Dimethyl sebacate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation (Mc Garry, 2013)

Dimethyl sebacate was assayed for mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254 induced rat liver post mitochondrial fraction (S-9), in two separate experiments.

The vehicle used was dimethyl sulphoxide (DMSO).

In the first experiment, evidence of toxicity was observed at 1581 µg/plate and above in strain TA102 in the absence and presence of S-9, and at 5000 µg/plate in strains TA98, TA100, TA1535 and TA1537 in the absence and presence of S-9.

In the second experiment, treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for strains TA98, TA100, TA1535 and TA1537 only. For strain TA102 the maximum test concentration was reduced to 2500 µg/plate based on strain specific toxicity observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges 78.13 – 2500 µg/plate or 156.3 – 5000 µg/plate, in order to examine more closely those concentrations of Dimethyl sebacate approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, evidence of toxicity was observed at 625 µg/plate and above in strain TA102 in the absence and presence of S-9, and at 1250 and/or 2500 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence and presence of S-9.

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Negative (vehicle) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies all fell within, or slightly above, acceptable ranges for negative control treatments, and were significantly elevated by positive control treatments.

Following Dimethyl sebacate treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no evidence of any Dimethyl sebacate mutagenic activity in this assay system.

It was concluded that Dimethyl sebacate did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), or toxic concentrations, in the absence and in the presence of a rat liver metabolic activation system (S-9).

Induction of micronuclei in vitro in Human lymphocytes (Llyod, 2013)

DimethylSebacatewas tested in anin vitromicronucleus assay accroding to OECD 487 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) fromAroclor1254-induced rats. The test article was formulated in anhydrous analytical grade dimethylsulphoxide(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 byphytohaemagglutinin(PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect ofDimethyl Sebacateon the replication index (RI). Micronuclei were analysed at three or four concentrations.

Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion ofmicronucleated binucleate(MNBN) cells in the vehicle cultures fell within current historical vehicle control (normal) ranges.MitomycinC (MMC) and Vinblastine (VIN) were employed asclastogenicandan eugenicpositive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as aclastogenicpositive 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 DimethylSebacatefor 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 DimethylSebacateshowed 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. 

Mutation at the Hprt locus of mouse Lymphoma (Massip, 2013)

Dimethyl sebacate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells according to OECD 476. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 71.97 to 2303 µg/mL (equivalent to 10 mM, an acceptable maximum concentration forin vitrogenetic toxicology studies according to current regulatory guidelines). The highest concentrations to give > 10% relative survival (RS) were 287.9 µg/mL in the absence of S-9 and 2303 µg/mL in the presence of S-9, which gave 84% and 70% RS, respectively.

In Experiment 1 twelve concentrations were tested, ranging from 50 to 500 µg/mL in the absence of S-9 and 100 to 2303 µg/mL in the presence of S-9. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 400 µg/mL in the absence of S-9 and 2303 µg/mL in the presence of S-9, which gave 12% and 89% RS, respectively.

In Experiment 2 twelve concentrations, ranging from 50 to 600 µg/mL, were tested in the absence of S-9 and nine concentrations, ranging from 150 to 2303 µg/mL were tested in the presence of S-9. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 400 µg/mL in the absence of S-9 and 2303 µg/mL in the presence of S-9, which gave 5% and 59% RS, respectively. Steep concentration-related toxicity was observed between 350 and 400 µg/mL (65% and 5% RS, respectively) in the absence of S-9, therefore both concentrations were analysed.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4 nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In Experiments 1 and 2 no statistically significant increases in mutant frequency were observed following treatment with Dimethyl sebacate at any concentration tested in the absence and presence of S-9. A significant linear trend was observed in Experiment 2 in the absence of S-9 which indicated that MF increased significantly with increasing test article concentration. However, as the comparison of the MF of each test article concentration compared to the concurrent vehicle control MF indicated there was no significant difference at any concentration tested, the significant linear trend obtained was therefore not considered biologically relevant. There were no other significant linear trends observed in either experiment.

It is concluded that Dimethyl sebacate did not induce mutation at thehprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in the absence of S-9 and up to a concentration equivalent to 10 mM in the presence of S-9, in two independent experiments.

