Hexanedioic acid, di-C16-18 (even numbered) alkyl esters

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Aug - 26 Sep 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Health Care Inspectorate of the Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

Type of assay:

other: In vitro mammalian cell gene mutation test using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Hexanedioic acid, di-C16-18-alkyl esters

EC Number:

296-703-7

EC Name:

Hexanedioic acid, di-C16-18-alkyl esters

Cas Number:

92969-90-9

Molecular formula:

not applicable, substance is UVCB

IUPAC Name:

Hexanedioic acid, di-C16-18 (even numbered)-alkyl esters

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

L5178Y/TK(+/-)-3.7.2C

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection (ATCC, Manassas, USA)
- Suitability of cells: The cell line is the recommended test system in international guidelines.
- Methods for maintenance in cell culture: Per culture 8 x 10E6 cells (10E6 cells/mL for 3 hour treatment) or 6 x 10E6 cells (1.25 x 10E5 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm² culture flasks at 37.0 ± 1.0 °C.

MEDIA USED
- Type and identity of media: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin. The growth and exposure medium contained 10 and 5% inactivated horse serum in addition, respectively.
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: Prior to dose range finding and mutagenicity testing the mouse lymphoma cells were grown for 1 day in growth medium containing 10E-4 M hypoxanthine (Sigma), 2 x 10E-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10E-5 M thymidine (Merck) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on growth medium containing hypoxanthine and thymidine only. After this period cells were returned to growth medium for at least 1 day before starting the experiment.

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and β-naphthoflavone.

Test concentrations with justification for top dose:

Experiment I
With and without S9 mix: 0.54, 1.7, 5.4, 17, 52, 164, 512 and 1600 µg/mL (3 h)

Experiment II
Without S9 mix: 0.54, 1.7, 5.4, 17, 52, 164, 512 and 1600 µg/mL (24 h)

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 10E6 cells /mL for 3 h treatment and 1.25 x 10E5 cells/mL for 24 h treatment

DURATION
- Exposure duration: 3 h with and without metabolic activation and 24 h without metabolic activation
- Expression time (cells in growth medium): 2 days
- Selection time: 11 or 12 days

SELECTION AGENT (mutation assays): Trifluorothymidine (5 µg/mL)

STAIN: MTT (0.5 µg/mL)

NUMBER OF REPLICATIONS: 1 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growth, cloning efficiency, relative survival and relative total growth

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency (MF) should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT 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 x 10E-6.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner (significant in a trend test). A Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction (ToxRat Professional v 3.2.1). An observed increase should be biologically relevant and was compared with the historical control data range.
A test item 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 item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

A Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction (ToxRat Professional v 3.2.1).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

at 1600 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

at 52 µg/mL after 24 h treatment

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

observed at 1600 µg/mL (3 h treatment); and observed at 164, 512 and 1600 µg/mL (24 h treatment)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH at a concentration of 512 µg/mL was 7.27 and in the solvent control 7.29. Therefore pH was not considered to have interfered with the results.
- Effects of osmolality: The osmolality at a concentration of 512 µg/mL was 0.440 Osm/kg and in the solvent control 0.449 Osm/kg. Therefore the osmolality was not considered to have interfered with the results.
- Precipitation: The test substance precipitated in the exposure medium at concentrations of 1600 µg/mL and above for the 3 h treatment and at concentrations ≥ 164 µg/mL for the 24 h treatment.

RANGE-FINDING/SCREENING STUDIES: In the dose range-finding test, L5178Y mouse lymphoma cells were treated with test substance concentrations of 17, 52, 164, 512 and 1600 μg/mL in the absence of S9-mix with 3 and 24 hour treatment periods; and in the presence of S9-mix with a 3-hour treatment period (see Table 1-2 under' Any other information on results incl. tables'). Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 1600 μg/mL, compared with the suspension growth of the solvent control, as measured after 3 and 24 hours of treatment. Based on this range-finding study, test substance concentrations of 0.54, 1.7, 5.4, 17, 52, 164, 512 and 1600 µg/mL were chosen for the mutation experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The mutation frequency of 710 (3 h, -S9), 1916 (3 h, +S9) and 681 (24 h, -S9) found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
3 h treatment without metabolic activation: Mean = 857; SD = 246; n = 110; Upper control limit (95% control limits) = 1425; Lower control limit (95% control limits) = 289
24 h treatment without metabolic activation: Mean = 688; SD = 187; n = 102; Upper control limit (95% control limits) = 1124; Lower control limit (95% control limits) = 253
3 h treatment with metabolic activation: Mean = 1710; SD = 815; n = 139; Upper control limit (95% control limits) = 4214; Lower control limit (95% control limits) = -793

- Negative (solvent/vehicle) historical control data: The mutation frequency of the solvent control cultures in the 3 h-treatment experiment observed in the absence of S9-mix (MF of 137) and in the presence of S9-mix (MF of 146) were just above the upper control limits. These limits are 95% control limits and a slightly higher response is within the expected response ranges, according to the author of the study report. Furthermore, the solvent control was tested in duplicates and the mean of the MF was within the historical control range and considered to be valid. The mutation frequency of 125 and 113 in the 24 h-treatment experiment was within the 95% control limits of the distribution of the historical control database, and valid.
3 h treatment without metabolic activation: Mean = 86; SD = 23; n = 220; Upper control limit (95% control limits) = 135; Lower control limit (95% control limits) = 37
24 h treatment without metabolic activation: Mean = 81; SD = 26; n = 202; Upper control limit (95% control limits) = 135; Lower control limit (95% control limits) = 28
3 h treatment with metabolic activation: Mean = 87; SD = 28; n = 273; Upper control limit (95% control limits) = 145; Lower control limit (95% control limits) = 28

OTHER:
Results
In the second mutation experiment (24 h treatment), the test substance induced an up to 2.3-fold increase in the mutation frequency at the TK locus at the non-precipitating dose level of 52 μg/mL (MF = 272). This result was above the global evaluation factor (GEF) of (245 x 10E6), and therefore indicative of a positive result. The test item showed up to 2.6- and 1.6-fold increases in the mutation frequency of the small and large colonies, respectively, compared with the mean mutation frequency of the small and large colonies of the respective solvent controls. At the precipitating dose levels from 164 μg/mL, an increase in the mutation frequency was observed. However, the MF was 176 x 10E6 at 164 μg/mL, 194 x 10E6 at 512 µg/mL and 192 x 10E6 at 1600 µg/mL, which is below the GEF. A statistical significant dose related trend (p<0.001) was observed following the 24-h treatment, and considered to be biologically relevant (the concentrations for which this trend was observed were not given in the study report) (see Table 3-4 under' Any other information on results incl. tables').

Any other information on results incl. tables

Table 1: Dose range-finding test: Cytotoxicity of the test substance (3 h treatment)

Dose (µg/mL)	Cell count after 24 h of subculture (cells/mL x 10^5)	Cell count after 48 h of subculture (cells/mL x 10^5)	Suspension growth (cells/mL x 10^5)	Relative suspension growth (%)
Without metabolic activation
DMSO	5.1	7.0	18	100
17	5.7	6.6	19	105
52	6.0	6.4	19	108
164 P	6.2	6.5	20	113
512 P	5.6	6.5	18	102
1600 P	5.3	6.8	18	101
With metabolic activation
Solvent control	3.9	6.7	13	100
17	4.7	6.8	16	122
52	4.1	7.6	16	119
164 P	4.7	6.1	14	110
512 P	4.5	7.0	16	121
1600 P	4.0	7.4	15	113

P = Precipitation

Table 2: Dose range-finding test: Cytotoxicity of the test substance (24 h)

Dose (µg/mL)	Cell count after 24 h of subculture (cells/mL x 10^5)	Cell count after 48 h of subculture (cells/mL x 10^5)	Suspension growth (cells/mL x 10^5)	Relative suspension growth (%)
Without metabolic activation
DMSO	10.5	6.2	42	100
17	10.1	6.5	42	100
52	9.8	6.4	40	95
164 P	9.1	6.1	36	86
512 P	9.6	6.0	37	88
1600 P	7.9	6.2	31	74

P = Precipitation

Table 3: Cytotoxic and mutagenic response of the test substance in the mouse lymphoma L5178Y test system (3 h treatment)

Dose (µg/mL)	Relative suspension growth (%)	Cloning efficiency day 2 (%)	Relative survival day 2 (%)	Relative total growth (%)	Mutation frequency per 10^6survivors total	Mean MF + GEF (126 x 10^-6)	GEF exceeding
Without metabolic activation
DMSO	100	76	100	100	137	247.5	-
DMSO		78			106
0.54	95	88	114	108	91	-	no
1.7	79	99	129	102	85	-	no
5.4	75	72	94	71	168	-	no
17	86	89	115	99	101	-	no
52	85	85	111	94	118	-	no
164	84	93	121	101	90	-	no
512	82	90	117	96	89	-	no
1600 P	77	85	111	85	101	-	no
MMS	78	58	76	59	710	-	yes
With metabolic activation
DMSO	100	80	100	100	144	271	-
DMSO		74			146
0.54	60	93	120	73	129	-	no
1.7	104	79	103	107	143	-	no
5.4	106	76	98	104	109	-	no
17	107	76	98	105	139	-	no
52	98	101	131	129	122	-	no
164	96	89	115	111	132	-	no
512	89	108	141	125	91	-	no
1600 P	80	108	141	113	89	-	no
MMS	36	36	47	17	1916	-	yes

P = Precipitation

MMS = Methylmethanesulfonate

CP = Cyclophosphamide

Table 4: Cytotoxic and mutagenic response of the test susbtance in the mouse lymphoma L5178Y test system (24 h treatment)

Dose (µg/mL)	Relative suspension growth (%)	Cloning efficiency day 2 (%)	Relative survival day 2 (%)	Relative total growth (%)	Mutation frequency per 10^6survivors total	Mean MF + GEF (126 x 10^-6)	GEF exceeding
Without metabolic activation
DMSO	100	97	100	100	125	245	-
DMSO		88			113
0.54	89	101	110	97	148	-	no
1.7	96	104	113	108	195	-	no
5.4	93	94	102	95	182	-	no
17	105	102	111	116	206	-	no
52	124	83	90	112	272	-	yes
164 P	114	88	95	108	176	-	no
512 P	123	91	99	122	194	-	no
1600 P	102	93	101	103	192	-	no
MMS	123	85	92	113	681	-	yes

P = Precipitation

MMS = Methylmethanesulfonate