Alcohols, C9-11-branched

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented stury report equivalent or similar to OECD guideline 487: GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Isotridecan-1-ol

EC Number:

248-469-2

EC Name:

Isotridecan-1-ol

Cas Number:

27458-92-0

Molecular formula:

C13H28O

IUPAC Name:

3,5,7 trimethyl decanol

Constituent 2

Reference substance name:

Isododecan-1-ol

Molecular formula:

C12H26O

IUPAC Name:

Isododecan-1-ol

Constituent 3

Reference substance name:

Isotetradecan-1-ol

Molecular formula:

C14H30O

IUPAC Name:

Isotetradecan-1-ol

Constituent 4

Reference substance name:

Isoundecan-1-ol

EC Number:

257-376-6

EC Name:

Isoundecan-1-ol

Cas Number:

51750-47-1

Molecular formula:

C11H24O

IUPAC Name:

3,5 dimethyl nonanol-1

Constituent 5

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H20

IUPAC Name:

water

Test material form:

liquid

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: F-12K Medium supplemented with 10% fetal bovine serum
- Properly maintained: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Aroclor induced rat liver

Test concentrations with justification for top dose:

Rangefinder cytotoxicity:
(+S9): (0.006, 0.02, 0.06, 0.2, 0.6, 2 ul/mL)
(-S9): (0.006, 0.02, 0.06, 0.2, 0.6, 2 ul/mL)
Micronucleus Test:
Short term exposure (0.004, 0.006, 0.008, 0.02, and 0.04 uL/ml)
Long term exposure (0.004, 0.006, 0.008, 0.02, and 0.04 uL/ml)

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO alone

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: cytosine B-D-arabinofuranoside and digitonin

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Cells were plated at a seeding density of 1.25x104 cells/well in 48-well plates for both cytotoxicity and test runs, allowed to grow for 24 hours post-seeding, and then exposed to the test article and controls. Test article was added to the wells with or without S9 fraction.
DURATION
Short Exposure:
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 21 hours
Long Exposure (absence of S9):
- Exposure duration: 21 hours
- Expression time (cells in growth medium): 21 hours
NUMBER OF REPLICATIONS:
- 3 replicate wells
STAIN
- Microflow Kit, using EMA (nucleic acid dye A solution), PeakFlow Green Flow Cytometry Reference Beads
DETERMINATION OF CYTOTOXICITY
- Method: assessment of relative increase in cell counts (RICC)

Evaluation criteria:

The data from the flow cytometer was collected and compared to the negative control. The sample in each well was analyzed for % of events that are EMA-positive total counts of nucleated events, total counts of hypodiploidy, total bead counts and total micronuclei counts. The data was assessed as follows:
-The percent hypodiploid counts in each well, percent micronuclei counts in each well, and the nuclei to bead ratio in each well was determined
-The fold change in hypodiploidy in each well, fold change in micronuclei in each well, and the fold change in %EMA positive cells in each well was determined
-The mean fold change in hypoploidy EMA positive cell and micronuclei was determined
-The cell viability was determined by dividing the mean nuclei-bead ratio of the test article by the mean nuclei-bead ratio of the negative control, and multiplying by 100
A test article is considered clearly positive if, in any of the experimental conditions examined: at least one of the test concentrations exhibits a statistically significant increase of 2-fold compared with the concurrent negative control, which is considered a biologically relevant induction in this system; if the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test; or if any of the results are outside the distribution of the historical negative control data.
A test article is considered clearly negative if, in all experimental conditions examined: none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control; or if there is no concentration-related increase when evaluated with an appropriate trend test.

Statistics:

For cytotoxicity the mean % viability and standard deviation were calculated. The mean of fold change vs vehicle control (negative) control was determined. A Student’s paired T-test was performed to assess statistical significance (p≤0.01) between each tested concentration and the negative (vehicle) control.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Short term exposure (0.04ul/mL)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Short term exposure - The test substance did not induce a statistically significant increase in micronuclei formation at any of the non-cytotoxic dose levels tested, with or without metabolic activation.
Long term exposure - The test substance did not induce a statistically significant increase in micronuclei formation at any of the dose levels tested without metabolic activation.

