4-Decenal, 9-hydroxy-5,9-dimethyl-

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:

From 2020-10-05 to 2021-03-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 476 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

1

Reference substance name:

9-hydroxy-5,9-dimethyldec-4-enal

EC Number:

940-437-2

Cas Number:

926-50-1

Molecular formula:

C12H22O2

IUPAC Name:

9-hydroxy-5,9-dimethyldec-4-enal

Test material form:

liquid

Details on test material:

Apearance: Colorless to ale yellow liquid
Odour: Floral, Green, Lily-of-the-valley, fatty odour
Condition of storage: 15-25°C, protected from light
Expiry date: 09 April 2021

Specific details on test material used for the study:

Date received: 02 September 2020

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Cells: L5178Y tk+/- (3.7.2C) mouse lymphoma cells were obtained from Dr Donald Clive, Burroughs Wellcome Co. Cells. Cells are stored as frozen stocks in liquid nitrogen. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and placed in an incubator set to 37ºC. When the cells were growing well, subcultures were established in an appropriate number of flasks.
- Type and identity of media:
RPMI 1640 media, containing L-glutamine and HEPES were prepared. Resulting mediums are referred to as RPMI A (0% v/v), RPMI 10 (10% v/v) and RPMI 20 (20% v/v). RPMI 5 consisted of RPMI 10 diluted with RPMI A [prepared as RPMI 10 but with no serum added] to give a final concentration of 5% serum.
RPMI A medium supplemented with 10% horse serum (heat inactivated) referred to as RPMI 10, is used for general cell culture, e.g. when growing cells up from frozen stocks.
Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free.
All cell cultures are maintained in an incubator set to 37ºC.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction obtained from Molecular Toxicology Incorporated, USA, was prepared from male Sprague-Dawley rats dosed with β-Naphthoflavone/Phenobarbital.
The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-10°C and thawed and reconstituted with purified water to provide a 10% S-9 mix 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 150 mM KCl or 10% S-9 mix respectively. The final S-9 volume in the test system was 1% (v/v). The final content per mL of the 10% S9 mix is: sodium phosphate buffer pH 7.4 (100 µmol), glucose-6-phosphate (5 µmol), NADP (4 µmol), MgCl2 (8 µmol), KCl (33 µmol), S9 mix (100 µL) and water to volume.

Test concentrations with justification for top dose:

- Range-Finder (3 hours; -S9 and +S9): 1.548 to 198.2 mg/mL for a final concentration range of 15.48 to 1982 µg/mL.
- Mutation experiment (3 hours; -S9): 2.500 to 35.00 mg/mL for a final concentration range of 25.00 to 350.0 µg/mL.
- Mutation experiment (3 hours; +S9): 5.00 to 50.00 mg/mL for a final concentration range of 50.00 to 500.0 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO. Test article stock solutions were prepared by formulating Mahonial under subdued lighting in DMSO, with the aid of vortex mixing, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 2.5 hours of initial formulation.

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Mahonial was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 200.6 mg/mL. The solubility limit in culture medium was below 501.5 µg/mL, as indicated by the appearance of precipitate at this concentration approximately 3 hours after test article addition, with warming at 37°C

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Negative (vehicle) controls comprised treatments with the vehicle DMSO diluted 100-fold in the treatment medium.

True negative controls:

no

Positive controls:

yes

Remarks:

100-fold dilution

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

in the absence of S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Negative (vehicle) controls comprised treatments with the vehicle DMSO diluted 100-fold in the treatment medium.

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

in the presence of S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

CYTOTOXICITY RANGE-FINDER EXPERIMENT
Following 3 hour treatment, cells were centrifuged (200 g) for 5 minutes, washed with tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10.
Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, for 8 days. Wells containing viable clones were identified by eye using background illumination and counted.

MUTATION EXPERIMENT
- Treatment of cell cultures:
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and placed in an incubator set to 37ºC. When the cells were growing well, subcultures were established in an appropriate number of flasks.
At least 10^7 cells in a volume of 17.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S-9 mix or 150 mM KCl was added.
After 3 hours in an incubator set to 37°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10 medium.
Cell densities were determined using a Coulter counter and, where sufficient cells survived, the concentrations adjusted to 2 x 10^5 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival.
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies (Brusick, 1986; Scott et al., 1991). Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.

- Plating for survival
Following adjustment of the cultures to 2 x 10^5 cells/mL after treatment, samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (7 days). Wells containing viable clones were identified by eye using background illumination and counted.

