2-Butenedioic acid (E)-, di-C16-18-alkyl esters

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

15 Feb 2013 - 11 Apr 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - guideline study. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test), adopted July 22, 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.49 (In vitro Mammalian Cell Micronucleus Test), adopted July 6, 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium fuer Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Didodecyl fumarate

EC Number:

219-280-2

EC Name:

Didodecyl fumarate

Cas Number:

2402-58-6

Molecular formula:

C28H52O4

IUPAC Name:

didodecyl but-2-enedioate

Details on test material:

- Name of test material (as cited in study report): Didodecyl fumarate; fumaric acid, di-dodecyl ester
- Molecular weight: 452.72 g/mol
- Physical state: solid
- Analytical purity: 93.8 area-% (GC-FID)
- Lot/batch No.: 0008043725
- Expiration date of the lot/batch: Dec 2013
- Stability under test conditions: not indicated by the sponsor
- Storage condition of test material: atroom temperature

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank's salts, glutamine and Hepes (25 mM). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL) and 10% (v/v) fetal bovine serum.
- 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:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated phenobarbital/beta-naphtoflavone

Test concentrations with justification for top dose:

- Experiment I - 4 h exposure (-S9): 0.6, 1.4, 3.6, 9.0, 22.5, 56.3, 140.8, 352.0, 880.0, and 2200.0 µg/mL (evaluated: 1.4, 9.0, 22.5, and 56.3 µg/mL)
- Experiment II - 24 h exposure (-S9): 0.6, 1.4, 3.6, 9.0, 22.5, 56.3, 140.8, 352.0, 880.0, and 2200.0 µg/mL (evaluated: 1.4, 9.0, 22.5, and 56.3 µg/mL)
- Experiment I - 4 h exposure (+S9): 0.6, 1.4, 3.6, 9.0, 22.5, 56.3, 140.8, 352.0, 880.0, and 2200.0 µg/mL (evaluated: 3.6, 9.0, 22.5, and 56.3 µg/mL)
- Experiment II - 4 h exposure (+S9): 0.6, 1.4, 3.6, 9.0, 22.5, 56.3, 140.8, 352.0, 880.0, and 2200.0 µg/mL (evaluated: 3.6, 9.0, and 22.5 µg/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: solubility of the test item

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: griseofulvin (24 h exposure)

Remarks:

-S9: mitomycin C 0.3 µg/mL (in deionised water), griseofulvin 0.8 µg/mL (in DMSO); +S9: cyclophosphamide 15.0 µg/mL (in saline)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 hours; Experiment II: 24 hours (-S9 mix) or Experiment I and II: 4 h (+S9 mix)
- Expression time (cells in growth medium): 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures in 2 independent experiments

NUMBER OF CELLS EVALUATED: 1000 cells/culture

DETERMINATION OF CYTOTOXICITY
- Method: proliferation index

Evaluation criteria:

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
- no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent control.
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.
If the above mentioned criteria for the test item are not clearly met, the test item will be classified as equivocal or a confirmatory experiment may be performed. However, results may remain questionable regardless of the number of times the experiment is repeated.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the Chi square test. Evaluation was performed only for test groups showing a higher number of micronucleated cells than the respective solvent control group.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Water solubility: Yes (phase at ≥3.6 µg/mL (-S9) and at ≥9.0 µg/mL (+S9))
- Precipitation: Yes (at the end of treatment at ≥56.3 µg/mL in Experiment I (+/-S9) and in Experiment II (-S9) and at the end of treatment at ≥22.5 µg/mL in Experiment II (+S9)
- Phase separation: Yes (at 9.0 µg/mL and above in all experiments)

HISTORICAL CONTROL DATA (SOLVENT CONTROL):
- 4 h treatment (-S9): 0.15–1.50% micronucleated cells
- 24 h treatment (-S9): 0.05–1.50% micronucleated cells
- 4 h treatment (+S9): 0.05 – 1.70% micronucleated cells

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Number of micronucleated cells; Experiment I - exposure period 4 h - without S9 mix

Treatment group	Concentration per mL	Σ of micronucleated cells/2000 cells	% of micronucleated cells	Mean Proliferation index
Solvent control (THF)	0.5%	15	0.75	2.94
Positive control (Mitomycin C)	0.3 µg	162	8.10	2.18
Test item	1.4 µg	16	0.80	2.85
	9.0 µg	18	0.90	2.84
	22.5 µg	19	0.95	2.92
	56.3 µg	10	0.50	2.85

 

Table 2: Number of micronucleated cells; Experiment I - exposure period 4 h - with S9 mix

Treatment group	Concentration per mL	Σ of micronucleated cells/2000 cells	% of micronucleated cells	Mean Proliferation index
Solvent control (THF)	0.5%	24	1.20	1.98
Positive control (Cyclophosphamide)	15.0 µg	236	11.80	1.60
Test item	3.6 µg	25	1.25	1.96
	9.0 µg	38	1.90	1.96
	22.5 µg	21	1.05	1.94
	56.3 µg	24	1.20	2.00

 

Table 3: Number of micronucleated cells; Experiment II - exposure period 24 h - without S9 mix

Treatment group	Concentration per mL	Σ of micronucleated cells/2000 cells	% of micronucleated cells	Mean Proliferation index
Solvent control (THF)	0.5%	10	0.50	2.75
Positive control (Griseofulvin)	8.0 µg	153	7.65	2.63
Test item	1.4 µg	9	0.45	2.76
	9.0 µg	7	0.35	2.78
	22.5 µg	11	0.55	2.81
	56.3 µg	7	0.35	2.72

 

Table 4: Number of micronucleated cells; Experiment II - exposure period 4 h - with S9 mix

Treatment group	Concentration per mL	Σ of micronucleated cells/2000 cells	% of micronucleated cells	Mean Proliferation index
Solvent control (THF)	0.5%	15	0.75	2.01
Positive control (Cyclophosphamide)	15.0 µg	49	2.45	1.42
Test item	3.6 µg	15	0.75	1.94
	9.0 µg	23	1.15	2.06
	56.3 µg	14	0.70	2.27

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative