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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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
1 (reliable without restriction)

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to 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

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
liquid: viscous
Specific details on test material used for the study:
Details of the test item provided by the Sponsor (Ref. TIDS):

Test Item Name


CAS Number

Molecular Formula

Molecular Weight

Batch/Lot Number

Analysed Purity
(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

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


Storage Location


Test Item Control Office, JRF


Species / strain
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:
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
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.

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.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocites cultured in vitro
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
Additional information on results:
The substance is not mutagenic on the tested conditions
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Applicant's summary and conclusion

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 


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.