Dimethyl sebacate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 November 2012 to 7 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully GLP compliant and in accordance with current test guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 487

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl sebacate

EC Number:

203-431-4

EC Name:

Dimethyl sebacate

Cas Number:

106-79-6

Molecular formula:

C12H22O4

IUPAC Name:

1,10-dimethyl decanedioate

Test material form:

other: Colourless solid to liquid (melting point 26°C)

Details on test material:

Name: Dimethyl Sebacate
CAS number: 106-79-6
Batch number: 120801
Description: colourless solid
Receipt date: 11 October 2012
Storage details: 15-25°C protected from light and with desiccant from 2 November 2012
Purity: 99.08% (assumed 100% for testing)
Expiry date: August 2013

Method

Target gene:

Human lymphocyte cultures

Species / strain

Species / strain / cell type:

lymphocytes: Human lymphocyte cultures

Details on mammalian cell type (if applicable):

Blood from two healthy, non-smoking male volunteers from a panel of donors at Covance was used for each experiment in this study. No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication. The measured cell cycle time of the donors used at Covance falls within the range 13 +/- 2 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within one day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.

Metabolic activation:

with and without

Metabolic activation system:

S-9 from male Sprague Dawley rats induced with Arcolor 1254

Test concentrations with justification for top dose:

Positive controls:
Mitomycin C: stock concentration, 0.060 and 0.080 mg/mL and final concentration, 0.60 and 0.80 µg/mL without metabolic activation
Cyclophosphamide: stock concentration, 0.625 and 1.25 mg/mL and final concentration, 6.25 and 12.50 µg/mL with metabolic activation
Vinblastine: stock concentration, 0.008, 0.010 and 0.012 mg/mL and final concentration, 0.08, 0.10 and 0.12 µg/mL without metabolic activation

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Remarks:

For concentrations see test concentrations section.

Positive control substance:

cyclophosphamide

mitomycin C

other: Vinblastine

Details on test system and experimental conditions:

No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication. The measured cell cycle time of the donors used at Covance falls within the range 13 +/- 2 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within one day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 9.0 mL pre-warmed (in an incubator set to 37 ± 1°C) HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin, so that the final volume following addition of S-9 mix/KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37 ± 1°C for approximately 48 hours and rocked continuously.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of micronucleated binucleate (MNBN) cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.

Statistics:

Results which only partially satisfied the above criteria were dealt with on a case by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990).

Results and discussion

Test results

Species / strain:

lymphocytes: Human lymphocyte cultures

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Treatment of cells with Dimethyl Sebacate for 3+21 hours and for 24+24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were similar to and not significantly higher than those observed in concurrent vehicle controls at any concentration analysed under either treatment condition. The MNBN cell frequencies of all treated cultures fell within the normal ranges.
Treatment of cells for 3+21 hours in the presence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p < 0.05), compared to those observed in concurrent vehicle controls, at all four concentrations analysed (400.0 to 1600 µg/mL, giving 5% to 55% reductions in RI). The MNBN cell frequencies exceeded the normal range in single cultures at 400.0 and 800.0 µg/mL and in both cultures at 1200 and 1600 µg/mL, with evidence of a concentration-related increase in MNBN cell frequency.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Data for 3+21 hour treatments -S-9, Range-Finder - male donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total Number of Cells	RI	Cytotoxicity (%)
Vehicle	A	47	151	2	200	0.78	-
	B	44	153	3	200	0.80
8.355	A	NS					-
13.93	A	NS					-
23.21	A	NS					-
38.68	A	NS					-
64.47	A	38	157	5	200	0.84	0
107.4	A	32	162	6	200	0.87	0
179.1	A	32	158	10	200	0.89	0
298.5	A	62	135	3	200	0.71	10
497.4	A	166	34	0	200	0.17	78
829.1	A	NE					-
1382	A	NE					-
2303	A	NE					-

 

NE = Not evaluated – no scoreable cells

NS = Not scored

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

 

Table 2: Data for 3+21 hour treatments +S-9, Range-Finder - male donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total Number of Cells	RI	Cytotoxicity (%)
Vehicle	A	46	150	4	200	0.79	-
	B	44	150	6	200	0.81
8.355	A	NS					-
13.93	A	NS					-
23.21	A	NS					-
38.68	A	NS					-
64.47	A	NS					-
107.4	A	NS					-
179.1	A	NS					-
298.5	A	40	153	7	200	0.84	0
497.4	A	45	151	4	200	0.80	1
829.1	A	57	138	5	200	0.74	8
1382	A	59	137	4	200	0.73	9
2303	A	93	107	0	200	0.54	33

 

Table 3: Data for 24+24 hour treatments -S-9, Range-Finder – male donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total Number of Cells	RI	Cytotoxicity (%)
Vehicle	A	21	145	34	200	1.07	-
	B	18	151	31	200	1.07
8.355	A	NS					-
13.93	A	NS					-
23.21	A	12	158	30	200	1.09	0
38.68	A	18	152	30	200	1.06	0
64.47	A	11	157	32	200	1.11	0
107.4	A	13	164	23	200	1.05	1
179.1	A	19	163	18	200	1.00	7
298.5	A	19	171	10	200	0.96	10
497.4	A	173	27	0	200	0.14	87
829.1	A	NE					-
1382	A	NE					-
2303	A	NE					-

 

NE = Not evaluated – no scoreable cells

NS = Not scored

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation

It is concluded that Dimethyl Sebacate showed evidence of inducing micronuclei in cultured human peripheral blood lymphocytes when tested for 3+21 hours in the presence of a rat liver metabolic activation system (S-9). In the same test system, Dimethyl Sebacate did not induce micronuclei when tested up to toxic concentrations for 3+21 hours and for 24+24 hours in the absence of S-9.

Executive summary:

Dimethyl Sebacate was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). The highest concentrations analysed in the Micronucleus Experiment were limited by toxicity and were determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatments were conducted (as detailed in Table A) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of Dimethyl Sebacate on the replication index (RI). Micronuclei were analysed at three or four concentrations and a summary of the data is presented in the table below:

Table A: Micronucleus Experiment – Results summary

Treatment	Concentration (mg/mL)	Cytotoxicity (%)^$	Mean MNBN cell frequency (%)	Historical(%)^#	Statistical significance
3+21 hour -S-9	Vehicle^a	-	0.45	0.10 – 1.00	-
	200.0	0	0.30		NS
	250.0	6	0.50		NS
	300.0	54	0.50		NS
	*MMC, 0.80	ND	9.45		p<0.001
3+21 hour +S-9	Vehicle^a	-	0.55	0.00 – 1.00	-
	400.0	5	1.15		p<0.05
	800.0	18	1.35		p<0.01
	1200	40	1.55		p<0.001
	1600	55	1.75		p<0.001
	*CPA, 12.5	ND	2.55		p<0.001
24+24 hour -S-9	Vehicle^a	-	0.40	0.10 - 1.10	-
	300.0	7	0.45		NS
	330.0	23	0.70		NS
	360.0	48	0.50		NS
	390.0	61	0.65		NS
	*VIN, 0.10	ND	8.10		p<0.001
^a      Vehicle control was DMSO *     Positive control ^#95^thpercentile of the observed range ^$      Based on replication index NS  Not significant ND Not determined

Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in the vehicle cultures fell within current historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and an eugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.

All acceptance criteria were considered met and the study was accepted as valid.

Treatment of cells with Dimethyl Sebacate for 3+21 hours and for 24+24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were similar to and not significantly higher than those observed in concurrent vehicle controls at any concentration analysed under either treatment condition. The MNBN cell frequencies of all treated cultures fell within the normal ranges.

Treatment of cells for 3+21 hours in the presence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p<0.05), compared to those observed in concurrent vehicle controls, at all four concentrations analysed (400.0 to 1600 µg/mL, giving 5% to 55% reductions in RI). The MNBN cell frequencies exceeded the normal range in single cultures at 400.0 and 800.0 µg/mL and in both cultures at 1200 and 1600 µg/mL, with evidence of a concentration-related increase in MNBN cell frequency.

It is concluded that Dimethyl Sebacate showed evidence of inducing micronuclei in cultured human peripheral blood lymphocytes when tested for 3+21 hours in the presence of a rat liver metabolic activation system (S-9). However, Dimethyl Sebacate did not induce micronuclei in the same test system when tested up to toxic concentrations for 3 + 21 hours and for 24 + 24 hours in the absence of S-9.