Any other information on results incl. tables

Table 1: Osmolality and pH of Exxal™ 13 at final concentrations in culture media (i.e. dosing solutions) were assessed in the presence and absence of S9.

-S9	Osmolality			pH
1% DMSO	494	493	493	8.68
2µL/mL Exxal 13	445	448	451	8.64

+ S9	Osmolality			pH
1% DMSO	470	467	465	8.59
2 µL/mL Exxal 13	437	435	435	8.56

Table 2: Cytotoxicity assessment of the test article in the rangefinder under a) short term exposure +S9 and b) long term exposure no S9.

a)

	MEAN	SD	SEM	%CV
Vehicle	100.0	12.1	7.0	12.1
1mM Digitonin	14.3	1.6	0.9	10.9
0.006µL/mL Exxal 13	61.5	11.3	6.5	18.3
0.02 µL/mL Exxal 13	64.5	4.6	2.7	7.2
0.06 µL/mL Exxal 13	58.5	5.9	3.4	10.1
0.2 µL/mL Exxal 13	51.9	12.7	7.3	24.4
0.6 µL/mL Exxal 13	41.1	7.2	4.2	17.6
2 µL/mL Exxal 13	10.4	2.3	1.3	21.9

b)

	MEAN	SD	SEM	%CV
Vehicle	100.0	3.8	2.2	3.8
1mM Digitonin	5.0	0.1	0.1	2.5
0.006µL/mL Exxal 13	87.9	20.2	11.7	23.0
0.02 µL/mL Exxal 13	75.1	24.2	14.0	32.2
0.06 µL/mL Exxal 13	5.0	0.3	0.1	5.1
0.2 µL/mL Exxal 13	5.2	0.5	0.3	9.9
0.6 µL/mL Exxal 13	5.5	0.2	0.1	4.0
2 µL/mL Exxal 13	4.8	0.7	0.4	14.8

Table 3: CHO-K1 cells were exposed to the a) positive control mytomycin C and b) the test article Exxal™ 13, for approximately 3 hours in the absence of S9, and then incubated for approximately 1.5 – 2.0 population doublings. Cells were assessed for micronuclei formation according to Section 12.j. Statistical significance if p ≤ 0.01.

a)

Exposure	Conc (µg/mL)	Mean Fold Change EMA	Mean Fold Change MN	Mean Fold Change Hypoploid	Cell Viability (% of control)	T-Test p value	Statistically Significant MN Induction?
Negative (Solvent) Control	0	1.00	1.00	1.00	100.00	N/A	N/A
Mitomycin C	0.2	0.78	1.87	1.29	94.2	0.0123	NO
	2	3.03	11.01	2.03	56.41	0.0001	YES

b)

Exposure	Conc (µg/mL)	Mean Fold Change EMA	Mean Fold Change MN	Mean Fold Change Hypoploid	Cell Viability (% of control)	T-Test p value	Statistically Significant MN Induction?
Negative (Solvent) Control	0	1.00	1.00	1.00	100.00	N/A	N/A
Exxal 13	0.004	1.05	1.19	1.38	96.05	0.1347	NO
	0.006	5.11	1.19	1.11	94.20	0.1609	NO
	0.008	1.90	1.22	1.20	99.38	0.1296	NO
	0.02	1.37	1.23	1.17	103.45	0.0754	NO
	0.04	15.49	1.74	1.74	13.69	0.0084	YES

Table 4: CHO-K1 cells were exposed to the a) positive control cyclophosphamide and b) the test article Exxal™ 13, for approximately 3 hours in the presence of S9, and then incubated for approximately 1.5 – 2.0 population doublings. Cells were assessed for micronuclei formation according to Section 12.j. Statistical significance if p ≤ 0.01.

a)

Exposure	Conc (µg/mL)	Mean Fold Change EMA	Mean Fold Change MN	Mean Fold Change Hypoploid	Cell Viability (% of control)	T-Test p value	Statistically Significant MN Induction?
Negative (Solvent) Control	0	1.00	1.00	1.00	100.00	N/A	N/A
Cyclo-phosphamide	2	0.95	1.88	0.83	107.64	<0.0001	YES
	5	1.17	3.07	1.60	69.23	<0.0001	YES

b)

Exposure	Conc (µg/mL)	Mean Fold Change EMA	Mean Fold Change MN	Mean Fold Change Hypoploid	Cell Viability (% of control)	T-Test p value	Statistically Significant MN Induction?
Negative (Solvent) Control	0	1.00	1.00	1.00	100.00	N/A	N/A
Exxal 13	0.004	0.94	1.04	0.91	109.63	0.3828	NO
	0.006	1.11	1.31	1.28	82.42	0.1101	NO
	0.008	1.15	1.03	1.14	90.76	0.7255	NO
	0.02	1.02	1.18	1.04	96.61	0.0129	NO
	0.04	0.57	1.04	0.92	175.40*	0.382	NO

* data shows there was no increase in RICC suggesting this mean cell viability is false

Table 5: CHO-K1 cells were exposed to the a) positive control cytosine β-D-arabinofuranoside and b) the test article Exxal™ 13, for approximately 1.5 – 2.0 population doublings in the absence of S9. Cells were assessed for micronuclei formation according to Section 12.j. Statistical significance if p ≤ 0.01.

a)

Exposure	Conc (µg/mL)	Mean Fold Change EMA	Mean Fold Change MN	Mean Fold Change Hypoploid	Cell Viability (% of control)	T-Test p value	Statistically Significant MN Induction?
Negative (Solvent) Control	0	1.00	1.00	1.00	100.00	N/A	N/A
Cytosineβ-D-arabino- furanoside	0.04	0.74	2.32	1.76	70.19	0.0009	YES
	0.4	1.42	5.52	2.63	39.50	0.0001	YES

b)

Exposure	Conc (µg/mL)	Mean Fold Change EMA	Mean Fold Change MN	Mean Fold Change Hypoploid	Cell Viability (% of control)	T-Test p value	Statistically Significant MN Induction?
Negative (Solvent) Control	0	1.00	1.00	1.00	100.00	N/A	N/A
Exxal 13	0.004	0.84	1.19	1.21	97.38	0.161	NO
	0.006	0.49	1.15	1.08	111.36	0.3034	NO
	0.008	0.51	1.13	1.20	98.76	0.2672	NO
	0.02	0.47	1.15	1.24	94.73	0.2479	NO
	0.04	1.83	1.10	0.85	41.68	0.5276	NO

Applicant's summary and conclusion

Conclusions:

The mammalian cell micronucleus test to assess the genotoxicity of Exxal 13 was negative.

Executive summary:

Exxal 13 was examined for its potential to induce the formation of micronuclei in Chinese Hamster Ovary (CHO) cells, at both short (with and without S9 metabolic activation) and long (without S9 metabolic activation) exposures. The doses were: short term exposure (0.004, 0.006, 0.008, 0.02, and 0.04 uL/ml) and long term exposure (0.004, 0.006, 0.008, 0.02, and 0.04 uL/ml).

Exxal 13 did not induce a statistically significant increase in the number of cells with micronuclei at any of the non-cytotoxic doses chosen with or without metabolic activation; significant cytotoxicity was observed at 0.02 ul/mL and above in the absence of S9, and at concentrations at and above 0.006ul/mL in the presence of S9. Under the conditions in this study, Exxal 13 was cytotoxic but negative for micronuclei formation in all three tested conditions (short term exposure with and without metabolic activation; long exposure without metabolic activation) for CHO cells.

The mammalian cell micronucleus test to assess the genotoxicity of Exxal 13 was negative.