2-Oxetanone, 3-C14-16-alkyl 4-C15-17-alkylidene derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reliable studies, showing negative results with and without metabolic activation, are available on genetic toxicity in bacteria and mammalian cells. In addition the substance did not induce chromosome aberrations in a chromosome aberration study wit and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 October 2019 to 09 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type: L5178Y/TK+/--3.7.2C mouse lymphoma cells
- Source of cells:
American Type Culture Collection, (ATCC, Manassas, USA) (2001)
- Suitability of cells:
Recommended test system in international guidelines (e.g. OECD)

For cell lines:
- Absence of Mycoplasma contamination:
checked
- Methods for maintenance in cell culture:
stored in the freezer (-150°C); Cell density was kept below 1 x 106 cells/mL
- Periodically ‘cleansed’ of spontaneous mutants: Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing hypoxanthine, aminopterine and thymidine to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only.

MEDIA USED
Growth medium
Basic medium (RPMI 1640 Hepes buffered medium), supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium
Cells were exposed to the test item in basic medium supplemented with 5%/10% (v/v)
heat-inactivated horse serum (R5-medium) for 3/24 hour exposure.
Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 µg/mL trifluorothymidine (TFT) (Sigma).
Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).

ENVIRONMENTAL CONDITIONS: All incubations were carried out in a humid atmosphere (actual range 34 - 98%) containing 5.0 ± 0.5% CO2 in air in the dark 35.4 - 37.6 °C.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

NA

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
- method of preparation of S9 mix : components contained per mL physiological saline: 1.63 mg MgCl2.6H2O ; 2.46 mg KCl ; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES (Life technologies).To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration S9 in the final culture medium: 4%
- quality controls of S9: filter sterilized

Test concentrations with justification for top dose:

DRF(3h): 3.9, 7.8, 15.6, 31.3 and 62.5 ug/mL with and without metabolic activation (2 highest concentration showed precipitate)
DRF(24h): 0.5, 1, 2, 4 and 8 ug/mL without metabolic activation (highest concentration showed precipitate)
First exp (3h): 0.3, 0.6, 1.2, 2.5, 5, 10, 15 and 35 ug/mL with and without metabolic activation(highest concentration showed precipitate)
Second exp (24h): 0.06, 0.1, 0.3, 0.5, 1, 2, 4 and 8 ug/mL without metabolic activation (highest concentration showed precipitate)

Vehicle / solvent:

hexane

Negative solvent / vehicle controls:

yes

Remarks:

hexane

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 5 selection plates for treatment groups and 10 for controls
- Number of independent experiments
: 2 (first exp 3 h with and without metabolic activation; sec exp 24 h without metabolic activation)

METHOD OF TREATMENT/ EXPOSURE:
- Initial cell density: exp 1: 1E06 cells/mL; exp 2: 1.25E05 cells/mL
- Test substance added in medium

FOR GENE MUTATION:
- Method used: microwell plates
- Selective agent: TFT 5 µg/mL
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 9.6 E05 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells (for positive controls 9.6 E05 cells per concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells)
- Expression time (cells in growth medium between treatment and selection):
cells were cultured for 2 days; thereafter plates for CEday2 and MF determinations were incubated for 11 or 12 days in presence of TFT.
- Selection time:
1.5-2 hours, stained by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
- Criteria for small (slow growing) and large (fast growing) colonies:
The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Methods: cloning efficiency; relative suspension growth (RSG)
- Scoring: visually (naked eye)

Evaluation criteria:

A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 (GEF) in a dose-dependent manner. An observed increase should be biologically relevant and will be 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 (GEF).

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

Positive controls validity:

valid

Additional information on results:

see detailed results in attached tables

Conclusions:

The substance is not mutagenic in the mouse lymphoma L5178Y test system both with and witout metabolic activation.

Executive summary:

The substance was tested for its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix) according to the procedures as described in OECD 490.

In the first experiment, the test item was tested up to concentrations of 35 µg/mL in the absence and presence S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix.

The test item precipitated in the culture medium at this dose level.

In the second experiment, the test item was tested up to concentrations of 8 µg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level. The test item precipitated in the culture medium at this dose level.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

In the absence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency.

In conclusion, the substance is not mutagenic in the mouse lymphoma L5178Y test system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 October 2002 - 14 January 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to internationally accepted guidelines and under GLP. No CoA attached to study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2A. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Adopted at Step 4 of the ICH process 19 July 1995.

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2B. Genotoxicity: A Standard Battery for Genotoxicity Testing for Pharmaceuticals. Adopted at Step 4 of the ICH process 16 July 1997.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name: Aquapel® 364
Source: Hercules Int. Ltd
Colour: White
Physical state: Waxy solid
Batch reference: 3LP1456
CTL test substance reference number: Y09622/002
Purity: 90.1% w/w
Storage conditions: At a temperature between 4°C and 25°C
Stability: Expiry date December 2004

No CoA attached to study report.

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Experiment 1: 5000, 2500, 1000, 500, 200, 100, 50, 10 µg/ml
Experiment 2: 2500, 1000, 500, 200, 100, 50, 10 µg/ml

For metaphase analysis 1000, 500, 200 µg/ml was selected based on observed precipitation at higher concentrations. Little or no reduction in mitotic activity was observed in either experiment at concentrations selected for chromosomal aberration analysis.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not given, standard solvent

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

-S9mix and +S9-mix DMSO: 10 µl/ml

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9-mix mitomycin C: 0.5 µl/ml. +S9-mix cyclophosphamide: 50 µg/ml.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours or 20 hours
- Expression time (cells in growth medium): total 68 hours
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): filtered Giemsa stain (10% Gurr's R66 in buffered [pH 6.8] double deionised water)

NUMBER OF REPLICATIONS: Duplicate human peripheral blood cultures were exposed.

NUMBER OF CELLS EVALUATED: the mitotic index was determined by examining 1000 lymphocytes per culture and calculating the percentage of cells in metaphase. 100 cells in metaphase, where possible, were analysed from each selected culture for the incidence of structural chromosomal
damage.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Other: The effect of Aquapel® 364 on the pH and osmolality of the culture medium was investigated, using single cultures containing medium only.

OTHER: none

Evaluation criteria:

The data have been interpreted as follows:
a) No statistically significant increase in the percentage of aberrant cells (at any concentration) above concurrent solvent control values - NEGATIVE.
b) A statistically significant increase in the percentage of aberrant cells above concurrent solvent control values, which falls within the laboratory solvent control range -NEGATIVE.
c) An increase in the percentage of aberrant cells, at least at one concentration, which is substantially greater than the laboratory historical solvent control values - POSITIVE.
d) A statistically significant increase in the percentage of aberrant cells which is above concurrent solvent values and which is above the historical solvent control range upper value but below that described in (c) may require further evaluation.

Statistics:

The Fisher Exact Probability Test (one-sided) was used to evaluate statistically the percentage of metaphases showing aberrations (excluding cells with only gap-type aberrations). Data from each treatment group, in the presence and absence of S9-mix, was compared with the respective solvent control group value.

Species / strain:

lymphocytes: human

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 effects up to 5000 µg/ml
- Effects of osmolality: no effects up to 5000 µg/ml
- Evaporation from medium: not determined
- Water solubility: not determined
- Precipitation: from 1000µg/ml precipitate was observed
- Other confounding effects: not determined

RANGE-FINDING/SCREENING STUDIES: mitotic index experiments see tables below.

COMPARISON WITH HISTORICAL CONTROL DATA: solvent and positive control values fell within historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Mitotic index:

-S9-mix Experiment 1			-S9-mix Experiment 2
Treatment	Mitotic index %	Mean % mitotic index	Treatment	Mitotic index %	Mean % mitotic index
Solvent control (10 µl/ml)	14.8	16.8	Solvent control (10 µl/ml)	10.0	10.4
	18.7			10.4
Aquapel^®  364			Aquapel^®  364
5000	a b		2500	a b
	a b			a b
2500	a c		1000	9.8	10.4
	a c			11.0
1000	21.6a	17.7	500	10.8	10.0
	13.8a			9.2
500	16.5	15.6	200	d
	14.6			d
200	d		100	10.8	10.4
	d			10.0
100	20.8	18.7	50	d
	16.5			d
50	d		10	d
	d			d
10	d
	d

