020)
- Number of passages if applicable: Dose range-finding test: 9; Main test: 10
- Cell cycle length, doubling time or proliferation index : 9.0 hours
- Periodically ‘cleansed’ of spontaneous mutants: Spontaneous mutant frequency: = 76.4 × 10−6

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
RPMI1640 media containing heat-inactivated horse serum at 0-20 v/v%, Penicillin-streptomycin, Sodium Pyruvate and 10% PLURONIC® F-68. L5178Y cells were seeded in tissue culture flasks or 96-well plates and statically cultured in a high humidity CO2 incubator (5% CO2, 37°C).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Oriental Yeast Co. Ltd.

- Method of preparation of S9 mix: S9: A supernatant fraction of a liver homogenate (9000 × g) obtained from 7-week-old male Sprague- Dawley rats pretreated with phenobarbital and 5,6-benzoflavone. S9 mix: S9 and Cofactor-C were thawed immediately prior to preparation and mixed at a ratio of 3/7(mL) to prepare the S9 mix. The S9 mix was stored in ice water until use.

- Final concentration of S9-fraction in cultures : 1.5%

Test concentrations with justification for top dose:

Preliminary tests: 0.0625, 0.125, 0.25, 0.5, 1, 3, 10 µg/mL.
Main tests: 0.0625, 0.125, 0.25, 0.5, 1 µg/mL.
Based on the results of the dose range-finding test, 1 µg/mL, at which precipitation of the test article was observed, was selected as the highest dose level.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water for Injection, JP

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Single (negative/positive controls, test item)
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 e6 cells/mL
- Test substance added in suspension

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hrs (+/-S9); 24 hrs (-S9)
- Harvest time after the end of treatment (sampling/recovery times):

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): 12 days
- Method used: microwell plates
- If a selective agent is used: Trifluorothymidine (600 μg/mL TFT, 0.5 mL for the negative control group and 0.25 mL for the other groups)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: The TFT-treated cell suspension was divided into each well (0.2 mL (2000 cells)/well) of two 96-well MF plates (4 plates for the negative control group).
- Criteria for small (slow growing) and large (fast growing) colonies: A large colony was defined as that covering 1/4 or more of the well’s diameter and a small colony as that covering less than 1/4 of the well’s diameter. It was specified as the criteria for morphological classification of colonies that the cell density is low and the cell margin is loose in a large colony and the cell density is high and the cell margin is well-defined in a small colony. However, the colonies were classified in priority to the colony size.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity:
In the short-term treatment (−S9 mix) at 0.0625-1 µg/mL, no dose-related cytotoxicity was noted and the RTG values were 92.8% to 108.9%.
In the short-term treatment (+S9 mix) at 0.0625-1 µg/mL, no dose-related cytotoxicity was noted and the RTG values were 78.7% to 106.5%.
In the continuous treatment at 0.0625-1 µg/mL, no dose-related cytotoxicity was noted and the RTG values were 95.9% to 139.6%.

Evaluation criteria:

• The results were judged to be positive or negative based on the T-MF values according to
the criteria for global evaluation factors (GEF) listed below.
• The GEF value in this study was 126 e-6.
• Negative:
1.When X < (GEF + T-MF in the simultaneous negative control group)
2.When marked dose-dependency is not noted
• Positive:
1.When (GEF + T-MF in the simultaneous negative control group) ≤ X is noted at 1 dose level or more
2.When a dose-dependent increase is noted

Statistics:

INATOX-DP (SAS) system: For statistical analysis and tabulation of data.
Microsoft Excel 2007: For statistical analysis (simple regression analysis).

For judgement of dose-dependency, simple regression analysis was performed on T-MF values between the negative control and test article groups at a significance level of 0.05. The dose levels at which excessive cytotoxicity (RTG < 10%) was noted were excluded from study evaluation.

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

3 hrs, 24 hrs

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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
- Water solubility: 0.7 mg/L
- Precipitation and time of the determination: Precipitation of the test article was observed at 1 µg/mL at initiation and termination of treatment in the main tests.

RANGE-FINDING/SCREENING STUDIES (if applicable): The RTG values at 0.0625-10 µg/mL were 78.8% to 107.3% in the short-term treatment (−S9 mix), 77.0% to 117.9% in the short-term treatment (+S9 mix) and 76.3% to 95.6% in the continuous treatment, indicating no clear dose-related cytotoxicity. Precipitation of the test article was observed at 1 µg/mL and above at initiation and termination of treatment.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
3 hrs – S9: In the negative control group, PE0, PE2 and T-MF were 89.3%, 69.2% and 130.6 × 10−6, respectively, which conformed to the criteria for validity of the study. In the positive control group, T-MF was 1197.1 × 10−6, indicating a marked positive reaction.
3 hrs +S9: In the negative control group, PE0 (cytotoxicity), PE2 (mutants) and T-MF were 109.1%, 67.2% and 147.4 × 10−6, respectively, which conformed to the criteria for validity of the study. In the positive control group, T-MF was 1653.4 × 10−6, indicating a marked positive reaction.
24 hr – S9: In the negative control group, PE0, PE2 and T-MF were 101.2%, 74.3% and 141.9 × 10−6, respectively, which conformed to the criteria for validity of the study. In the positive control group, T-MF was 1316.0 × 10−6, indicating a marked positive reaction.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency:
3 hrs - S9: No dose-related cytotoxicity was noted and the RTG values were 92.8% to 108.9%.
3 hrs +S9: No dose-related cytotoxicity was noted and the RTG values were 78.7% to 106.5%
24hrs -S9: No dose-related cytotoxicity was noted and the RTG values were 95.9% to 139.6%.

- Genotoxicity results:
3 hrs -S9: T-MF values at 0.0625-1 µg/mL were 134.8 × 10−6 to 151.7 × 10−6, in which neither increases exceeding GEF (126 × 10−6) as compared to the T-MF value in the negative control group (130.6 × 10−6) at any dose level nor dose-dependency was noted
3hrs +S9: T-MF values at 0.0625-1 µg/mL were 59.2 × 10−6 to 174.8 × 10−6, in which neither increases exceeding GEF (126 × 10−6) as compared to the T-MF value in the negative control group (147.4 × 10−6) at any dose level nor dose-dependency was noted.
24 hrs -S9: T-MF values at 0.0625-1 µg/mL were 89.5 × 10−6 to 131.1 × 10−6, in which no increases exceeding GEF (126 × 10−6) were noted as compared to the T-MF value in the negative control group (141.9 × 10−6) at any dose level. Dose-dependent decreases were noted in the T-MF values in the test article groups, but not considered to be a biologically significant change.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
Not provided

Conclusions:

In conclusion, the test item is considered to be non-mutagenic with and without metabolic activation in this in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

In a mammalian cell gene mutation assay [MLA; EF20382], mouse lymphoma L5178Y cells cultured in vitro were exposed to the test item (92.4%) in water at concentrations of 0, 0.0625, 0.125, 0.25, 0.5, 1 µg/mL for 3 hours with phenobarbital and 5,6-benzoflavone -induced rat liver S9 metabolic activation and 3 and 24 hrs without metabolic activation.
There was no cytotoxicity noted but the test item was assayed up to precipitating concentrations. The positive and negative controls induced the appropriate response. Neither increases exceeding the global evaluation factor (GEF, 126 × 10−6) in the total colony mutant frequency (T-MF) values of the test article groups as compared to the T-MF values in the negative control group nor dose-dependent increases were noted in any of the treatment methods. In conclusion, the test item was considered not to possess the gene mutagenic potential to L5178Y cells under the conditions of this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 11 2021-June 10 2021

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Provided by Sponsor, Lot number 91112Y
- Purity92.4%.:


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
The test substance was put into an airtight container and stored at room temperature in the test substance storage room(permissible range：from 10℃ to 30℃) .
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stability


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): the test substance was weighted, it was crushed using an agate mortar. Purity was corrected.

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
- Normal cell cycle time (negative control): About 15 hours

For cell lines:
- Absence of Mycoplasma contamination: Confirmed as negative
- Number of passages if applicable: 13 for cell growth inhibition test; 7 for chromosomal aberration test; Passaged twice or three times a week.
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes: 25 per cell

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: L-Glutamine (final concentration：0.292g/L) and sodium hydrogen carbonate(final Concentration: approximately 1.95g/L) were added to Eagle's minimum essential medium (Lot number 766006), Nissui Pharmaceutical) and basal medium (MEM) was prepared. This medium was then supplemented with 10% heat-inactivated NBCS (NBCS of Lot number 18H495 (SAFC Biosciences) and NBCS of Lot number S17420S0750 (Bio West) were mixed in a ratio of 4:1). 5% CO2, humid conditions; 37℃

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Ieda Trading Corporation ((RAA202011A (prelim test); RAA202101A (chr. abb. test)))

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL;

Main tests:
6 hrs – S9: 50, 100, 150, 200, 250, 300, 350, 400 μg/mL;
6 hrs +S9: 100, 200, 400, 500, 600, 700, 800, 900 μg/mL;
24 hrs -S9: 15, 30, 40, 50, 60, 70, 80, 90, 100 μg/mL.