- Expression period:
Cultures were maintained in flasks for a period of 7 days during which the hprt mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask.

- Plating for viability:
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10^5 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (10 days). Wells containing viable clones were identified by eye using background illumination and counted.

- Plating for 6TG resistance:
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10^5 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 µg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (13 days). Wells containing viable clones were identified by eye using background illumination and counted.

NUMBER OF REPLICATIONS: Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

A maximum concentration of 1982 µg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to 10 mM and also a precipitating treatment concentration. Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.
The data presented for Mutation Experiment were derived from a repeat experiment. In the initial Mutation Experiment, no colonies were observed on the vehicle control mutation plates, therefore scoring was aborted. The experiment was invalidated and data from this experiment are not further reported.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the vehicle control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical vehicle control database (mean MF +/- 2 standard deviations).
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 that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Key result

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes in pH were observed in the Range-Finder at the highest concentration tested (1982 µg/mL), compared to the concurrent vehicle controls.
- Effects of osmolality: No marked changes in osmolality were observed in the Range-Finder at the highest concentration tested (1982 µg/mL), compared to the concurrent vehicle controls.
Preliminary solubility data indicated that Mahonial was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 200.6 mg/mL. The solubility limit in culture medium was below 501.5 µg/mL, as indicated by the appearance of precipitate at this concentration approximately 3 hours after test article addition, with warming at 37°C. A maximum concentration of 1982 µg/mL was therefore selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to 10 mM and also a precipitating treatment concentration.
- Evaporation from medium: not applicable
- Water solubility: not soluble in water
- Precipitation: yes
- Other confounding effects: none

PRELIMINARY TOXICITY TEST (see Table 7.6.1/1 below)
In the cytotoxicity Range-Finder Experiment, eight concentrations were tested in the absence and presence of S-9, ranging from 15.48 to 1982 µg/mL (equivalent to 10 mM at the highest concentration tested and also a precipitating concentration).
Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations tested in the absence and presence of S-9 (495.5 to 1982 µg/mL).
Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration in the absence and presence of S-9 (1982 µg/mL). The highest concentrations to give >10% RS were 123.9 µg/mL in the absence of S-9 and 247.8 µg/mL in the presence of S-9, which gave 49% and 71% RS, respectively.

MAIN TEST (see Table 7.6.1/2 below)
The cell concentration was confirmed to be 1.2 x 10^6 cells/mL (i.e. 24 x 10^6 cells treated per cIn the Mutation Experiment twelve concentrations, ranging from 25 to 350 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9, were tested.
Upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations tested in the presence of S-9 (450 and 500 µg/mL).
Following the 3 hour treatment incubation period, no precipitation was observed in the absence or presence of S-9. Seven days after treatment, the highest concentration in the absence of S-9 (350 µg/mL) and the highest three concentrations in the presence of S-9 (420 to 500 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, the lowest concentration in the absence of S-9 (25 µg/mL) was not selected as there were sufficient concentrations to define the toxicity profile. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 280 µg/mL in the absence of S-9 and 390 µg/mL in the presence of S-9, which gave 15% and 14% RS, respectively.

MUTATION RESULTS
The MF could not be calculated for B[a]P at 4 µg/mL in the presence of S-9 because there were no colonies detected on the viability plates (due to probable technical error). However, the data were sufficiently robust for B[a]P at 6 µg/mL, therefore the acceptance criteria were met and the study was accepted as valid.

When tested up to toxic concentrations, no statistically significant increases in MF were observed following treatment with Mahonial at any concentration tested in the absence and presence of S-9. There was a statistically significant linear trend (p≤0.01) in the absence of S-9 but the MF values in all test article-treated cultures were not significantly higher than the concurrent vehicle control MF and were within the historical vehicle control range, therefore this observation was considered not biologically relevant. The result was considered negative under both treatment conditions.

Any other information on results incl. tables

Table 7.6.1/1: RS values - Range-Finder experiment - 3-hour treatments in the absence and presence of S9

cf. attached full study report

Concentration (µg/mL)	Percent relative survival (% RS)
	- S9 mix	+ S9 mix
0 15.48 30.97 61.94 123.9 247.8 495.5 P 991 P 1982 P, PP	100 81 76 55 49 9 0 0 0	100 81 102 78 84 71 0 0 0

P: precipitation noted at time of treatment

PP: precipitation noted at end of treatment incubation period

 

Table 7.6.1/2: Summary of Mutation Data - 3 Hour Treatments in the Absence and Presence of S-9

cf. attached full study report

 

3-hour Treatment -S9 mix			3-hour Treatment +S9 mix
Concentration µg/mL	% RS	MF*	Concentration µg/mL	% RS	MF*
0	100	2.83	0	100	3.35
50	80	1.76 NS	50	87	2.97 NS
100	68	2.35 NS	100	86	1.98 NS
125	52	3.48 NS	150	87	3.60 NS
150	57	4.36 NS	200	73	3.15 NS
175	55	3.12 NS	250	64	5.66 NS
200	42	2.25 NS	300	59	2.46 NS
220	39	5.29 NS	330	61	4.29 NS
240	27	4.88 NS	360	23	3.77 NS
260	25	5.86 NS	390	14	1.70 NS
280	15	5.35 NS
NQO 0.15	52	23.20
NQO 0.20	47	17.93	B[a]P 6	70	15.14

 

Test for Linear trend

- S9 mix		+ S9 mix
Slope Variance b²/Sb	1.06E-08 1.24E-17 8.998**	Slope Variance b²/Sb	-8.91E-10 5.29E-18 0.150

 

*6-TG resistant mutants/10^6 viable cells 7 days after treatment
%RS: Percent relative survival adjusted by post treatment cell counts

*, **, *** Test for linear trend: χ² (one-sided), significant at 5%, 1% and 0.1% level respectively
NS: Not significant

 

Table 7.6.1/3: Historical control ranges

The historical control ranges for the last 20 experiments performed in this laboratory are as follows:

 

Vehicle controls

S9 mix	Mean MF (mutants per 10^6 viable cells)	MF range* (mutants per 10^6 viable cells)
- S9 mix + S9 mix	4.40 4.53	1.09 to 7.70 0.77 to 8.30

*Range = Mean ± 2 x SD.

 

Positive controls

Control concentration	S9 mix	Mean MF (mutants per 10^6 viable cells)	MF range* (mutants per 10^6 viable cells)
NQO 0.15 µg/mL NQO 0.20 µg/mL	- S9 mix - S9 mix	20.55 30.60	5.89 to 35.22 16.37 to 44.82
B[a]P 2 µg/mL B[a]P 3 µg/mL	+ S9 mix + S9 mix	22.13 26.90	2.50 to 41.76 1.88 to 51.92

*Range = Mean ± 2 x SD.

 

The mean mutant frequency values described above differ from those stated in the historical control ranges attached which are based on the entire historical control ranges for this assay (updated after each experiment).

Applicant's summary and conclusion

Conclusions:

Under the test conditions, it is concluded that Mahonial did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

In an in vitro mammalian cell mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, mouse lymphoma L5178Y cells were exposed to the test item.The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by a β-Naphthoflavone/Phenobarbitalinduced rat liver post-mitochondrial fraction (S-9). The test article was formulated in
anhydrous analytical grade dimethyl sulphoxide (DMSO). Three-hour exposures were used both with and without activation (S9) in all tests.

 

In the cytotoxicity Range-Finder Experiment, eight concentrations were tested in the absence and presence of S-9, ranging from 15.48 to 1982 µg/mL (equivalent to 10 mM at the highest concentration tested and also a precipitating concentration). The highest concentrations to give >10% relative survival (RS) were 123.9 µg/mL in the absence of S-9 and 247.8 µg/mL in the presence of S-9, which gave 49% and 71% RS, respectively.

In the Mutation Experiment twelve concentrations, ranging from 25 to 350 µg/mL in the absence of S-9 and from 50 to 500 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 280 µg/mL in the absence of S-9 and 390 µg/mL in the presence of S-9, which gave 15% and 14% RS, respectively.

 

Vehicle and positive control treatments were included in the 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 one or both concentrations of the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore, the study was accepted as valid.

When tested up to toxic concentrations, no statistically significant increases in MF were observed following treatment with Mahonial at any concentration tested in the absence and presence of S-9. There was a statistically significant linear trend (p≤0.01) in the absence of S-9 but the MF values in all test article-treated cultures were not significantly higher than the concurrent vehicle control MF and were within the historical vehicle control range, therefore this observation was considered not biologically relevant. The result was considered negative under both treatment conditions.

 

It is concluded that Mahonial did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint.