Carbamic acid, N-[(1R)-2-hydroxy-1-phenylethyl]-, 1,1-dimethylethyl ester

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:

24th September 2018 to 29th October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: PRC-MEP, The Guidelines for the Testing of Chemicals (Health Effect, 2nd Edition), 476 'In vitro Mammalian Cell Mutation Tests (2013)

GLP compliance:

yes

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:

Carbamic acid, N-[(1R)-2-hydroxy-1-phenylethyl]-, 1,1-dimethylethyl ester

EC Number:

600-273-1

Cas Number:

102089-74-7

Molecular formula:

C13H19NO3

IUPAC Name:

Carbamic acid, N-[(1R)-2-hydroxy-1-phenylethyl]-, 1,1-dimethylethyl ester

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: TK± (3.7.2C0)

For cell lines:
- Absence of Mycoplasma contamination: Affirmed
- Periodically ‘cleansed’ of spontaneous mutants: Yes

MEDIA USED
- Type and composition of media: RPMI1640 plus horse serum (1-20%), Penicillin-Streptomycin 1% and sodium pyruvate 2%.
- CO2 concentration: 4-5%
humidity level,
- Temperature: 35.6 - 37.3 oC

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 prepared in-house

Test concentrations with justification for top dose:

In a preliminary test, the cells were exposed to 2000, 400, 80, 16 and 3.2 µg/ml for 3 and 24 hours with and without metabolic activity. The results showed that 2000 and 400 µg/ml had precipitation at the beginning and end of the test. There was no test item precipitation observed in the other doses.
Six exposure concentrations of 2000, 667, 222, 74, 25 and 8.2 µg/ml were chosen for the definitive test.

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

yes

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 per concentration: duplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 6 x 10 5
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours with metabolic treatment, 3 hours without metabolic treatment, 3 hours without metabolic treatment

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 24 hours
- Method used: microwell plates for the mouse lymphoma assay.
- Selective agent: Trifluorothymidine (TFM), 3ug/ml, incubated for 11 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 0.2ml of cell culture.

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

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mutant frequency (MF)

Evaluation criteria:

When the IMF (Induced Mutant Frequency) at one or several doses is more than 126 x 10 6 and the increase is concentration related and can be replicated, the result is evaluated as positive.
If this criteria is not met, the result is evaluated as negative.

The increase is dose-related or reproducible.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The RTG of the cells exposed for 3 hours in the absence of S9 mix at the doses of 10, 4, 1.6, 0.64, 0.26 and 0.1μg/ml were 25%, 37%, 60%, 96%, 90% and 91%. The  RTG of the cells exposed for 3 hours in the presence of S9 mix at the doses of 10, 4, 1.6, 0.64, 0.26 and 0.1 μg/ml were 38%, 45%, 56%, 76%, 105% and 107%.The  RTG of the cells exposed for 24 hours in the absence of S9 mix at the doses of 10, 4, 1.6, 0.64, 0.26 and 0.1 μg/ml were 34%, 53%, 61%, 81%, 76% and 80%.

The results of mutant frequency showed that the IMF of all cells cultures at all doses exposed for 3 and 24 hours in the presence and absence of S9 were less than 126 x 106.

Applicant's summary and conclusion

Conclusions:

It can be concluded that the test item Boc-phenyl glycinol is non-mutagenic to the L5178Y mouse lymphona cells under the test conditions of this study. 

Executive summary:

This study was performed to evaluate Boc-phenyl Glycinol for its ability to cause gene mutations in vitro in cultured mammalian cells after being exposed for 3 hours with and without metabolic action and for 24 hours without metabolic action. The test method was designed to be compatible with OECD Guideline No. 490 'In vitro Cell Gene Mutation tests using the Thymidine Kinase Gene' (July 29, 2016). 

The mouse lymphona L5178Y (TK^± (3.7.2C0)) cells were exposed to six exposure concentrations, 2000, 667, 222, 74, 25 and 8.2 µg/ml at the treatment conditions outlined above. The positive and solvent (DMSO) controls were included at the same time. Plating Efficiency (PE) of the cells after expression was determined and the relative total growth (RTG) was calculated to evaluate cytotoxicity. The mutant frequency (MF) and the induced mutant frequency (IMF) above solvent control of each culture, the induced mutant frequency of small clones (IMFsc) of the highest evaluatable concentration and all controls were determined after selection with 5-tri-fluorothymidine (TFT). For the determination of cloning efficiency as well as the selection of mutants, the microwell technoique was applied.  

In this test, the results of the solvent and positive controls met all quality criteria, so the sensitivity of the assay and the efficacy of the S9 mix could be validated. In the cell cultures of the definitive test series, 2000 and 667 µg/ml had precipitation at the beginning and end of the test. There was no test item precipitation observed at any of the remaining test concentrations either at the beginning or the end of the treatment. 

The cytotoxicity results were as follows: The RTG of the cells exposed for 3 hours in the absence of S9 mix at doses of 2000, 667, 222, 74, 25 and 8.2 µg/ml were 11.53%, 35.89%, 73.47%, 86.16%, 73.71% and 91.29% respectively.  The RTG of the cells exposed for 3 hours in the presence of S9 mix at doses of 2000, 667, 222, 74, 25 and 8.2 µg/ml were 11.12%, 52.50%, 89.40%, 80.87%, 89.05% and 97.59% respectively. The RTG of the cells exposed for 24 hours in the absence of S9 mix at doses of 2000, 667, 222, 74, 25 and 8.2 µg/ml were 3.3%, 13.18%, 73.27%, 87.82%, 86.28% and 83.02% respectively.

The results of mutant frequency showed that the induced mutant frequency of all cultures exposed for 3 and 24 hours were consistently less than the average background mutant frequency of 126 x 10^-6. 

The results of this study are negative, so it can be concluded that the test item Boc-phenyl glycinol is non-mutagenic to the L5178Y mouse lymphona cells under the test conditions of this study.