Cytotoxicity was observed in all treatment methods. Therefore, the doses above were set in the main tests to obtain the dose at which RPD or RICC was 40% or more and 50% or less.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:0.5% w/v methylcellulose (MC) solution

- Justification for choice of solvent/vehicle: The test substance was insoluble in distilled water and 0.5% w/v CMC solution at 20.0mg/mL and in DMSO and acetone at 200mg/mL. The test substance was suspended in 0.5% w/v MC solution at 20.0mg/mL. This formulation was considered to be stable from the facts on account of neither change in color, exothermic reaction nor gas generation at room temperature until 2 hours after preparation. Therefore， the 0.5% w/v MC solution was selected as a vehicle，

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.5% w/v% MC solution

True negative controls:

no

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: 1 (6 hrs +/-S9; 24 hrs-S9)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 5 x 10~3
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 6 hrs +/-S9; 24 hrs-S9
- Harvest time after the end of treatment (sampling/recovery times): 6 hrs: 18 hrs further incubation in fresh medium; 24 hrs: It was predicted that precipitation of the test substance prevents specimens observation in the 24 hours continuous treatment, medium exchange was carried out before the addition of demecolcine solution.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Fifty microliters of 10 ug/mL Demecolcine solution was added to each dish at 2 hours before the end of the culture.

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): At the end of the culture, leftover cells after cell count were collected by a centrifugation at 1000 rpm for 5 minutes and were treated hypotonically with 3 mL of 0.075 mol/L KCl at 37℃ for 15 minutes. After the hypotonic treatment， the cells were pre-fixed once with approximately 0.3 mL of fixative solution (methanol:acetic acid=3:1) and were completely fixed twice with 3mL of fixative solutions. Then, the cell suspension was prepared with appropriate amount of a fixative solution， the suspension was dropped onto a glass slide. Four specimens per dose (two specimens per dish) were prepared. 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).

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
Structural: Three hundred metaphase cells per dose (75 cells per specimen) containing 25 ± 2 chromosomes were observed using a microscope
Numerical: 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.

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): The total number of cells with structural aberrations excluding gaps and the number of aberrant cells in each aberration category shown below were recorded. 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 (di centric,ring and translocation)
(5) Fragmentation
- Determination of polyploidy: 38 or more chromosomes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative population doubling (RPD); relative increase in cell count (RICC).

- Any supplementary information relevant to cytotoxicity:
In all treatment methods, the cytotoxicity was observed. In “-S9mix”, the lowest dose at which RPD was 40% or more and 50% or less was selected as the maximum dose and the following doses were selected for observation. In “+S9mix”and 24 hours continuous treatment, the lowest dose at which RICC was 40% or more and 50% or less was selected as the maximum dose and the following doses were selected for observation:

6hrs - S9: 100, 200, 300 µg/mL
6 hrs +S9; 200, 400, 600 µg/mL
24 hrs - S9: 15, 50 and 60 µg/mL

Evaluation criteria:

Regarding the frequencies of cells with chromosomal aberrations in the test substance doses，
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 in the test substance doses，
the test substance was judged to be positive in cases of c) toe) or f) were fulfilled：
c) outside the distribution of the historical data of the negative control group
d) the doses of the test substance exhibit 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 method was performed in order to compare the number of cells with structural
aberrations and numerically aberrant cells. Significance level of these tests was both sides
of 1% and 5%，

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 atleast one of the test substance doses exhibited a statistically signficant
increase compared with the negative control group in the Fisher's exact test the dose-
dependency was evaluated by using Cochran-Armitage trend test.

Species / strain:

Chinese hamster lung (CHL/IU)

Remarks:

6 hrs

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

300 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung (CHL/IU)

Remarks:

6 hrs

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

600 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung (CHL/IU)

Remarks:

24 hrs

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

60 µg/mL

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
- Precipitation and time of the determination: At the start and the end of the treatment and at the end of the culture, precipitation of the test substance were observed in preliminary and main tests.

RANGE-FINDING/SCREENING STUDIES (if applicable): Cytotoxicity was observed in all treatment methods. Therefore, the doses were set in the main tests to obtain the dose at which RPD or RICC was 40% or more and 50% or less.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Yes
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: No
- Statistical analysis; p-value if any: Only positive controls were significant (p<0.01) compared to controls.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index.
Cell proliferation criteria in the negative control met the criteria in the testing facility.