Short description of key information:
There are 3 tests performed in vitro according to GLP procedures and one in vivo micronucleus test by oral route in rats accroding to OECD 414. An in vitro micronucleus test (Llyod, 2013) performed according to OECD 487 which was positive in the presence of S9. An Ames test in bacteria performed accroding to OECD 471 which was negative and an in vitro Hprt performed in mouse Lymphoma test (OECD 476) wich was negative in both absence of presence of metabolic activation. Based on the available results, an in vivo test was performed to conclude on the genotoxicity potential of the substance. In the in vivo micornucleus test in ras performed according to OECD 414, the DMS did not induce damage to the chromosome or the mitotic apparatus of rat bone marrow cells.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

other: OECD guideline 487

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Human lymphocyte cultures

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

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).

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'.

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

 

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. 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 October 2012 to 3 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

mouse lymphoma L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5 ± 1% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9), prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Positive controls
4-nitroquinoline 1-oxide (NQO), stock solution: 0.015 and 0.020 mg/mL and final concentration: 0.15 and 0.20 µg/mL, no metabolic activation
Benzo[a]pyrene (B[a]P), stock solution: 0.200 and 0.300 mg/mL and final concentration: 2.00 and 3.00 µg/mL with metabolic activation

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 71.97 to 2303 µg/mL.

In Experiment 1 twelve concentrations were tested, ranging from 50 to 500 µg/mL in the absence of S-9 and 100 to 2303 µg/mL in the presence of S-9.
In Experiment 2 twelve concentrations, ranging from 50 to 600 µg/mL, were tested in the absence of S-9 and nine concentrations, ranging from 150 to 2303 µg/mL were tested in the presence of S-9.

Vehicle / solvent:

DMSO diluted 100 fold in the treatment medium.

Untreated negative controls:

yes

Remarks:

DMSO diluted 100 fold in the treatment medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Remarks:

For positive control concentrations see test concentrations

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: Not applicable
- Exposure duration: 7 days
- Expression time (cells in growth medium): 7 days

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p < 0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p < 0.05).
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Chi squared test used (in tables as ¿²)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 71.97 to 2303 µg/mL (equivalent to 10 mM, an acceptable maximum concentration for in vitro genetic toxicology studies according to current regulatory guidelines). Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations in the absence and presence of S-9 (575.8 to 2303 µg/mL) at the time of treatment. No precipitate was observed following the treatment incubation period. The highest concentrations to provide 10% RS were 287.9 µg/mL in the absence of S-9 and 2303 µg/mL in the presence of S-9, which gave 84% and 70% RS, respectively.

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

RS Values - Range-Finder Experiment

Treatment (µg/mL)	-S-9 % RS	+S-9 % RS
0	100	100
71.97	114	97
143.9	98	123
287.9	84	88
575.8  P	0	46
1152  P	0	74
2303  P	0	70

%RS                       Percent relative survival adjusted by post treatment cell counts

P                             Precipitation observed at the time of treatment

 

Summary of mutation data

Experiment 1 (3 hour treatment in the absence and presence of S-9)

Treatment (µg/mL)		-S-9			Treatment (µg/mL)		+S-9
		%RS	MF§				%RS	MF§
0		100	4.64		0		100	3.82!
50		95	2.86	NS	300		97	1.14	NS
100		83	2.98	NS	400	P	96	3.17	NS
200		68	1.50	NS	450	P	96	3.92	NS
260		50	1.80	NS	500	P	68	2.60	NS
290	P	24	2.62	NS	550	P	103	0.93	NS
300	P	50	2.98	NS	600	P	96	2.04	NS
320	P	45	3.83	NS	800	P	105	1.35	NS
350	P	17	4.00	NS	1000	P	102	2.23	NS
400	P	12	2.53	NS	1500	P	82	2.59	NS
					2303	P	89	2.51	NS
Linear trend			NS		Linear trend			NS
NQO					B[a]P
0.15		43	57.64		2		90	12.50
0.2		33	27.34		3		66	28.07

 

Experiment 2 (3 hour treatment in the absence and presence of S-9)

Treatment (µg/mL)		-S-9			Treatment (µg/mL)		+S-9
		%RS	MF§				%RS	MF§
0		100	2.75		0		100	3.75
50		108	2.50	NS	150		88	3.89	NS
100		94	4.14	NS	300		90	4.84	NS
200		96	3.28	NS	400	P	91	6.22	NS
300		86	6.80	NS	500	P	82	5.64	NS
325	P	80	5.61	NS	750	P	76	6.85	NS
350	P	65	6.70	NS	1000	P	78	4.18	NS
400	P	5	5.02	NS	1500	P	60	5.52	NS
					2000	P	56	2.89	NS
					2303	P	59	6.15	NS
Linear trend			**		Linear trend			NS
NQO					B[a]P
0.15		67	30.91		2		84	36.54
0.2		49	29.38		3		54	43.39

§                             6-TG resistant mutants/10⁶ viable cells 7 days after treatment.

