Diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bis(2-ethylhexyl) azelate was tested negative in an Ames test (with and without metabolic activation in Samonella typhimurium TA 98, TA100, TA1535 and TA 1537 and E.coli WP2 urv A (Miwa 2004).

In a mouse lymphoma study according to OECD 476 diester of isodecanol with azelaic acid was tested in ethanol up to precipitating concentrations. No increased incidence of mutations was observed in presence and absence of metabolic activation (Verspeek 2010).

In a chromosome aberration assay in Chinese Hamster Lung cells bis(2-ethylhexyl) azelate was tested up to cytotoxic concentration in presence and absence of metabolic activation. No increased number of aberrations was observed at any of the doses tested (Miwa 2004).

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His operon (S. typhimurium)
Trp operon (E. coli)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

Range finding test:
1.22, 4.88, 19.5, 78.1, 312.5, 625, 1250 and 5000 µg/plate with and without metabolic activation
Main test:
312.5, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF2; 0.01 µg/plate: TA100, WP2uvrA; 0.1 µg/plate: TA98); sodium azide (SA; 0.5 µg/plate: TA1535); 9-aminoacridine (9AA; 80 µg/plate: TA1537); +S9: 2-aminoanthracene (2AA; up to 10 µg/plate all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

If the number of colonies with revertants was as double as that of control group and dose response was observed, the interpretation of the result was positive.

Statistics:

Mean values and standard deviation were calculated.

Species / strain:

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

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

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: There were colorless clear fine oil drop-like precipitations and oil membrane-like precipitations on the surface of agar in any test concentration.

RANGE-FINDING: No cytotoxicity was observed in a range finding study with doses up to 5000 µg/plate.

 Table 1. Test results of experiment 1 (plate incorporation)

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type		cross-linking type	Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
–	0 (aqua dest.)	102 ± 8.1	9 ± 2.6	35 ± 2.6	20 ± 3.5	5 ± 0.6
–	0 (vehicle)	110 ± 14	13 ± 6.2	27 ± 2.1	29 ± 4.2	7 ± 2.3
–	312.5	100 ± 9.3P	8 ± 2.1P	25 ± 7.0P	23 ± 0.6P	13 ± 3.1P
–	625	106 ± 2.6P	11 ± 2.6P	31 ± 6.5P	23 ± 4.2P	11 ± 0.6P
–	1250	112 ± 12.1P	10 ± 1.2P	26 ± 5.5P	29 ± 2.1P	6 ± 0.6P
–	2500	94 ± 2.5P	9 ± 2.5P	29 ± 2.6P	27 ± 4.0P	10 ± 3.5P
–	5000	92 ± 11.5P	7 ± 1.0P	24 ± 1.5P	23 ± 5.0P	4 ± 0.6P
Positive controls, -S9 mix	Name	AF2	SA	AF2	AF2	9AA
	Concentrations (μg/plate)	0.01	0.5	0.01	0.1	80
	Mean No. of colonies/plate (average of 3 ± SD)	548 ± 7.8	525 ± 7.1	132 ± 6.5	421 ± 11.2	334 ± 87.7
+	0 (medium)	106 ± 12.1	11 ± 0.6	24 ± 3.5	30 ± 3.0	13 ± 1.0
+	0 (vehicle)	105 ± 12	10 ± 4.0	33 ± 7.2	27 ± 3.5	16 ± 3.8
+	312.5	103 ± 8.1P	7 ± 4.4P	30 ± 6.1P	25 ± 4.6P	13 ± 1.7P
+	625	96 ± 8.1P	7 ± 2.6P	31 ± 4.6P	31 ± 8.0P	11 ± 1.5P
+	1250	97 ± 15.6P	9 ± 2.6P	31 ± 5.0P	31 ± 6.4P	14 ± 1.5P
+	2500	96 ± 9.7P	6 ± 3.2P	33 ± 2.6P	25 ± 7.5P	11 ± 2.0P
+	5000	90 ± 3.2P	7 ± 4.2P	32 ± 5.8P	32 ± 5.5P	12 ± 2.9P
Positive controls, +S9 mix	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1	2	10	0.5	2
	Mean No. of colonies/plate (average of 3 ± SD)	1036 ± 46.9	327± 18.4	969 ± 142.3	437 ± 30.3	181 ± 29.5

AF2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = sodium azide

P = Precipitate

Table 2. Test results of experiment 2 (plate incorporation)

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type		cross-linking type	Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
–	0 (aqua dest.)	132 ± 15.5	8 ± 1.0	39 ± 2.5	23 ± 8.1	9 ± 5.5
–	0 (vehicle)	131 ± 14.5	9 ± 3.1	41 ± 6.1	31 ± 6.9	9 ± 3.0
–	312.5	129 ± 17.1P	7 ± 2.1P	38 ± 7.0P	27 ± 4.2P	10 ± 1.5P
–	625	124 ± 5.2P	7 ± 1.2P	33 ± 7.8P	30 ± 11P	14 ± 5.2P
–	1250	127 ± 10.4P	8 ± 1.5P	34 ± 4.0P	30 ± 2.1P	13 ± 1.0P
–	2500	128 ± 16.8P	8 ± 3.8P	38 ± 3.2P	25 ± 1.0P	13 ± 3.6P
–	5000	124 ± 4.0P	10 ± 2.5P	38 ± 0.0P	27 ± 4.0P	10 ± 2.5P
Positive controls, - S9	Name	AF2	SA	AF2	AF2	9AA
	Concentrations (μg/plate)	0.01	0.5	0.01	0.1	80
	Mean No. of colonies/plate (average of 3 ± SD)	569 ± 27.5	589 ± 36.5	137 ± 17.6	446 ± 19.1	411 ± 100.1
+	0 (medium)	137 ± 16.1	7 ± 2.5	33 ± 3.2	31 ± 7.8	12 ± 3.0
+	0 (vehicle)	132 ± 5.2	7 ± 3.2	46 ± 7.2	31 ± 6.1	16 ± 2.1
+	312.5	125 ± 22.4P	7 ± 1.5P	39 ± 2.6P	23 ± 1.2P	12 ± 1.5P
+	625	123 ± 10.0P	8 ± 2.1P	41 ± 5.1P	31 ± 2.5P	14 ± 6.5P
+	1250	125 ± 14.0P	9 ± 3.2P	40 ± 5.7P	23 ± 4.5P	14 ± 4.2P
+	2500	116 ± 3.5P	8 ± 2.1P	39 ± 12.5P	31 ± 1.2P	17 ± 5.1P
+	5000	121 ± 7.6P	11 ± 3.8P	46 ± 4.0P	35 ± 2.5P	13 ± 2.1P
Positive controls, + S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1	2	10	0.5	2
	Mean No. of colonies/plate (average of 3 ± SD)	999 ± 117.8	289 ± 6.1	995 ± 77.9	420 ± 33.7	135 ± 13.1

AF2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = sodium azide

P = Precipitate

Conclusions:

negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung (CHL/IU) cell

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagles's MEM modified with NaHCO3 and HCl (pH 7.0 - 7.1). Fetal calf serum was added up to 10%.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

6 h treatment: 150, 300, 600, 1200, 2400 µg/mL with and without metabolic activation.
24 h treatment: 37.5, 75, 150, 300, 600 µg/mL without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: Mitomycin C (MMC, 0.1 µg/mL ); +S9: Dimethylnitrosamin (DMN; 500 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 h and 24 h

NUMBER OF REPLICATIONS: duplicates in single experiment

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

Evaluation was made on the basis of incidence as; -: negative (less than 5.0%); ±: equivocal (5.0% or higher to less than 10.0%); +: positive (10.0% or higher)

Species / strain:

mammalian cell line, other: Chinese hamster lung (CHL/IU) cell

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: There were colorless clear fine oil drop-like precipitations and oil membrane-like precipitations in the culture fluid in petri plate at the concentrations of 1200 µg/mL or more.

RANGE-FINDING: The maximum concentration was established, based on the growth inhibition test. In this test, 50% growth inhibition was observed at 1859.0 µg/mL with S9 and at 1555.5 µg/mL without S9 for 6 h short treatment and 534.2 µg/mL without S9 for 24 h continuous treatment.

Table 1. Test results

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 6 h, without S9 mix
Medium		99	0.5	0.5
Ethanol		100	1	1
MMC	0.1	83	55.5	55.5
Test substance	150	99	0	0
	300	88	0	0
	600	70	0.5	0.5
	1200 P	51	1	1
	2400 P	39	0.5	0.5
Exposure period 6 h, fixation time 24 h, with S9 mix
Medium		100	0	0
Ethanol		100	0	0
DMN	500	78	65.5	65.5
Test substance	150	99	1	1
	300	80	0	0
	600	64	0.5	0.5
	1200 P	48	0	0
	2400 P	35	0	0
Exposure period 24 h, fixation time 24 h, without S9 mix
Medium		97	0.5	0.5
Ethanol		100	1	1
MMC	0.05	86	44.5	44.5
Test substance	37.5	97	1.5	1.5
	75	98	0	0
	150	87	0	0
	300	56	1	1
	600	35	0	0

MMC: Mitomycin C

DMN: Dimethylnitrosamine

P: Colorless clear fine oile drop-like precipitations were noted in the culture fluid in petri plate.

Conclusions:

negative with and without metabolic activation

Reference 3

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:

7 Jan - 16 Feb 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with acceptable restrictions; analytical purity of test substance not indicated by the sponsor

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Analytical purity of test substance not indicated

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: (-S9): methylmethanesulfonate: 15 and 5 µg/mL; (+S9): cyclophosphamide: 7.5 µ/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
1st experiment: 3 h exposure with and without S9 mix
2nd experiment: 3 h exposure with S9 mix and 24 h without S9 mix
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days
- Fixation time: 14 to 16 days

SELECTION AGENT (mutation assays): 5 µg/mL TFT (trifluorothymidine)

NUMBER OF CELLS EVALUATED: five 96-well microtiter plates with 2000 cells per well; each in 2 individual experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:

A test substance is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency (MF) of more than 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of more than 126.
b) The results are confirmed in an independently repeated test.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 (Moore M.M., 2006).
The mutation frequency was expressed as the number of mutants per 10^6 viable cells. The plating efficiencies of both mutant and viable cells (CE day 2) in the same culture were determined and the mutation frequency (MF) was calculated as follows: MF = {-ln P(0)/number of cells plated per well}/CE day2 x 10^6. Small and large colony mutation frequencies were calculated in an identical manner.

Species / strain:

mouse lymphoma L5178Y cells

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Diisodecyl azelate precipitated in the exposure medium at concentrations of 100 µg/mL and above. Diisodecyl azelate was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 µg/mL.

RANGE-FINDING/SCREENING STUDIES: In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating cells with a number of test substance concentrations increasing with approximately half log steps. In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 333 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the soIvent-treated control cultures were between the
minimum and maximum value of the historical control data range.

Table 1: Dose range finding test: Cytotoxicity (3 hours treatment)

Dose     (µg/mL)	Cell count after 3 h of treatment (cells/mLx1E6)	Cell count after 24 h of subculture (cells/mLx1E6)	Cell count after 48 h of subculture (cells/mLx1E6)	Suspension growth   (%)	Relative suspension growth (%)
Without S9 mix
SC	6.5	5.1	7.8	166	100
3	6.7	5.6	8.0	191	115
10	6.2	5.4	8.0	173	104
33	6.3	5.4	7.6	167	101
100	6.4	4.9	7.6	153	92
333	6.4	4,9	7.8	148	89
With S9 mix
SC	6.6	5.4	7.0	161	100
3	6.4	5.6	7.0	159	99
10	7.2	5.5	7.0	179	112
33	6.9	5.6	7.5	184	115
100	7.2	5.0	7.2	166	103
333	7.0	4.8	7.1	153	95

SC = solvent control (ethanol)

 

Table 2: Dose range finding test: Cytotoxicity (24 hours treatment)

Dose     (µg/mL)	Cell count after 24 h of treatment (cells/mLx1E6)	Cell count after 24 h of subculture (cells/mLx1E6)	Suspension growth   (%)	Relative suspension growth (%)
Without S9 mix
SC	6.5	6.3	33	100
3	6.1	6.4	31	94
10	5.7	5.6	25	77
33	6.3	5.6	28	84
100	6.9	5.4	30	90
333	6.6	5.2	27	83

SC = solvent control (ethanol)

 

Table 3: Main test: Results

Dose     (µg/mL)	Relative suspension growth (%)	Cloning efficiency (%)	Relative survival (day 2) (%)	Relative total growth (%)	Total mutation frequency (1/1xE6)
Without metabolic activation: 3 hours treatment
SC1	100	104	100	100	70
SC2		116			64
0.03	104	105	96	100	62
0.1	104	111	101	105	72
0.3	104	88	80	83	71
1	111	95	87	96	65
3	106	90	82	87	83
10	98	108	98	97	71
100	106	107	97	102	63
333	96	94	85	82	74
MMS	63	84	76	48	717
With 8% metabolic activation: 3 hours treatment
SC1	100	83	100	100	103
SC2		84			98
0.03	103	83	99	102	92
0.1	94	88	105	98	86
0.3	87	97	116	101	83
1	99	86	104	103	93
3	97	85	102	99	101
10	96	95	115	110	73
33	96	90	108	104	82
100	97	101	121	117	73
CP	54	62	75	40	1064
Without metabolic activation: 24 hours treatment
SC1	100	90	100	100	73
SC2		95			76
0.03	104	93	100	104	59
0.1	100	89	96	96	66
0.3	103	89	96	98	62
1	105	95	103	108	73
3	102	91	99	101	54
10	83	108	117	97	63
100	102	99	107	110	59
333	98	93	100	98	71
MMS	83	70	76	63	910
With 12% metabolic activation: 3 hours treatment
SC1	100	98	100	100	124
SC2		91			119
0.03	105	91	96	101	103
0.1	116	98	104	120	101
0.3	110	115	121	133	109
1	111	99	105	117	145
3	115	98	104	119	106
10	113	88	92	105	110
33	105	90	95	100	94
100*	110	84	89	97	163
CP	68	72	77	52	1201

SC = solvent control; MMS = methylmethansulfonate; CP = cyclophosphamide

Conclusions:

negative with and without metabolic activation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Based on data on two analogue substances and in a weight of evidence approach, it can be concluded that diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid is not mutagenic or clastogenic (see also rationale for read-across in section 13)

Justification for classification or non-classification

Based on the outcome of the available studies on the analogue substances, no classification for diesters of alcohols, C7-9-iso-, C8-rich, 2-ethylhexyl and nonanedioic acid is considered according to EC No 1272/2008.