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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results from the chromosome aberration, AMES, and in vitro mammalian cell gene mutation tests there was no indication of genetic toxicity.

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:

24th September 2018 to 29th October 2018

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)

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

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)

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.

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.

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. 

 

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 20 2016 to July 15 2016

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:

The Guidelines for the Testing of Chemicals Health Effects

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

other: S. typhimurium TA 97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Source: Research Institute for Liver Diseases (Shanghai) Co, LTD
Treatment: Metabolic activation system was prepared from the livers of male SD rat inducing by PCBs (Aroclor 1254)
Storage: stored at -80C
Before the used, made S9 and coenzyme system (S9 mixture).

Test concentrations with justification for top dose:

Based on the prelimary experiment, the doses of this test were 156, 312, 625, 1250, and 2500ug/plate. Blank control, solvent control (dimethylsulfoxide) and positive control had been included. Triplicate cultures were made per dose in the test.

Vehicle / solvent:

Dimethylsulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other: sodium p (dimethylamino)benzenediazo sulfonate, methyl sulfonic acid methyl ester, cyclophosphamide, 2-aminofluorene

Evaluation criteria:

A test substance is deemed to provide evidence of mutagenic potential if a significant dose-related increase in the number of revertant colonies is obtained in two separate experiments, and the increase in the number of revertant colonies is at least twice the concurrent solvent control group value.

The test substance should be detected by five strains. as long as there is a test strain, either with S9 or without S9 is positive, can determine the test sample is positive in Ames Test.

According to the guidelines of OECD471 if the test results are negative or questionable results it should be repeated. In this test, formal test results were negative, so the test was repeated.

Key result

Species / strain:

other: TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

other: TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Based on the test results, under the current test conditions, N-Boc-D-phenylglycinol is not mutagenic in the tester strains TA97a, TA98, TA100, TA102, and TA1535.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 07 2016 to July 05 2016

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

Source: Cell Bank of the Preservation Committee for Typical Cultures, Chinese Academy of Sciences.
Culture Media: Roswell Park Memorial Institute Medium 1640
Culture Conditions: The cells were cultured in an incubator at 37C with 5% CO2 concentration.

Metabolic activation:

with and without

Metabolic activation system:

Source: Research Institute for Liver Diseases Co Ltd.
Treatment: Metabolic Activation system was prepared from the livers of male SD rats induced by polychlorinated benzenes

Test concentrations with justification for top dose:

Based on the result of the preparatory test, the IC50 value of N-Boc-D-phenylglycinol was determined to be 0.38mg/mL, the IC50 value is used as the highest dose and the other rates were set at the 1/2 and 1/4 of the highest dose level according to the generally accepted procedures for in Vitro Mammalian Chromosome Aberration Test such as OECD guideline 473. Therefore the dosages used in this test were 0.38mg/mL, 0.19mg/mL, and 0.095mg/mL respectively.

Vehicle / solvent:

DMSO (dimethylsulfoxide)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Evaluation criteria:

At least one of the test concentrations exhibits a statistically signifigant increase compared with the concurrent negative control. The difference between the exposure group and the control group had used the chi square test to evaluate it whether has statistically signifigant.

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The induced chromosome aberration rates of the CHL cells with metabolic activation system (S9), after 6 hours treatment by N-Boc-D-Phenylglycinol at a dose of 0.38, 0.19, and 0.095 mg/ml, were determined to be 1.67%, 1.00%, and 1.33%; and 1.00&, 1.00%, and 0.67% respectively after 6 hours of treatment without metabolic activation system (S9); and 2.67%, 2.00%, and 1.67% respectively after 24 hours treatment without the metabolic activation system (S9). Compared with the negative control groups, there was no statistically signifigant difference observed (P>0.05). The chromosome aberrations rate excluding the gap of the positive control group was determined to be 19.00% for the 6 hours of treatment with metabolic activation system(S9); 23.00% for the treatment of 6 hours without metabolic activation system (S9); and 27.33% for the treatment of 24 hours without metabolic activation system (S9), respectively. Compared with the negative control group, there was statistically signifigant difference observed (P<0.05).

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Based on the test results, under the current test conditions, N-Boc-D-Phenylglycinol does not induce chromosome aberration in cultured Chinese Hamster Lung Fibroblast cells (CHL) both with and without metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available