%RS                       Percent relative survival adjusted by post treatment cell counts.

NS                          Not significant.

P                             Precipitation observed at the time of treatment.

!                              Based on one replicate only due to suspected technical dilution error (zero counts obtained on viability plates of one replicate).

*, **, ***                               Test for linear trend: ¿² (one-sided), significant at 5%, 1% and 0.1% level respectively.

 

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that Dimethyl sebacate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in the absence of S-9 and up to a concentration equivalent to 10 mM in the presence of S-9, in two independent experiments.

Executive summary:

Dimethyl sebacate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 71.97 to 2303 µg/mL (equivalent to 10 mM, an acceptable maximum concentration for in vitro genetic toxicology studies according to current regulatory guidelines). The highest concentrations to give > 10% relative survival (RS) were 287.9 µg/mL in the absence of S-9 and 2303 µg/mL in the presence of S-9, which gave 84% and 70% RS, respectively.

In Experiment 1 twelve concentrations were tested, ranging from 50 to 500 µg/mL in the absence of S-9 and 100 to 2303 µg/mL in the presence of S-9. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 400 µg/mL in the absence of S-9 and 2303 µg/mL in the presence of S-9, which gave 12% and 89% RS, respectively.

In Experiment 2 twelve concentrations, ranging from 50 to 600 µg/mL, were tested in the absence of S-9 and nine concentrations, ranging from 150 to 2303 µg/mL were tested in the presence of S-9. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 400 µg/mL in the absence of S-9 and 2303 µg/mL in the presence of S-9, which gave 5% and 59% RS, respectively. Steep concentration-related toxicity was observed between 350 and 400 µg/mL (65% and 5% RS, respectively) in the absence of S-9, therefore both concentrations were analysed.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4 nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In Experiments 1 and 2 no statistically significant increases in mutant frequency were observed following treatment with Dimethyl sebacate at any concentration tested in the absence and presence of S-9. A significant linear trend was observed in Experiment 2 in the absence of S-9 which indicated that MF increased significantly with increasing test article concentration. However, as the comparison of the MF of each test article concentration compared to the concurrent vehicle control MF indicated there was no significant difference at any concentration tested, the significant linear trend obtained was therefore not considered biologically relevant. There were no other significant linear trends observed in either experiment.

It is concluded that Dimethyl sebacate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in the absence of S-9 and up to a concentration equivalent to 10 mM in the presence of S-9, in two independent experiments.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 October 2012 to 22 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Mutation experiment 1: 5, 15.81, 50, 158.1, 500, 1581, 5000 µg/plate
Mutation experiment 2: 78.13, 156.3, 312.5, 625.0, 1250, 2500, 5000 µg/plate (78.13 µg/plate for strain TA 102; 5000 µg/plate for strains TA 98, TA 100, TA 153 and TA 1537 only)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-nitrofluorene

Remarks:

Final concentrations: 2-nitrofluorene (2NF) 5 µg/plate; Sodium azide (NaN3) 2 µg/plate; 9-aminoacridine (AAC) 50 µg/plate; Mitomycin C (MMC) 0.2 µg/plate; Benzo[a]pyrene (B[a]P) 10 µg/plate and 2-aminoanthracene (AAN) 5 and 20 µg/plate

Details on test system and experimental conditions:

Metabolic activation system
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The batches of MolToxTM S 9 were stored frozen in aliquots at <-50°C, and thawed just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin O dealkylase activities).

Treatments were carried out both in the absence and presence of S 9 by addition of either buffer solution or 10% S-9 mix respectively.
10% S-9 mix Buffer solution
Sodium phosphate buffer pH 7.4 100 µMoles 100 µMoles
Glucose-6-phosphate (disodium) 5 µMoles -
b-Nicotinamide adenine dinucleotide 4 µMoles -
phosphate (NADP) (disodium)
Magnesium chloride 8 µMoles -
Potassium chloride 33 µMoles -
Water To volume To volume
S-9 100 µL -

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p < 0.01) which was concentration related.
2. The positive trend/effects described above were reproducible.
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.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

Not applicable

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that Dimethyl sebacate did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines) or toxic concentrations, in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

Dimethyl sebacate was assayed for mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254 induced rat liver post mitochondrial fraction (S-9), in two separate experiments.

