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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

950-492-4

Cas Number:

2361378-62-1

Molecular formula:

C21-23H39-43NO3

IUPAC Name:

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

impurity 1

Reference substance name:

Amides, C16-18, N-(hydroxyethyl)

EC Number:

309-819-0

EC Name:

Amides, C16-18, N-(hydroxyethyl)

Cas Number:

101226-97-5

IUPAC Name:

Amides, C16-18, N-(hydroxyethyl)

impurity 2

Reference substance name:

Zirconium compounds

IUPAC Name:

Zirconium compounds

Test material form:

solid: granular

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

Method

Target gene:

TK +/- locus

Species / strain

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

Controls

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.

Results and discussion

Test results

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

Applicant's summary and conclusion

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.