Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The studies performed concludedthat the substance is non mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February to April 2019
Reliability:
1 (reliable without restriction)
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:
Details of the test item provided by the Sponsor (Ref. TIDS):

Test Item Name
POLIOL MB 600

IUPAC Name
2,6-bis[[bis(2-hidroxietil)amino]metil]-4-nonil-fenol

CAS Number
20073-51-2

Molecular Formula
C25H46N2O5

Molecular Weight
454.652

Batch/Lot Number
7590

Analysed Purity
99.6%
(Refer certificate of analysis in (APPINDIX 9)

Manufactured by
Sistemas Ecologicos De Poliuretano, SL

Supplied to JRF by
Sistemas Ecologicos De Poliuretano, SL

Date of Manufacture
December 12, 2018

Date of Expiry
December 12, 2019

Appearance
Viscous Liquid, Dark yellow, slightly like formaldehyde

Other Characteristics
Specific gravity: 1085 g/L, pH: 7

As per the instruction received from the Sponsor on storage of the test item, the test item was stored :

Storage Temperature : Room Temperature
Storage Condition: Protected against moisture
Storage Container: In original container as supplied by the Sponsor

Sponsor

Storage Location

:

Test Item Control Office, JRF


Species / strain / cell type:
lymphocytes: human peripheral blood lymphocites cultured in vitro
Details on mammalian cell type (if applicable):
human peripheral blood lymphocites cultured in vitro
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Absence of Metabolic Activation
60 µL of 0.5 mg/mL of Mitomycin-C added to 940 µL of sterile distilled water. 80 µL of this was used for 8 mL of treatment medium (0.3 µg/mL).

Presence of Metabolic Activation
20.0 mg of Cyclophosphamide dissolved in 5 mL of sterile distilled water (Phase I) to obtain required concentration (Stock - 4 mg/mL). 80 µL of this stock was used for 8 mL of treatment medium (40 µg/mL).

Test concentrations with justification for top dose:
5 ug/mL, 10 ug/mL, 20 ug/mL, 40ug/mL, 60ug/mL, 80 ug/mL.
No relevant influence of the test item on pH value or osmolality was observed in the absence (Phase I and II) and the presence of metabolic activation (Phase I). Precipitation was not observed up to 80 µg/mL, in the absence and 125 µg/mL, in the presence of the metabolic activation at 0 and 4 hour after incubation in Phase I and 24 hour in Phase II in absence of metabolic activation.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
The test system used for the in vitro chromosomal aberration test was human peripheral blood lymphocytes, as recommended by the OECD and other regulatory authorities. The selection criteria for volunteers were as per JRF standard operating procedure. Blood was drawn from healthy, 26 and 25 years old female volunteer for the cytotoxicity test and main study, respectively, by venous puncture using heparinised syringe (Heparin obtained from Biological E. Limited, Hyderabad). The donor selected were non-smoker, non-alcoholic, free from drug, radiation and chemical exposure. A trained medical laboratory technician collected the blood by vein puncture using a 21 G needle attached to a 50 mL disposable syringe.

Evaluation criteria:
Chromosomal Aberrations
Mitotic index was scored for all the six test concentrations and based on mitotic index; three suitable concentrations were selected for assessment of chromosomal aberrations. All slides, including those of positive and negative controls, were independently coded prior to microscopic analysis for chromosomal aberrations. 300 well spread metaphases (150/replicate) were scored for the structural chromosomal aberrations per concentration and controls.

Only cells containing 46 ± 2 chromosomes were examined for structural changes. A smaller number (e.g. 50 metaphases per slide) of metaphases were analyzed in slides showing higher frequency (more than 20%) of aberrant cells. Gaps, breaks, fragments, exchanges, multiple aberration and deletions were recorded with their numbers and frequencies for all the treated and control cultures separately. In addition, 100 metaphases/replicate were examined for polyploidy.

The number of metaphases with only gap were recorded but not considered to calculate total aberrations and percent aberrant cells.

Statistics:
Gaps and polyploidy were not included in the calculation of total aberration frequency. Data on percent aberrant cells and polyploidy were subjected to Shapiro-Wilk’s test for normality and Bartlett’s test to assess homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test (Gad and Weil, 1994). Where the data did not meet suitable homogeneity of variance, Student's t-test was performed to determine the level of significance between negative control, three selected test concentrations (selected based on the mitotic index data) and positive controls. Where the data show significance in Shapiro-Wilk’s test, Chi-square test for trend analysis was performed to determine the significance between negative controls, three selected test concentrations and positive controls.

Key result
Species / strain:
lymphocytes: human peripheral blood lymphocites cultured in vitro
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The substance is not mutagenic on the tested conditions
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
From results of this study, it is concluded that POLIOL MB 600 did not show any potential to induce chromosomal aberration, either in the absence or presence of the metabolic activation under the present experimental conditions and is considered to be negative for clastogenicity.
Executive summary:

In a mammalian chromosomal aberration test, human peripheral blood 


lymphocytes, 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). 


 

Based  on  results  of  preliminary  solubility  and  cytotoxicity  tests,  80  µg/mL,  in  the  absence  of  the 


metabolic activation in Phase I and II and 125 µg/mL in the presence of the metabolic activation (2% v/v 


S9 mix) were selected as the highest test concentration for the main study. POLIOL MB 600 was tested in 


two phases, with (2% v/v S9 mix) and without metabolic activation (4h exposure) and second phase (24h 


exposure)  without  metabolic  activation.  Hence,  human  peripheral  blood  lymphocyte  cultures  were 


exposed to POLIOL MB 600 at six concentrations (two cultures/concentration in each experiment) from 5 


to 80 µg/mL, in the absence in Phase I and II and 3.91 to 125 µg/mL in the presence of the metabolic 


activation. 


POLIOL MB 600 did not induce any statistically significant or biologically relevant increase in the 


number of percent aberrant cells, in the absence and presence of  S9-mix  for  the  short  term  (phase-I,  4  


hours) and in the absence of S9, long term (phase-II, 24 hours) exposure period. No effect of POLIOL 


MB 600 on the number of polypoid cells was observed, in the absence (phase I and phase II) and presence 


of S9-mix (phase I) in both phases. All negative controls were comparable to historical control limits and 


positive controls showed an increase in the incidence of cells with chromosomal aberrations. 


 

All criteria for a valid study were met, as described in the study plan. From results of this study, it is 


concluded that POLIOL MB 600 did not show any potential to induce chromosomal aberrations either in 


the absence or presence of the metabolic activation system, under the described experimental conditions 


and is considered to be negative for clastogenicity. 


 

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)
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
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 / 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.
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.
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
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. 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February - March 2019
Reliability:
1 (reliable without restriction)
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
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: This strains are unable to synthetize histidine.
Metabolic activation:
with and without
Metabolic activation system:
S9 Fraction and S9 Mix
Test concentrations with justification for top dose:
Initial toxicity-mutation test
doses of 5000, 1500, 500, 150, 50, 15, 5, 1.5 and 0 ug/plate.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2- aminoanthracene
Evaluation criteria:
Once criteria for a valid assay had met, responses overved in the assay were evaluated. The conditions necessary for determining a positive result were: there sould be a dose related increase in the mean revertants per plate of at lesast one tester strain over the range tested and/or at one or more doses of the test item etiehr in the absecne or presence of the metabolic activation system.
Five analysable doses were available to evaluate assay data. Cytotoxicity is detectabe as decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background llawn.
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
Based on results of this study, it is concluded that POLIOL MB600 is non-mutagenic to any strain of Salmonella typhimurium, viz., TA1537, TA1535,TA98, TA100, and TA102, when tested under the specified conditions.
Executive summary:

EXECUTIVE SUMMARY:  


 

This study was performed to evaluate the mutagenic activity of POLIOL 


MB 600, using the bacterial reverse mutation test, on five histidine deficient mutant tester strains of 


Salmonella typhimurium (i.e., TA1537, TA1535, TA98, TA100 and TA102). 


 

POLIOL  MB  600  was  tested  in two  independent  experiments  –  first,  in the  absence  of  metabolic 


activation and the second in the presence of metabolic activation. Bacterial cultures were exposed to 


POLIOL MB 600 at 8 concentration levels (two plates/concentration) ranging from 1.5 to 5000 µg/plate 


in the initial toxicity-mutation test. Normal growth, in the background lawn, was observed, up to 500 


µg/plate in TA98 and TA102, in the absence and presence (5% v/v S9 mix) of the metabolic activation. 


Normal growth was observed up to 50 µg/plate in the absence and 150 µg/plate, in the presence of the 


metabolic activation (5% v/v S9 mix) in TA1537, TA1535, and TA100. Partial inhibition and complete 


inhibition was observed up to 1500 and 5000 µg/plate, respectively, in the absence and presence of the 


metabolic activation (5% v/v S9 mix) in TA102. Complete inhibition was observed between 150 and 


5000 µg/plate, in absence; 500 and 5000 µg/plate, in the presence of the metabolic activation (5% v/v S9 


mix) in TA1537. Normal background lawn pattern was observed in TA98 in absence and presence of 


metabolic activation (5% v/v S9 mix) up to the tested concentration of 500 µg/plate.  


 

Partial inhibition was observed at 150 µg/plate in absence and 500 µg/plate, in the presence of the 


metabolic activation (5% v/v S9 mix) in the TA1535 and TA100. Complete inhibition was observed 


between 500 and 5000 µg/plate, in the absence and 1500 and 5000 µg/plate, in presence of the metabolic 


activation (5% v/v S9 mix) in TA1535 and TA100. Complete inhibition was observed between 1500 and 


5000 µg/plate, in absence and presence of the metabolic activation (5% v/v S9 mix) in TA98.  


 

No increase (i.e., no mutagenic effect) was observed either in the absence or presence of the metabolic 


activation system (5% v/v S9 mix) in tester strains in the number of revertant colonies. A confirmatory 


mutation test was conducted with an increased S9 concentration, i.e., 10% v/v S9 mix and concentration 


spacing modification, to confirm the negative results obtained in the initial toxicity mutation test.  

Bacterial cultures were exposed to POLIOL MB 600 at 7 concentration levels (three plates/concentration) 


ranging from 5.86 to 375 µg/plate, in the absence and presence of the metabolic activation (10 % v/v S9 


mix) for TA1537. Bacterial cultures were exposed to POLIOL MB 600 at 7 concentration levels (three 


plates/concentration) ranging from 11.72 to 750 µg/plate, in the absence and presence of the metabolic 


activation (10 % v/v S9 mix) for TA1535, TA98, and TA100. Bacterial cultures were exposed to POLIOL 


MB 600 at 7 concentration levels (three plates/concentration) ranging from 23.44 to 1500 µg/plate, in the 


absence and presence of the metabolic activation (10 % v/v S9 mix) for TA102. Revertant colonies were 


scored, after 48 hours of incubation at 37 ± 1 °C. 


POLIOL MB 600 did not induce any significant increase in the number of revertants, in trials with and 


without S9 mix, in any tester strain. Values for the negative control were within the historical control 


ranges of the laboratory. Positive controls showed an increase in the number of revertant colonies, 


demonstrating the efficiency of the test system.  


 

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


the  specified  experimental  conditions,  POLIOL  MB  600  is  concluded  to  be  non-mutagenic  in  the 


Bacterial Reverse Mutation Test using Salmonella typhimurium. 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

As the three studies concluded that the substance is non mutagenic we decdided to not classify the substance.