All Dimethyl sebacate treatments in this study were performed using formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO).

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Dimethyl sebacate at 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate, plus negative (vehicle) and positive controls. Following these treatments, evidence of toxicity was observed at 1581 µg/plate and above in strain TA102 in the absence and presence of S-9, and at 5000 µg/plate in strains TA98, TA100, TA1535 and TA1537 in the absence and presence of S-9.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for strains TA98, TA100, TA1535 and TA1537 only. For strain TA102 the maximum test concentration was reduced to 2500 µg/plate based on strain specific toxicity observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges 78.13 – 2500 µg/plate or 156.3 – 5000 µg/plate, in order to examine more closely those concentrations of Dimethyl sebacate approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, evidence of toxicity was observed at 625 µg/plate and above in strain TA102 in the absence and presence of S-9, and at 1250 and/or 2500 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence and presence of S-9.

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Negative (vehicle) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies all fell within, or slightly above, acceptable ranges for negative control treatments, and were significantly elevated by positive control treatments.

Following Dimethyl sebacate treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no evidence of any Dimethyl sebacate mutagenic activity in this assay system.

It was concluded that Dimethyl sebacate did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), or toxic concentrations, in the absence and in the presence of a rat liver metabolic activation system (S-9).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The objective of the in vivo micronucleus study was to evaluate according to OECD 474 the potential of the test item, DIMETHYL SEBACATE, to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of rats.

 

A preliminary dose range finding test was performed to define the dose-levels to be used for the main study (micronucleus test).

 

In the main study (micronucleus test), Sprague-Dawley male rats received two oral treatments of DIMETHYL SEBACATE at the dose-levels of 500, 1000 and 2000 mg/kg/day, at a 24-hour interval. Five rats were treated with the low and intermediate dose-levels, and eight rats were treated with the high dose-level to ensure sufficient animals were available for micronucleus analysis.

One group of five males received the vehicle (corn oil) under the same experimental conditions, and acted as vehicle control group. One group of five males received the positive control item (cyclophosphamide) once by oral route at the dose-level of 15 mg/kg/day.

Moreover, for the evaluation of plasma level of the test item, three satellite males received two oral treatments 24 hours apart, of the test item at the highest tested dose-level of 2000 mg/kg/day.

 

Animals from the test item-treated groups and from the vehicle control group were sacrificed between 18 and 24 hours after the last treatment and animals from the positive control group were sacrificed between 18 and 24 hours after the single treatment. Bone marrow smears were then prepared.

 

For each animal of each group, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 4000 Polychromatic Erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocytes ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

 

Results

 

Mortality and clinical signs in the main study (micronucleus test)

No unscheduled mortality was observed in any groups during the main study.

A reflux at dosing was observed on Day 2 in one of the supernumerary males treated at 2000 mg/kg/day.

No other clinical signs were recorded in any other treated animals.

 

Body weight

No relevant changes in body weight were observed in treated groups in comparison to the vehicle group.

 

Cytogenetic test results

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data.

Moreover, cyclophosphamide induced a highly significant increase in the frequencies of MPE, indicating the sensitivity of the test system under our experimental conditions tested. The study was therefore considered to be valid.

 

The mean values of the PE/NE ratio in the groups treated with the test item were not statistically significantly different from that of the respective vehicle control animals.

 

The mean values of MPE in the groups administered the test item at 500, 1000 and 2000 mg/kg/day were similar to those of the vehicle control animals. No statistically significant difference was noted, no dose-response relationship was evidenced and all the frequencies obtained remained within the vehicle historical range. Therefore, these results met the criteria of a negative response.

 

Conclusion

 

Under the experimental conditions of the study, the test item DIMETHYL SEBACATE did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells, after two oral administrations, 24 hours apart, at dose-levels of 500, 1000 and 2000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Although the in vitro micronucleus test performed according to OECD 487 was positive with metabolic activation, the Ames test (OECD 471) , the gene mutation test (OECD 476) and the in vivo micronucleus test (OECD 474) were negative, therefore no classification for mutagenicity is warranted.