[No public or meaningful name is available]

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:

January - April 2019

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

2,6-bis[[bis(2-hydroxyethyl)amino]methyl]-4-nonylphenol

EC Number:

243-500-6

EC Name:

2,6-bis[[bis(2-hydroxyethyl)amino]methyl]-4-nonylphenol

Cas Number:

20073-51-2

Molecular formula:

C25H46N2O5

IUPAC Name:

2,6-bis{[bis(2-hydroxyethyl)amino]methyl}-4-nonylphenol

Test material form:

liquid: viscous

Method

Target gene:

In a mammalian cell gene mutation assay [hprt locus], CHO-K1 cells cultured in vitro were exposed to Poliol MB600 at different concnetrations, in the absence and presence of the metabolic activation (2% v/v S9 mix) for a period of 4 hours and 5 min.
Cells deficient in Hypoxanthine guanine Phosphoribsyl Transferase (HPRT), due to mutation, are resistant to the cytotoxic effects of the purine analogue (6-thioguanine). HPRT proficent cellls are sensitive to 6-thioguanine which causes inhibition of cellular metabolism and halts further cell division. HPRT deficient cells are presumed arise through mutation at the hprt locus; they cannot metabolize 6- thioguanine and thus survive and grow in its presence.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1 cell line (free from mycoplasma contamination), a sub clone of chinese hamster ovary cell line obtained from the Japanese Collection of Research Bioresources (JCRB), maintained in the Mutagenicity Section at Jai Research Foundation was used for this study. The cells were grown as monolayer in disposable tissue culture flasks. The cell line free from mycoplasma contamination was used in the study. cultures were freee from any contamination during the conduct of the study. CHO-K1 cell lien passage number 36 was used in cytotoxicity test and passage number 24 was used in main study.

Additional strain / cell type characteristics:

other: Cells deficient in Hypoxanthine-guanine Phosphoribosyl tansferase

Cytokinesis block (if used):

Not used

Metabolic activation:

with and without

Metabolic activation system:

The in vitro test systems lack the necessary oxidative enzyme systems for metabolizing foreign
compounds to electrophilic metabolites capable of reacting with DNA. Sometimes these foreign

compounds, when reacting with a mammalian enzyme system, yield mutagenic metabolic

products.

In order to test these indirectly acting mutagens, a metabolically active extract of rat liver (treated

with Aroclor 1254) called S9 fraction is used.

The S9 fraction procured from M/s G.P. Meshram, Nagpur (Lot N° MWR/ARI/S9F/01/18

APPENDIX 7) was used in this assay. The S9 fraction is buffered and supplemented with the

essential co-factors -NADP, KCL and Glucose-6- phosphate to form the “S9 mix”. The S9

fraction was used at a concentration of 2% v/v in the final culture medium for main study. The

details of preparation of medium containing S9 fraction are given in APPENDIX 4.

Test concentrations with justification for top dose:

Cultures were exposed to POLIOL MB 600 at 6 dose-levels (two cultures/dose-level) from 10 to 80 µg/mL of culture medium, selected from cytotoxicity test, in the absence and presence of the metabolic activation (2% v/v S9 mix) for a period of 4 hours and 5 min

Vehicle / solvent:

Considering molecular weight of test item as 454.652 and purity 99.6% (provided by sponsor),
solubility was tested at 200000 µg/mL. Test item was insoluble in distilled water while soluble in
dimethyl sulfoxide. Therefore dimethyl sulfoxide was selected as the vehicle for the cytotoxicity
test and main study.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

CHO-K1 cell line (free from mycoplasma contamination), a sub clone of Chinese hamster ovary cell line obtained from the Japanese Collection of Research Bioresources (JCRB), maintained in the Mutagenicity Section at Jai Research Foundation was used for this study. The cells were grown as monolayer in disposable tissue culture flasks. The cell line free from mycoplasma contamination was used in the study. Cultures were free from any contamination during the conduct of the study. CHO-K1 cell line passage number 36 was used in cytotoxicity test and passage number 24 was used in main study.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if all the following criteria are met, in any of the experimental conditions examined:
a. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
b. The increase is concentration-related when evaluated with an appropriate trend test.
c. Any of the results are outside the distribution of the historical negative control data (e.g. Poisson based 95% control limit).
When all of these criteria are met, the test item then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test item was considered clearly negative if, in all experimental conditions examined:
d. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
e. There is no concentration-related increase when evaluated with an appropriate trend test.
f. All results are inside the distribution of the historical negative control data (e.g. Poisson- based 95% control limit).
The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

From results of this study, it is concluded that POLIOL MB 600 did not show any potential to induce gene mutation, at the hprt locus of CHO-K1 cells, either in the absence or presence of the metabolic activation (2% v/v S9 mix), under the present experimental conditions.

Executive summary:

EXECUTIVE SUMMARY: In a mammalian cell gene mutation assay [hprt  locus],  CHO-K1  cells  

cultured in  vitro were exposed to POLIOL MB 600 at different concentrations, in the absence and 

presence of the metabolic activation (2% v/v S9 mix) for a period of 4 hours and 5 min. 

 

Cultures were exposed to POLIOL MB 600 at 6 dose-levels (two cultures/dose-level) from 10 to 80 

µg/mL of culture medium, selected from cytotoxicity test, in the absence and presence of the metabolic 

activation (2% v/v S9 mix) for a period of 4 hours and 5 min.  

 

A  significant  dose-related  increase  in  mutation  frequency  was  not  observed  in  any  treatment 

concentration from 10 to 80 µg/mL of culture medium, in the absence and presence of the metabolic 

activation system (2% v/v S9 mix), and induced mutation frequency was comparable to the negative 

control group. All negative controls were within historical limits and positive controls showed an increase 

in mutation frequency. No relevant influence of the test item, on pH or osmolality, was observed either in 

the absence or presence of the metabolic activation. 

 

All criteria for a valid study were met, as described in the study plan. Based on results from this study, it 

is concluded that POLIOL MB 600 does not have potential to induce gene mutation at the hprt locus of 

CHO-K1 cells, either in the absence or presence of the metabolic activation system (2% v/v S9 mix), 

under  present experimental conditions.