Disodium sebacate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No data are available for the test article. However, closely related chemicals gave negative results when tested in an Ames test, chrmomsomal aberration study and in an HPRT study. In addition, no findings were reported in an in vivo micronuclues test. The test article is therefore considered to be not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

According to Ames et al., Mut. Res., 31, 1975

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Tokyo Kasei Kogyo Co. LTD. Tokyo, Japan.

Target gene:

S. typhimurium: his-
E. coli: trp-

Species / strain / cell type:

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

Remarks:

S. typhimurium TA 1538 / E.coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1, 5, 10, 50, 100, 500, 1000, and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2 -(2 furyl)-3 -(5 -nitro-2 -furyl) acrylamide (AF2)

Remarks:

0.01 µg/plate for TA100, 0.05 µg/plate for WP 2 uvrA and TA 98, without S9-mix

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

5 µg/plate for TA1535 and for TA1535, without S9-mix

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for TA1537, without S9-mix

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.25 µg/plate for TA1538, without S9-mix

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

5 µg/plate for TA1535 and WP2 uvrA, with S9-mix

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

5 µg/plate for TA100, TA98, TA1537, TA1538, with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- preincubation, added to a minimal glucose agar plate

DURATION
- Preincubation period: 20 min

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

SUMMARY OF RESULTS

Compound	Dose (µg/plate)	TA100		TA1535		WP2 uvrA		TA98		TA1537		TA1538
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
		Revertant Colonies
DMSO		150	154	30	15	30	34	32	42	18	22	22	28
AF-2	0.01	501
AF-2	0.05					1082		278
ENNG	5.0			1101
9AC	80.0									889
4NQO	0.25											270
B(a)P	5.0		1084						809		313		354
2AA	5.0				440		359
Sebacic Acid	1	130	130	29	9	17	26	43	49	17	23	23	38
	5	119	138	34	11	24	22	52	50	13	25	26	38
	10	131	130	26	15	23	27	39	46	14	24	31	33
	50	125	123	34	13	18	24	47	56	11	24	27	33
	100	136	119	36	16	26	29	50	47	13	29	36	39
	500	140	122	28	13	24	32	42	56	11	18	20	34
	1000	127	132	26	12	27	23	34	45	18	12	28	33
	5000	112	134	36	14	24	27	45	47	12	20	30	32

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Test (HPRT)

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from male Wistar rats

Test concentrations with justification for top dose:

0.1, 0.25, 0.5, 1, 2.5, 5, 7.5, and 10 mM without metabolic activation
0.5, 0.75, 1, 2, 5, 4, 7, 8.5, and 10 mM with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

no metabolc activation was used

Qualifier:

no guideline followed

Principles of method if other than guideline:

Human embryonic lung fibroblast cultures (WI-38) were suspended in tissue culture medium and plated. The test compound was added at three dose levels using three bottles for each level, 24 hours after plating. A preliminary determination of tissue culture toxicity was performed (cytotoxic effects were observed at 400 mg/l). Cells were incubated at 37 degree Celsius and examined twice daily to determine when an adequate number of mitoses were present. Cells were harvested and fixed (3:1 absolute methanol : glacial acetic acid). The specimens were centrifuged, decanted, and suspended in acetic acid-orcein stain and dropped on a slide. The preparations were examined by microscopy. Cells in anaphase were observed for non-disjunction as indicative of cytogenetic damage. Analyzed aberrations include bridges, pseudochiasmata, multipolar cells, and acentric fragments. The positive control was triethylene melamine (TEM) and the negative control was saline. 100 cells were investigated per dose.

GLP compliance:

not specified

Remarks:

pre-GLP

Type of assay:

other: in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: human fibroblasts (WI-38)

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 2, 20, and 200 mg/L

Untreated negative controls:

yes

Remarks:

saline

Positive controls:

yes

Positive control substance:

triethylenemelamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h

STAIN (for cytogenetic assays): acetic acid-orcein

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 cells were investigated/dose.

DETERMINATION OF CYTOTOXICITY
- Any supplementary information relevant to cytotoxicity: Cytotoxic effects were observed at 400 mg/L in a preliminary tissue culture toxicity test.

- OTHER: Cells in anaphase were observed for non-disjunction as indicative of cytogenetic damage. Analyzed aberrations include bridges, pseudochiasmata, multipolar cells, and acentric fragments.

Key result

Species / strain:

mammalian cell line, other: human fibroblasts (WI-38)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 400 mg/L, preliminary Cytotoxicity test

Untreated negative controls validity:

valid

Positive controls validity:

valid

Negative and positive controls were functional. The negative controls contained two cells with bridges one of which contained an acentric fragment. The test compound was negative except for one cell which contained a bridge at the high dose level. In summary, the compound produced no significant aberration.