- Genotoxicity results (for both cell lines and lymphocytes)
The frequencies of structural and numerical aberrations for the test item were within the range of the historical data of the negative controls for all treatments. Therefore the test item was not mutagenic in the assay.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Historical control data (the latest 20 studies) completed by February 2021 were provided.
- Negative (solvent/vehicle) historical control data: Historical control data (the latest 20 studies) completed by February 2021 were provided.

Conclusions:

In an in vitro cytogenicity (chromosome aberration) study in Chinese hamster lung fibroblasts (CHL/IU) cells, the test item did not induce structural or numerical aberrations.

Executive summary:

In an in vitro cytogenicity (chromosome aberration) study in mammalian cells (K06-1691), Chinese hamster lung fibroblasts (CHL/IU) cells were exposed to the test item in 0.5% w/v methylcellulose (MC) solution at concentrations of 50, 100, 150, 200, 250, 300, 350, 400 μg/mL (6 hrs) and 15, 30, 40, 50, 60, 70, 80, 90, 100 μg/mL (24 hrs) without Phenobarbital/ 5,6-benzo flavone-induced rat liver S9 metabolic activation and 100, 200, 400, 500, 600, 700, 800, 900 μg/mL (6 hrs) with metabolic activation.

 

The test item was tested up to cytotoxic levels. Positive controls induced the appropriate response. The test material did not induce any statistically significant increases in the frequency of cells with structural or numerical aberrations in the presence or absence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 3, 2020 -March 25, 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: test material provided by sponsor:91112Y
- Purity.:92.4%;

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:
Insoluble in water and dimethyl sulfoxide(DMSO) at 50mg/mL, soluble and stable in acetone.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Oriental Yeast Co.， Ltd. (Lot No.20011711); Phenobarbital & 5,6-Benzoflavone-induced rat liver.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): sterile performed, the prepared S9 mix wa kept on an ice-bath until use(0.5mL/plate).

Test concentrations with justification for top dose:

Preliminary toxicity test: 4.88, 19.5, 78.1, 313, 1250, 5000 ug/plate.

Mutagenicity test 1 (+/-S9): 2.44， 4.88， 9.77， 19.5， 39.1, 78.1 ug/plate for TA 98 strain of-S9 mix，
0.610， 1.22， 2.44， 4.88， 9.77， 19.5， 39.1 ug/plate for TA 100， TA 1535， TA 1537 strains of -S9 mix，
19.5， 39.1， 78.1， 156.3. 312.5， 625 ug/plate for TA 98， TA 100， TA 1535， TA 1537 strains of + S9 mix and WP2urA strain of ±S9mix.

Mutagenicity test (-S9) 2: 2.44， 4.88， 9.77， 19.5， 39.1 ug/plate for TA 98 strain of-S9 mix，
0.610, 1.22, 2.44， 4.88， 9.77 ug/plate for TA 100， TA 1535， TA 1537 strains of -S9 mix.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: According to the information provided by the study sponsor，the solubility of the test substance was insoluble in water and DMSO at 50mg/ml, soluble and stable in acetone at 100 mg/ml. From the above results, acetone was selected as a solvent for the test.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments:2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: (20 minutes at 37.0℃)
- Exposure duration/duration of treatment: 48h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: stereomicroscope(×40）;

Evaluation criteria:

Determined as positive(+) if the revertant colony numbers of the test substance group increases two or more times than that of the negative control group and reproducibility or dose dependency was observed for the increase.

Statistics:

Statistical analysis were not used for evaluation of the resulis.

Species / strain:

S. typhimurium TA 100

Remarks:

Expt I & II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

156 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Expt 1 & II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

156 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Remarks:

Expt I & II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

156 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Remarks:

Expt I & II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

156 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Remarks:

Expt I&II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

156 µg/plate

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
- Precipitation and time of the determination:
Preliminary toxicity test: Precipitations of the test item were observed at 313 µg/plate to above of ±S9mix .
Mutagenicity test 1 AND 2: Precipitations of the test item were observed at 156 µg/plate to above of ± S9mix, no cytotoxicity was observd in any strains of ± S9mix


RANGE-FINDING/SCREENING STUDIES (if applicable):
There was no increase in the number of revertant colonies for each strain compared to the negative controls, with or without metabolic activation. The positive controls of each strain showed marked increase in the number of revertant colonies compared to that of the corresponding negative control of each strain.


STUDY RESULTS
- Concurrent vehicle negative and positive control data: There was no increase in the number of revertant colonies for each strain compared to the negative controls, with or without metabolic activation. The positive controls of each strain showed marked increase in the number of revertant colonies compared to that of the corresponding negative control of each strain (Mutagenicity test 1 & 2).

