Didodecyl fumarate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the available data the members of the PFAE fumarates category are considered to be non genotoxic.

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Feb 2013 - 11 Apr 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline Study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

not applicable

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

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.

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'.

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

 

 

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Justification for grouping of substances and read-across

The PFAE fumarates (Polyfunctional Aliphatic Ester) category consists of six members, which are either well-defined mono-constituent substances or related UVCB substances, with varying fatty alcohol chain lengths. The distinguishing feature of this category of chemicals is that its members are diester derivatives of fumaric acid (CAS 110-17-8). The alcohol moiety of the dicarboxylic esters generally falls in the C8-C22 carbon number range, including linear, even numbered alcohols. 

In order to avoid the need to test every substance for every endpoint, the category concept is applied for the assessment of environmental fate, environmental toxicity and human health hazards. Thus where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group by inter- or extrapolation to the target substances in the group (read-across approach) applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. Structural similarities and similarities in properties and/or activities of the source and target substances in the category are the basis of read-across.

The available studies providing information on the human health hazard assessment within the PFAE fumarates category were conducted with the category member Didodecyl fumarate (CAS 2402-58-6). This substance was selected for testing, because it represents the category member with the shortest fatty alcohol side chain, and consequently with the lowest molecular weight, which is regarded as worst-case approach in terms of hazard assessment of the PFAE fumarates for the local as well as for systemic effects.

Furthermore, the category is supported by another polyfunctional aliphatic ester, namely Bis(2-ethylhexyl) adipate (CAS 103-23-1). This supporting chemical is used to cover toxicological endpoints, exclusively. The read across of Bis(2-ethylhexyl) adipate (CAS 103-23-1) to the PFAE fumarate category is justified due to the similar structural and physico-chemical properties, as well as their toxicological, and ecotoxicological profiles.

A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Sections 7.1 and 13) and within Chapter 5.1 of the CSR.

Endpoint specific data matrix:

ID #	CAS	Genetic Toxicity in vitro: gene mutation in bacteria	Genetic Toxicity in vitro: cytogenicity in mammalian cells	Genetic Toxicity in vitro: gene mutation in mammalian cells	Genetic toxicity in vivo
1	2402-58-6	Experimental result: Negative	Experimental result: Negative	Experimental result: Negative	--
2	10341-03-4	RA: CAS 2402-58-6	RA: CAS 2402-58-6	RA: CAS 2402-58-6	--
3	68610-90-2	RA: CAS 2402-58-6	RA: CAS 2402-58-6	RA: CAS 2402-58-6	--
4	68921-51-7	RA: CAS 2402-58-6	RA: CAS 2402-58-6	RA: CAS 2402-58-6	--
5	68921-52-8	RA: CAS 2402-58-6	RA: CAS 2402-58-6	RA: CAS 2402-58-6	--
6	68921-53-9	RA: CAS 2402-58-6	RA: CAS 2402-58-6	RA: CAS 2402-58-6	--

Genetic toxicity (mutagenicity) in bacteria in vitro

Only one in vitro study investigating mutagenicity to bacteria is available for the PFAE fumarate category.

CAS 2402-58-6

The potential mutagenicity of Didodecyl fumarate (CAS 2402-58-6) was tested in a bacterial gene mutation assay according to OECD 471 (Woitkowiak, 2013). In two independent experiments, the S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A were exposed to the test substance at concentrations of 33, 100, 333, 1000, 2700 and 5400 µg/plate. Both experiments were performed in the absence or presence of a liver microsomal activation system (S9 mix) using either the plate incorporation or the pre-incubation method (20 min pre-incubation time). No cytotoxicity was noted in the treated bacteria strains up to precipitating concentrations when compared to controls. Precipitation of the test substance was observed at 333 µg/plate onward with and without S9 mix. The mean number of revertant colonies per plate was not significantly increased at any test concentration. The solvent and the positive control substances proved the validity of the study for each tester strain.

Under the conditions of this assay, Didodecyl fumarate was found to be non-mutagenic in S. typhimurium strains (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A, both in the presence and absence of S9 mix.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

Only one in vitro study on the induction of cytogenicity in mammalian cells is available for the PFAE fumarate category.

CAS 2402-58-6

The potential cytogenicity of Didodecyl fumarate was tested in the in vitro mammalian cell micronucleus test (Bohnenberger, 2013), conducted according to OECD 487, and in compliance with GLP. Chinese hamster lung fibroblasts (V79) were treated with the test item dissolved in tetra hydro furan (THF) both in the presence and absence of a metabolic activation system (S9 mix). Two independent experiments were conducted with concentrations of 0.6-2200.0 µg/mL: in Experiment I the cells were exposed to the test substance for 4 h with and without S9 mix; in Experiment 2 exposure was conducted for 24 h without S9 mix and for 4 h with S9 mix. In every case, duplicate cultures were prepared. After fixation and staining with Giemsa, 1000 cells/culture were scored and examined for micronucleated cells. Cytotoxicity was determined by means of proliferation index. There were no statistically significant cytotoxic and genotoxic properties noted at any dose level tested up to precipitating concentrations. Appropriate solvent and positive controls were included into this test and gave the expected results: the solvent control data were within the range of the historical control data, and a significant increase in micronucleated cells treated with the positive control substances proved the sensitivity of the test. The test item Didodecyl fumarate is therefore concluded to be not genotoxic under the conditions of this test.

In conclusion, Didodecyl fumarate was not clastogenic to Chinese hamster lung fibroblasts (V79) with or without exogenous metabolic activation system

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

Only one in vitro study on the induction of mutagenicity in mammalian cells is available for the PFAE fumarate category.

CAS 2402-58-6

An in vitro mammalian cell gene mutation assay (HPRT assay) with Didodecyl fumarate (CAS 2402-58-6) was performed according to OECD 476 and in compliance with GLP (Wollny, 2013). Mutations at the HPRT locus of Chinese hamster lung fibroblasts (V79) were investigated in two independent experiments at test substance concentrations of 1, 3, 9, 27 and 81 µg/mL. The cells were exposed both with and without metabolic activation (S9-mix) for 4 h each in the first experiment and for 4 and 24 h with or without S9-mix in the second experiment, respectively. The cells were allowed to express their genotype for 7-9 days, followed by a selection period of 8 days in the presence of 6-TG. The test substance did not induce a significant increase in the mutant frequency at any test substance concentration with or without metabolic activation. There was no cytotoxicity noted at any concentration tested. Test substance precipitation was noted at 27 µg/mL and above in all experimental parts; in addition small droplets were noted as phase separation. Appropriate positive and solvent controls were included into the test and gave the expected results. In conclusion, the test substance was not mutagenic to Chinese hamster lung fibroblasts (V79) in the HPRT assay with or without metabolic activation up to precipitating concentrations.

 

Genetic toxicity in vivo

No in vivo studies on genetic toxicity are available for the PFAE fumarate category. Available study is on adipate ester.

 

Conclusion for genetic toxicity

Data investigating genetic toxicity in vitro within the fumerate category are available only for Didodecyl fumarate (CAS 2402-58-6). In summary, Didodecyl fumarate (CAS 2402-58-6) was tested negative for mutagenicity to bacteria in the Amest test, negative for cytogenicity in the in vitro mammalian cell micronucleus test and negative for mutagenicity to mammalian cells in the HPRT test

Therefore, based on the available data the members of the PFAE fumarates category are considered to be non genotoxic.

A detailed reference list is provided in the technical dossier (see IUCLID, section 13) and within CSR.

Justification for selection of genetic toxicity endpoint
GLP study, supported by others certified studies

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the group concept is applied to the members of the PFAE fumarate category, data will be generated from representative reference substance(s) within the category to avoid unnecessary animal testing. Additionally, once the group concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the group concept, all available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.