Dose level	Mitotic index**	Number of cells	% cells with acentric fragments	% cells with bridges	% multipolar cells	% cells other aberrations*	% cells with aberrations++
2	1	100	0	0	0	0	0
20	1	100	0	0	0	0	0
200	1	100	0	1	0	0	1
negative control	1	100	1	2	0	0	3
positive control	1	100	3	12	0	0	15

*Cells that have polyploidy (P), pulverization (pp), fragments (f) or greater than 10 aberrations (a)

** Percent of cells in mitosis : 200 cells observed/dose level

++ Duplicate aberrations in a single cell will cause this to be a % less than a summation of the % aberration seen

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In a mammalian bone marrow chromosome aberration test similar to OECD Guideline 475 in rats (Litton, 1974), adipic acid was administered a single dose of 5000 mg/kg bw. Adequate positive and negative controls were valid. The mitotic indices were considered to be within the normal limits of the controls and there was no evidence for chromosomal damage.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 5 treated and 3 control animals were used. Animals were killed 6, 24 and 48 hours after a single administration in the acute study. In the subacute study 5 doses, 24 hours apart, were administered and animals were killed 6 hours after the last dose. Four hours after the last compound administration, and two hours prior to killing, each animal was given 4 mg/kg bw of colcemid intraperitoneally in order to arrest the bone marrow cells in mitosis. The marrow "plug" was removed and aspirated into Hanks' balanced salt solution. The specimen were centrifuged and resuspended in hypotonic 0.5% KCl. The specimens were placed in a 37 degree Celsius water bath in order to swell the cells. Following centrifugation the cells were resuspended in a fixative (3:1 absolute methanol : glacial acetic acid) and again centrifuged. Cells were resuspended and placed at 4 degree Celsius overnight. The following day cells were again centrifuged and freshly prepared fixative was added. The suspension was dropped onto a slide and ignited by an alcohol burner and allowed to flame. Slides were stained with 5% Giemsa solution. The preparations were examined by microscopy. The chromosomes of each cell were counted and only diploid cells were analyzed. They were scored for chromatid gaps and breaks, chromosome gaps and breaks, reunions, cells with greater than ten aberrations, polyploidy, pulverization, and other chromosomal aberrations which were observed. Fifty metaphase spreads were scored per animal. Mitotic indices were obtained by counting at least 500 cells and the ratio of the number of cells in mitosis / the number of cells observed was expressed as the mitotic index. Negative and positive (TEM) controls were run in each experiment. Two tests were performed at different time intervals.

GLP compliance:

not specified

Remarks:

pre-GLP

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

not specified

Sex:

male

Route of administration:

oral: gavage

Duration of treatment / exposure:

Acute study: single dosing; subacute study: once a day for 5 consecutive days

Frequency of treatment:

Acute study: single dosing; subacute study: once a day for 5 consecutive days

Post exposure period:

Animals were killed  6, 24 and 48 hours after a single administration in the acute study. In  the subacute study 5 doses, 24 hours apart, were administered and animals  were killed 6 hours after the last dose. 

Remarks:

Doses / Concentrations:
Test 1: acute and subacute: 3.75, 37.5, 375 mg/kg bw/day
Test 2: acute 5000 mg/kg bw and subacute 2500 mg/kg bw/day

No. of animals per sex per dose:

Groups of 5 treated/dose and 3 control male animals were used.

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylenemelamine (TEM)

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Four hours after the last compound administration, and two hours prior to sacrifice, each animal was given 4 mg/kg bw of colcemid intraperitoneally in order to arrest the bone marrow cells in mitosis. The marrow "plug" was removed and aspirated into Hanks' balanced salt solution. The specimen were centrifuged and resuspended in hypotonic 0.5 % KCl. The specimens were placed in a 37 °C water bath in order to swell the cells. Following centrifugation the cells were resuspended in a fixative (3:1 absolute methanol : glacial acetic acid) and again centrifuged. Cells were resuspended and placed at 4 °C overnight. The following day cells were again centrifuged and freshly prepared fixative was added. The suspension was dropped onto a slide and ignited by an alcohol burner and allowed to flame. Slides were stained with 5 % Giemsa solution.

METHOD OF ANALYSIS:
The preparations were examined by microscopy. The chromosomes of each cell were counted and only diploid cells were analyzed. They were scored for chromatid gaps and breaks, chromosome gaps and breaks, reunions, cells with greater than ten aberrations, polyploidy, pulverization, and other chromosomal aberrations which were observed. Fifty metaphase spreads were scored per animal. Mitotic indices were obtained by counting at least 500 cells and the ratio of the number of cells in mitosis / the number of cells observed was expressed as the mitotic index.

