2-Butenedioic acid (Z)-, ester with 1,2-propanediol, compd. with 2-(dibutylamino)ethanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome aberration test in vitro

Test material

Test material information

Constituent 1

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium). During the treatments, the serum content of the medium was reduced to 5 (v/v) % (DMEM-5).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix ( induced rat liver by Phenobarbital (PB) and β-naphthoflavone (BNF))

Test concentrations with justification for top dose:

Assay 1: (3 hours)
without S9-mix: 1500, 1000, 750, 500, 250, 120, 62.5 and 31.25 μg/mL
with S9-mix: 3000, 2500, 2000, 1000, 500, 250 and 125 μg/mL

Assay 2:
20 hours, without S9-mix: 3000, 2500, 2000, 1500, 1000, 500, 250 and 125 μg/mL
3 hours, with S9-mix: 1500, 1000, 750, 500, 250, 120, 62.5 and 31.25 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: appropriate for formulation and its dilution; compatible to the test system

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9-mix; dissolved in DMEM; 0.4 μL/mL (28-hour harvesting time) or 1.0 μL/mL (20-hour harvesting time) [

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix; dissolved in 0,9% NaCl; 6.0 μg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:3 and 20 hours
- Fixation time (start of exposure up to harvest of cells): 20 and 28 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.2 μg/mL)
STAIN (for cytogenetic assays): 5 % Giemsa solution

NUMBER OF CELLS EVALUATED: 200 cells (metaphases)/dose

DETERMINATION OF CYTOTOXICITY
- Method: determination of cell concentration by use of a haemocytometer

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.

Statistics:

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Assay 1: without S9-mix: > 500 μg/mL; with S9-mix: > 250 μg/mL. Assay 2: No significant increase in the number of cells with structural chromosome aberrations with or without S9-mix.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9-mix: > 500 (250) μg/mL; with S9-mix: > 2000 (1500) μg/mL, in brackets values of Assay 2

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Summarized results of the concentration selection cytotoxicity assays

	Dose(μg/mL)	Testgroup without S9-mix Relative survival (%) (Assay A/B)	Testgroup with S9-mix Relative survival (%) (Assay A/B)
Untreated control		84/127	125/101
Vehicle control 1%(v/v) Distilled water		100/100	100/100
Vehicle control 2% v/v Distilled water		100/100	100/100
WS400402	5000	0/0	2/0
	2500	0/0	41/18
	1250	7/4	73/63
	625	29/21	86/91
	312,5	67/53	88/94
	156,25	74/95	107/99
	78,13	76/99	112/101
	39,06	80/107	100/96

Time of Treatment/Sampling: 3h/20h

No insolubility of test material was detected at the end of the treatment period in the final treatment medium; there were no large changes in pH and osmolality either.

 

Table 2: Cytotoxicity results of the main experiments

	Dose(μg/mL)	Testgroup without S9-mix Rel. survival (%) Assay 1 3h/20h*	Testgroup without S9-mix Rel. survival (%) Assay 2 20h/28h*	Dose(μg/mL)	Testgroup with S9-mix Rel. survival (%) Assay 1 3h/20h*	Testgroup with S9-mix Rel. survival (%) Assay 2 3h/28h*
Vehicle control 2% v/v Distilled water		100	100		100	100
WS400402	1500	0	0	3000	23	11
	1000	2	1	2500	25	17
	750	13	3	2000	40	48
	500	37	12	1500	-	36
	250	73	41	1000	62	59
	125	97	65	500	76	83
	62,5	100	89	250	81	97
	31,25	101	80	125	95	90
Positive control	-	1μL/mL EMS 80	1μL/mL EMS 48	-	6 μg/mL CP 51	6 μg/mL CP 52

*Treatment time/sampling 

Table 3: Summary table of Chromosome Aberration Assay 1

Concentration (μg/mL)	Time of Treatment  /Sampling	Relative # Survival (%)	Mean% aberrant cells###
WS400402without metabolic activation (-S9)
Vehicle(solvent)control	3h/20h	100	1.0
1500	3h/20h	0	NE
1000	3h/20h	2	NE
750	3h/20h	13	NE
500	3h/20h	37	18.1***
250	3h/20h	73	2.0
125	3h/20h	97	3.0
62.5	3h/20h	100	NE
31.25	3h/20h	101	NE
Positive control	3h/20h	80	12.4***
WS400402with metabolic activation (+S9)
Vehicle(solvent)control	3h/20h	100	2.0
3000	3h/20h	23	NE
2500	3h/20h	25	NE
2000	3h/20h	40	60.0***
1000	3h/20h	62	12.3***
500	3h/20h	76	3.0
250	3h/20h	81	11.0**
125	3h/20h	95	NE
Positive control	3h/20h	51	96.8***

Vehicle (solvent) control: 2% (v/v) Distilled water Positive control (-S9):Ethyl methanesulfonate,1µL/mLPositive control (+S9): Cyclophosphamide, 6µg/mL

NE: notevaluated

#: compared to the vehicle (solvent) control

##:in the final treatment medium at the end of the treatment

###:excluding gaps

 

**: p<0.01comparing numbers of aberrant cells excluding gaps with corresponding vehicle (solvent)control

***:p<0.001comparing numbers of aberrant cells excluding gaps with corresponding vehicle (solvent)control

Table 4: Summary table of Chromosome Aberration Assay 2

Concentation (μg/mL)	Time of Treatment/Sampling	Relative # Survival (%)	Mean% aberrant cells###
WS400402without metabolic activation (-S9)
Vehicle (solvent) control	20h/28h	100	2.5
1500	20h/28h	0	NE
1000	20h/28h	1	NE
750	20h/28h	3	NE
500	20h/28h	12	NE
250	20h/28h	41	1.5
125	20h/28h	65	2.0
62.5	20h/28h	89	1.0
31.25	20h/28h	80	NE
Positive control	20h/28h	48	50.8***
WS400402with metabolic activation (+S9)
Vehicle(solvent)control	3h/28h	100	3.0
3000	3h/28h	11	NE
2500	3h/28h	17	NE
2000	3h/28h	28	NE
1500	3h/28h	36	7.0
1000	3h/28h	59	4.5
500	3h/28h	83	4.0
250	3h/28h	97	NE
125	3h/28h	90	NE
Positive control	3h/28h	52	68.2***

Vehicle (solvent) control: 2% (v/v) Distilled water

Positive control (-S9): Ethyl methanesulfonate,0.4µL/mLPositive control (+S9):Cyclophosphamide,6µg/mL

NE: not evaluated

#:compared to the vehicle (solvent)control

##:in the final treatment medium at the end of the treatmentment

###:excluding gaps

 

***:p<0.001comparing numbers of aberrant cells excluding gaps with corresponding vehicle (solvent) control.

None of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations in Assay 2 with or without metabolic activation although a dose related increase was observed across all the concentrations tested with metabolic activation.

Polyploid and endoreduplicated metaphases

Polyploid metaphases (1-4) were found in some cases in the vehicle (solvent) control, positive control or test material treated samples.No endoreduplicated metaphases were found in the two main tests except of one sample (in Assay 2 at 1500 μg/mL concentration with metabolic activation two endoreduplicated metaphases were found, all other values: 0).

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

In conclusion, WS400402 test item induced a significant level of chromosome aberrations in the performed experiments with and without metabolic activation. Therefore, WS400402 is considered clastogenic in this test system.