Ames test:
- Signs of toxicity: none
- Individual plate counts: Yes
- Mean number of revertant colonies per plate and standard deviation: Mean and SD not provided.

Each strain confirmed the reproducibility, the numbers of revertant colonies in the negative and positive controls of each strain were within Mean ±3 standard deviation of the historical control data, contamination was not observed in the sterility test therefore, it was judged that the study met the validity criteria.

Conclusions:

In a reverse gene mutation assay in bacteria, the test item was considered non-mutagenic.

Executive summary:

In a reverse gene mutation assay in bacteria (USA-R-20053), strains of S. typhimurium TA 98, TA 100, TA1535, TA1537 and E. coli WP2uvrA were exposed to the test item (92.4%) in acetone at concentrations of 9.77 -313 µg/plate ± S9 in experiment 1 and 2. Both experiments were carried out via the pre-incubation method (20 mins at 37 °C) and metabolic activation was phenobarbital & 5,6-benzoflavone-induced rat liver S9.

 

In mutagenicity test 1 and 2, no cytotoxicity were observed , but precipitations of the test item were observed at 156 µg/plate to above of ± S9 mix for all strains.

 

The positive controls of each strain showed a marked increase in the number of revertant colonies compared to that of the corresponding negative control of each strain. There was no increase in the number of revertant colonies for each strain compared to the negative controls in test-item treated strains, with or without metabolic activation, in either experiment. Under the conditions of this study, the test item is considered non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test):

 

There is one gene mutation study (Bacterial Reverse Mutation Assay/Ames test) with the test item available.

 

In a reverse gene mutation assay in bacteria ( Bacterial reverse mutation test, OECD471/GLP), strains of S. typhimurium TA 98, TA 100, TA1535, TA1537 and E. coli WP2uvrA were exposed to the test item (92.4%) in acetone at concentrations of 9.77 -313 µg/plate ± S9 in experiment 1 and 2. Both experiments were carried out via the pre-incubation method (20 mins at 37 °C) and metabolic activation was phenobarbital & 5,6-benzoflavone-induced rat liver S9.

 

In mutagenicity test 1 and 2, no cytotoxicity were observed , but precipitations of the test item were observed at 156 µg/plate to above of ± S9 mix for all strains.

 

The positive controls of each strain showed a marked increase in the number of revertant colonies compared to that of the corresponding negative control of each strain. There was no increase in the number of revertant colonies for each strain compared to the negative controls in test-item treated strains, with or without metabolic activation, in either experiment. Under the conditions of this study, the test item is considered non-mutagenic.

 

In vitro cytogenicity (chromosome aberration) study in mammalian cells

 

In an in vitro cytogenicity (chromosome aberration) study in mammalian cells (OECD 473/GLP), Chinese hamster lung fibroblasts (CHL/IU) cells were exposed to the test item in 0.5% w/v methylcellulose (MC) solution at concentrations of 50, 100, 150, 200, 250, 300, 350, 400 μg/mL (6 hrs) and 15, 30, 40, 50, 60, 70, 80, 90, 100 μg/mL (24 hrs) without Phenobarbital/ 5,6-benzo flavone-induced rat liver S9 metabolic activation and 100, 200, 400, 500, 600, 700, 800, 900 μg/mL (6 hrs) with metabolic activation.

The test item was tested up to cytotoxic levels. Positive controls induced the appropriate response. The test material did not induce any statistically significant increases in the frequency of cells with structural or numerical aberrations in the presence or absence of metabolic activation.

 

Gene mutation(Mouse Lymphoma TK Assay)

 

In a mammalian cell gene mutation assay [MLA; OECD 490/GLP], mouse lymphoma L5178Y cells cultured in vitro were exposed to the test item (92.4%) in water at concentrations of 0, 0.0625, 0.125, 0.25, 0.5, 1 µg/mL for 3 hours with phenobarbital and 5,6-benzoflavone -induced rat liver S9 metabolic activation and 3 and 24 hrs without metabolic activation. There was no cytotoxicity noted but the test item was assayed up to precipitating concentrations. The positive and negative controls induced the appropriate response. Neither increases exceeding the global evaluation factor (GEF, 126 × 10−6) in the total colony mutant frequency (T-MF) values of the test article groups as compared to the T-MF values in the negative control group nor dose-dependent increases were noted in any of the treatment methods.  In conclusion, the test item was considered not to possess the gene mutagenic potential to L5178Y cells under the conditions of this study.

Justification for classification or non-classification

Based on the available information in the dossier, the test item does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.