Key result

Sex:

male

Genotoxicity:

negative

Negative controls validity:

valid

Positive controls validity:

valid

Test I (3.75, 37.5 and 375 mg/kg bw/day dosing):

Acute study: The negative control group cells contained no aberrations. The compound produced no aberrations except for one cell containing a break in the 6-hour sample of the intermediate dose level. The expected severe chromosomal damage was observed for the positive control group (triethylenemelamine treated animals). The mitotic indices were within normal limits. Negative and positive controls were functional.

Subacute study (5 days): The negative control group and the low level test group contained no aberration. The intermediate level contained one cell with a reunion and one cell that was polyploid. The highest level contained three cells with breaks and one fragment. These were considered to be within the normal limits of the historical negative controls of the laboratory. Negative control was functional. No positive control was performed.

Test 2:

Acute study: Adipic acid was administered at a single dose of 5000 mg/kg bw. The compound produced no aberrations except for 3 cells with polyploidy (2 in the 6-hour sample and 1 in the 24-hour). Neither the variety nor the number of these aberrations differed significantly from the negative controls (polyploidy observed in 4 cells). Negative and positive controls were functional.

Subacute study (5 days, 2500 mg/kg bw/day). Only 218 metaphases have been evaluated. The compound produced no aberrations except for 1 cell with polyploidy. Polyploidy was also observed in the negative control group. These are considered to be within the normal limits of the historical negative controls. Negative control was functional, no positive control.

In summary, adipic acid can be considered non-mutagenic in the cytogenetic test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The genotoxicity potential of the test article was assessed with data available for structurally close related compounds (sebacic acid, adipic acid).

AMES TEST

In an Ames test (Shimizu, 1985), no mutagenicity was observered after exposure of S. typhimurium TA 1535, TA 1537, TA 98, TA 1538, TA 100, and E.coli WP2 uvrA with up to 5000 µg/plate sebacic acid with or without metabolic activation. Sebacic Acid disolved in DMSO was tested using the preincubation method in duplicates. Vehicle controls and adequate positive control substances were used concurrently. No cytotoxicity was observed within the used concentration range. No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of Sebacic Acid either with or without metabolic activation. In a second Ames test following OECD guideline 471 (Safepharm Laboratories Limited, 1992), similar results were obtained. In this assay, Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with Sebacic Acid by the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh chemical solutions. The solvent (acetone) control plates gave counts of revertant colonies within the normal range. All positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system. Sebacic Acid caused no reduction in the growth of the bacterial lawn at any of the dose levels employed in all of the strains of Salmonella used. Sebacic Acid was, therefore, tested up to the maximum recommended dose level of 5000 pg/plate. No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of Sebacic Acid either with or without metabolic activation. Sebacic Acid was found to be non-mutagenic under the conditions of this test.

CHROMOSOME ABERRATION IN VITRO

In a mammalian chromosome aberration test (Litton Bionetics, 1974, cited in OECD SIDS for dicarboxylic acid category, July 2001), no abberation was observed after exposure of human embryonic lung fibroblast cells (WI-38) with up to 200 mg/L adipic acid without metabolic activation. Cytotoxicity was observed at 400 mg/L. The positive and negative controls were functional.

GENE MUATATION MAMMALIAN CELLS

Adipic acid was investigated in an OECD TG 476 study in Chinese hamster V79 cells in the absence and in the presence of metabolic activator (S9) up to concentrations of 10 mM. No precipitation of the test item was noted in any experiment; no biological relevant growth inhibition was observed with and without metabolic activation. In both experiments no biologically relevant increase of mutants was found after treatment with the test item. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequencies. In conclusion, adipic acid is considered to be non-mutagenic in the HPRT locus using V79 cells.

CHROMOSOME ABERRATION IN VIVO

In a chromosome abberration test (Litton Bionetics, 1974) male rats were gavaged with adipic acid; either once with up to 5000 mg/kg bw or once per day on 5 consecutive days with up to 2500 mg/kg bw. 200 - 500 metaphase chromosomes of bone marrow cells per dose were scored for chromatid gaps and breaks, chromosome gaps and breaks, reunions, cells with more than 10 aberrations, polyploidy, pulverization and other chromosomal aberrations. The mitotic indices for all dose groups were considered to be within the normal limits of the controls and there was no evidence of chromosomal damage. The positive control groups, performed only during the acute studies, were functional.

Justification for classification or non-classification

The available experimental test data with the source substances sebacic acid or adipic acid are reliable and suitable for classification purposes under Regulation 1272/2008. In vitro results with sebacic acid were negative as well as in vitro and in vivo results with adipic acid. As a result the target substance is also not considered to be classified for genitic toxicity under Regulation (EC) No. 1272/2008, as amended for the ninth time in (EC) No. 2016/1179.