Reaction mass of (9-acetoxy-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undec-3-yl)methyl palmitate and (9-acetoxy-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undec-3-yl)methyl stearate

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:

13 Jul 2020 - 03 Sep 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Test material

Test material information

Constituent 1

Reference substance name:

[9-(acetyloxy)-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undecan-3-yl]methyl hexadecanoate; [9-(acetyloxy)-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undecan-3-yl]methyl octadecanoate

EC Number:

936-831-9

Molecular formula:

Unspecified

IUPAC Name:

[9-(acetyloxy)-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undecan-3-yl]methyl hexadecanoate; [9-(acetyloxy)-3,8,10-triethyl-7,8,10-trimethyl-1,5-dioxa-9-azaspiro[5.5]undecan-3-yl]methyl octadecanoate

Details on test material:

Physical state, appearance: liquid, yellowish to brownish, clear
Storage conditions: Room temperature

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
The CHO (Chinese hamster ovary) cell line is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.

MEDIA USED
Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS).
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

The S9 fraction was prepared according to Ames et al. At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days.
24 hours after the last administration, the rats were sacrificed and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawed at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors) in the pre-experiment and main experiments. This preparation, the S9 mix (10% S9 fraction), was kept on ice until used. The concentrations of the cofactors in the S9 mix were:
− MgCl2 8 mM
− KCl 33 mM
− glucose-6-phosphate 5 mM
− NADP 4 mM
− phosphate buffer (pH 7.4) 15 mM

Test concentrations with justification for top dose:

0.47 μg/mL, 0.94 μg/mL, 1.88 μg/mL, 3.75 μg/mL, 7.50 μg/mL, 15.00 μg/mL, 30.00 μg/mL, 60.00 μg/mL
Following the requirements of the current international guidelines and the ICPEMC Task Group a test substance should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. In case of toxicity, the top dose should result in approximately 10-20% relative survival (adjusted cloning efficiency), but not less than 10%. For relatively insoluble test substances at least one concentration should be scored showing no precipitation in culture medium at the end of the exposure period.
In the pre-test for toxicity based on the purity and the molecular weight of the test substance 2200.0 μg/mL (approx. 3.5 mM) was used as top concentration both with and without S9 mix at 4 hour exposure time. The pre-test was performed following the method described for the main experiment. The relative survival (RS) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses. Precipitation of the test substance in the vehicle acetone was not observed macroscopically up to the highest required concentration of 220.0 mg/mL (stock solution). In culture medium, test substance precipitation occurred by the end of treatment at concentrations of 17.25 μg/mL and above in the absence and presence of S9 mix. Based on the data and the observations from the pre-test and taking into account the current guidelines, the following doses were selected in this study.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
Due to the insufficient solubility of the test substance in culture medium HAM`s F12, acetone was selected as vehicle, which has been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available. The final concentration of the vehicle acetone in culture medium was 1% (v/v).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: two flasks
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 20x1e6 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 6-8 days
- Selection time (if incubation with a selective agent): 6 – 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days
- If a selective agent is used: 6-thioguanine, 10 µg/ml
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: for selection of the mutants, two 175 cm² flasks with 2x1e6 cells each from every treatment group were seeded. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Relative survival after treatment (Cloning efficiency 1 [CE1] adjusted by cell loss):
For the determination of the influence of the test substance after the exposure period, 200 cells per concentration were reserved from the treated cells and were seeded in petri dishes (60 mm diameter) and coated with 5 mL Ham's F12 medium incl. 10% (v/v) FCS in parallel to the 1st passage directly after test substance incubation.
Cloning efficiency 2 (CE2; viability):
For the determination of the mutation rate after the expression period, two aliquots of 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two petri dishes (60 mm diameter) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS.
In all cases, after seeding the flasks or petri dishes were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted. The absolute and relative cloning efficiencies (%) were calculated for each test group.
The test cultures of all test substance concentrations were examined microscopically for cell morphology and cellular attachment at the end of the exposure period, which is a further indication for cytotoxicity.

Evaluation criteria:

A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

A linear dose-response was evaluated by testing for linear trend. The dependent variable was the corrected mutant frequency and the independent variable was the dose.
The calculation was performed using EXCEL function RGP.
The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report.
A pair-wise comparison of each test group with the control group was carried out using Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using EXCEL function HYPGEOM.VERT.
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: pH values were not relevantly influenced by test substance treatment.
- Data on osmolality: Osmolality values were not relevantly influenced by test substance treatment.

CELL MORPHOLOGY
After 4 hours treatment either in the absence or presence of metabolic activation, the cell morphology and attachment of the cells was adversely influenced (grade > 2) in any test group tested for gene mutations.

CYTOTOXICITY
There was no decrease in the number of colonies as described by the relative survival in the presence and absence of S9 mix up to the highest evaluated concentrations for gene mutation.

STUDY RESULTS
In the absence of metabolic activation, the values for the corrected mutation frequencies (MFcorr.) ranged between 0.37 – 2.38 per 1e6 cells); the respective vehicle control had 2.78 per 1e6 cells. The obtained values were within the range of the 95% vehicle control limit (without S9 mix: MFcorr.: 0.00 – 6.21 per 1e6 cells). A concentration related increase in the mutant frequencies was not observed and no statistically significant increase in the mutant frequencies was determined.
In the presence of metabolic activation, the values for the corrected mutation frequencies ranged between MFcorr.: 0.35 – 3.18 per 1e6 cells; the respective vehicle control value had 1.32 per 1e6 cells. The obtained values were within the range of the 95% vehicle control limit (with S9 mix: MFcorr.: 0.00 – 7.02 per 1e6 cells. A concentration related increase in the mutant frequencies was not observed and no statistically significant increase in the mutant frequencies was determined.
The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 237.12 per 1e6 cells; with S9 mix: MFcorr.: 73.61 per 1e6 cells) were clearly within our historical positive control data range (without S9 mix: MFcorr.: 42.47 – 419.90 per 1e6 cells; with S9 mix: MFcorr.: 21.52 – 270.48 per 1e6 cells).

Any other information on results incl. tables

SUMMARY OF RESULTS

						Cytotoxicity***
Exp	Exposure period [h]	Test groups [μg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	RS [%]	CE2 [%]
1	4	Vehicle control^1	-	n.d.	2.78	100.0	100.0
		0.47	-	-	n.c.	163.3	n.c.
		0.94	-	-	n.c.	139.0	n.c.
		1.88	-	-	1.64	149.2	93.8
		3.75	-	-	0.71	132.4	87.3
		7.50	-	-	0.37	133.1	83.0
		15.00	-	+	2.38	105.2	77.8
		30.00	-	+	n.c.^1	n.c.^1	n.c.^1
		60.00	-	+	n.c.^1	n.c.^1	n.c.^1
		Positive control^2	-	n.d	237.12^s	106.2	70.7
1	4	Vehicle control^1	+	n.d.	1.32	100.0	100.0
		0.47	+	-	n.c.	90.4	n.c.
		0.94	+	-	n.c.	88.9	n.c.
		1.88	+	-	3.18	84.4	93.1
		3.75	+	-	0.74	123.1	89.5
		7.50	+	-	0.35	97.6	92.8
		15.00	+	+	0.43	92.8	75.7
		30.00	+	+	n.c.^1	n.c.^1	n.c.^1
		60.00	+	+	n.c.^1	n.c.^1	n.c.^1
		Positive control^3	+	n.d.	73.61^s	84.4	71.1

* Macroscopically visible precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle/negative control

^s Mutant frequency statistically significantly higher than corresponding control values (p ≤ 0.05)

n.c. Culture was not continued since a minimum of only four analysable concentrations is required

n.c.¹ Culture was not continued since only one concentration beyond the solubility limit is required

n.d. Not determined

¹ Acetone 1% (v/v)

² EMS 400 μg/mL

³ DMBA 1.25 μg/mL

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. One experiment was carried out, in the absence and presence of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Acetone was used as vehicle. According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested. The highest tested concentration (60.00 μg/mL) was based on test substance precipitation in culture medium. Test groups printed in bold type were evaluated for gene mutations:

without S9 mix 0; 0.47; 0.49; 1.88; 3.75; 7.50; 15.00; 30.00; 60.00 μg/mL

with S9 mix 0; 0.47; 0.49; 1.88; 3.75; 7.50; 15.00; 30.00; 60.00 μg/mL

Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. Dose selection for genotoxicity testing was based on the solubility properties of the test substance in culture medium. The highest evaluated concentration showed clear test substance precipitates in culture medium macroscopically at the end of exposure period in the presence and absence of metabolic activation. In the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.