Hexanoic anhydride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - May 2017

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 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Hexanoic anhydride

EC Number:

218-121-4

EC Name:

Hexanoic anhydride

Cas Number:

2051-49-2

Molecular formula:

C12H22O3

IUPAC Name:

hexanoyl hexanoate

Test material form:

liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: over 4 hours at room temperature in the light (Watson P, Study Number FP 57HY Envigo Report 13 April 2017)


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: All formulations were prepared freshly before treatment and used within two hours of preparation.

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany
- Suitability of cells: used successfully for many years in in vitro experiments; high proliferation rate, and a reasonable plating effeiciency
- Cell cycle length, doubling time or proliferation index: doubling time approximately 13 hours
- Methods for maintenance in cell culture if applicable: stocks are stored in liquid nitrogen; before freezing each batch is screened for mycoplasm contamination and
checked for karyotype stability
- Modal number of chromosomes: 22 +/- 1

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimal essential medium (MEM) with different supplements; incubations were done at 37°C in a humidified atmosphere with 1.5% carbon dioxide.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: not specified

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Exp. I, (4 hrs): 3.9, 7.8, 15.5, 31.3, 62.5, 125, 250, 500,1000 and 2000μg/mL (without and
with S9 mix)
Exp. IIA, (24 hrs): 62.5, 125, 250, 500,1000 and 2000 μg/mL (without S9 mix)
Exp. IIA, (4 hrs): 62.5, 125, 250, 500,1000 and 2000 μg/mL (with S9 mix)
Exp. IIB (4 hrs): 62.5, 125, 250, 500,1000 and 2000 μg/mL (with S9 mix)

The highest applied concentration was chosen with respect to the current OECD TG 487.

The following concentrations were selected for reading in all experiments: 500, 1000, 2000 µg/mL

Vehicle / solvent:

DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Griseofulvin

Details on test system and experimental conditions:

- Culture conditions: Thawed stock cultures were propagated at 37 °C in 80 cm^2 plastic flasks. About 5 x 10^5 cells per flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL) and 10 % (v/v) fetal bovine serum (FBS). The cells were sub-cultured twice a week.
Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.25 % (w/v) in Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes containing microscopic slides. Into each chamber 1.0 x 10^5 – 1.5 x 10^5 cells were seeded. For RICC (relative increase in cell count) determination, 16600 cells per well (Exp. I), 16720 cells per well (exp. IIA) and 15600 cells per well (Exp. IIB) were seeded in duplicates in a 24-well-plate.
All incubations were done at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

- TREATMENT:

Pre-experiment:
A preliminary cell growth inhibition test (determination of proliferation index) was performed to determine the concentrations to be used in the main experiment. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 24 hrs after start of the exposure.

Pulse exposure: The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4. The cells were then cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 20 hours.

Continuous exposure (without S9 mix) : The culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item. The medium was not changed until preparation of the cells.

Preparation of micronuclei: The cells were treated on the slides in the chambers with deionised water for 1 – 1.5 min at 37 °C. Afterwards the cells were fixed twice with a mixture of ethanol and glacial acetic acid (3+1 parts, respectively) containing 1.25 % formaldehyde. The slides were stained with Giemsa, mounted after drying and covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.

Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. Per culture at least 1000 cells from clones with 2 - 8 cells were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells.

Cytotoxicity was assessed by the determination of the relative increase in cell counts (RICC).

RICC = (increase in number of cells in treated cultures (final - starting)) diveded by (increase in number of cells in control cultures (final - starting)) multiplied by 100

Cytotoxicity [%] = 100 – RICC

In addition, cytotoxicity was assessed via counting the number of clones consisting of 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), and 5 - 8 cells (c8) among the cells that were scored for the presence of micronuclei. These clusters represent the cells that have divided 1, 2, or 3 times within the experiment. From these data, a proliferation index (PI) was calculated (see formula below). Only those cultures were evaluated which showed a PI > 1.3, in order to guarantee a sufficient cell proliferation during treatment and recovery.

PI = [(c1 x 1) + (c2 x 2) + (c4 x 3) + (c8 x 4)] / [c1 + c2 + c4 + c8]
PI = Proliferation index
cx = Number of clones with x cells (with x: 1, 2, 4, or 8)

Evaluation criteria:

Acceptability criteria:
The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range.
− The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase.
− Cell proliferation criteria in the solvent control are considered to be acceptable.
− All experimental conditions described in details on test system and conditions were tested unless one exposure condition resulted in a clearly positive result.
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.
− The criteria for the selection of top concentration are consistent with those described above.

Evaluation Criteria:
Clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

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:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant influence
- Effects of osmolality: no relevant influence
- Precipitation: phase separation was observed in all experimental parts at 2000 µg/mL at the end of treatment

Any other information on results incl. tables

In all experimental parts in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration, which showed phase separation.

In Experiment I and IIA, in the absence of S9 mix, no relevant increases in micronucleated cells were observed after treatment with the test item.     
In Experiment IIA in the presence of S9 mix, two statistically significant increases in micronucleated cells (1.45 and 2.05 %) were observed after treatment with 500 and 1000 µg/mL, respectively. The second value exceeded the range of the laboratory historical solvent control (0.20 – 1.72 % micronucleated cells).      
In the confirmatory experiment IIB, the values of all evaluated concentrations were within the range of the laboratory historical solvent control data (0.20 – 1.72 % micronucleated cells) and no statistical significance was observed after treatment with the test item.

Table            Summary of results

Exp.	Preparation	Test item	Proliferation	RICC	Cytotoxicity	Micronucleated
	interval	concentration	Index	in %	in %	Cells*
		in µg/mL				in %
Exposure period 4 hrs without S9 mix
I	24 hrs	Solvent control^1	2.66	100.00	0.00	0.90
		Positive control^2	2.31	74.06	25.94	4.05^S
		500	2.65	97.63	2.38	0.60
		1000	2.60	95.56	4.44	0.70
		2000^PS	2.61	109.34	n.c.	0.70
Exposure period 24 hrs without S9 mix
IIA	24 hrs	Solvent control^1	3.06	100.00	0.00	0.45
		Positive control^3	2.54	78.20	21.80	8.40^S
		500	2.80	71.93	28.07	0.45
		1000	2.47	73.18	26.82	0.60
		2000^PS	2.93	72.55	27.45	0.60
Exposure period 4 hrs with S9 mix
IIA	24 hrs	Solvent control^1	2.02	100.00	0	0.75
		Positive control^4	1.33	36.25	63.75	14.80^S
		500	1.79	72.18	27.82	1.45^S
		1000^#	1.93	82.44	17.56	2.05^S
		2000^PS	1.84	79.68	20.32	0.95
IIB	24 hrs	Solvent control^1	2.40	100.00	0.00	1.25
		Positive control^5	1.40	45.21	54.79	7.65^S
		500	2.24	84.67	15.33	1.70
		1000	2.38	104.03	n.c.	1.40
		2000^PS	2.52	106.69	n.c.	1.65

*      The number of micronucleated cells was determined in a sample of 2000 cells

^#      The number of micronucleated cells was determined in a sample of 4000 cells

n.c. Not calculated as the RICC is equal or higher than the solvent control value

^S       Number of micronucleated cells statistically significantly higher than corresponding control values

^PS     Phase separation was observed at the end of treatment

¹         DMSO                    0.5 % (v/v)

²         Mitomycin C         0.1 µg/mL

³         Griseofulvin         8.0 µg/mL

⁴         CPA                      10.0 µg/mL

⁵         CPA                      15.0 µg/mL

 

* The number of micronucleated cells was determined in a sample of 2000 cells

# The number of micronucleated cells was determined in a sample of 4000 cells

S Number of micronucleated cells statistically significantly higher than corresponding control values

PS Phase separation was observed at the end of treatment

n.c. Not calculated as the RICC is equal or higher than the solvent control value

1 DMSO 0.5 % (v/v)

2 Mitomycin C 0.1 μg/mL

3 Griseofulvin 8.0 µg/mL

4 CPA 10.0 μg/mL

5 CPA 15.0 µg/mL

Applicant's summary and conclusion

Conclusions:

negative

Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in Chinese hamsterV79cellsin vitroin three independent experiments. The highest applied concentration in this study (2000 µg/mL of the test item) was chosen with respect to the current OECD Guideline 487. In all experimental parts in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration, which showed phase separation. Under the experimental conditions reported, the test item did not induce micronuclei as determined by thein vitromicronucleus test in Chinese hamsterV79cells. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.