+S9-mix Experiment 1			+S9-mix Experiment 2
Treatment	Mitotic index %	Mean % mitotic index	Treatment	Mitotic index %	Mean % mitotic index
Solvent control (10 µl/ml)	17.2	17.4	Solvent control (10 µl/ml)	12.4	11.6
	17.5			10.8
Aquapel^®  364			Aquapel^®  364
5000	a b		2500	a c
	a b			a c
2500	a		1000	10.4	10.2
	a			10.0
1000	12.8a	13.5	500	11.2	12.0
	14.1a			12.8
500	17.2	16.9	200	d
	16.6			d
200	d		100	9.4	10.5
	d			11.5
100	15.1	16.8	50	d
	18.4			d
50	d		10	d
	d			d
10	d
	d

a – precipitate visible on slides

b - dark cytoplasms and tight metaphases

c - Precipitate partially obscuring the metaphases

d - Mitotic index not required for selection of concentrations for analysis

Mean chromosomal aberrations

-S9-mix treatment	Mean % aberrant cells excluding gaps	Mean % mitotic index
Experiment 1
Solvent control 10µl/ml	0.5	16.8
Mitomycin C 0.5µg/ml	68.00**	6.6*
Aquapel^® 364
1000 µg/ml	0.00	17.7
500 µg/ml	2.00	15.6
100 µg/ml	1.00	18.7
Experiment 2
Solvent control 10µl/ml	1.00	10.4
Mitomycin C 0.2 µg/ml	26.00**	7.4*
Aquapel^® 364
1000 µg/ml	1.50	10.4
500 µg/ml	0.50	10.0
100 µg/ml	0.50	10.4

+S9-mix treatment	Mean % aberrant cells excluding gaps	Mean % mitotic index
Experiment 1
Solvent control 10µl/ml	0.00	17.4
Cyclophosphamide 50µg/ml	48.00**	7.8*
Aquapel^® 364
1000 µg/ml	0.50	13.5
500 µg/ml	1.00	16.9
100 µg/ml	1.50	16.8
Experiment 2
Solvent control 10µl/ml	1.50	11.6
Cyclophosphamide 50µg/ml	40.00**	6.0*
Aquapel^® 364
1000 µg/ml	0.00	10.2
500 µg/ml	0.00	12.0
100 µg/ml	2.50	10.5

** Statistically significant increase in the percentage of aberrant cells at p<0.01 using Fisher's Exact Test (one-sided).

* Positive control mitotic index and % aberrant cells are determined from a single culture.

Conclusions:

Interpretation of results: negative

It is concluded that, under the conditions of this assay, Aquapel® 364 is not clastogenic to cultured human lymphocytes treated in vitro in either the presence or absence of S9-mix.

Executive summary:

The study wasperformed according to internationally accepted guidelines and under GLP. Aquapel^® 364 was evaluated for its clastogenic potential in an in vitro cytogenetic assay using human lymphocytes in two separate experiments treated in the presence and absence of a rat liver-derived metabolic activation system (S9-mix). In Experiment 1 cultures were treated for a period of 3 hours both in the presence and absence of S9-mix. In Experiment 2 cultures were treated for a period of 3 hours in the presence of S9 -mix and 20 hours in the absence of S9-mix. Cultures were harvested 68 hours after culture initiation. Cultures treated with Aquapel^® 364 at the following concentrations were selected for chromosomal aberration analysis along with the appropriate solvent and positive control cultures: 1000, 500, 100 µg/ml.

The highest concentration selected for chromosomal aberration analysis was limited by the solubility of the test substance in the culture medium. Concentrations above 1000 µg/ml were considered not to be suitable for analysis due to precipitated test substance present on the slides obscuring the metaphases or too few metaphases for analysis. No statistically or biologically significant increases in the percentage of aberrant cells, compared to the solvent control values, were recorded in cultures from either experiment treated in either the presence or absence of S9-mix. The sensitivity of the test system, and the metabolic activity of the S9-mix employed, were clearly demonstrated by the increases in the percentage of aberrant cells induced by the positive control agents, mitomycin C and cyclophosphamide.

It is concluded that, under the conditions of this assay, Aquapel^® 364 is not clastogenic to cultured human lymphocytes treated in vitro in either the presence or absence of S9-mix.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 August 2019 to 05 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium His+, Escherichia Coli Trp+

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.

Test concentrations with justification for top dose:

Test 1: TA 100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate; TA1535, TA1537 and TA 98: 1.7, 5.4, 17, 52, 164 and 512 µg/plate
Test 2: TA 100, TA1535, TA1537, TA 98 and WP2uvrA : 1.7, 5.4, 17, 52, 164 and 512 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: hexane
- Justification for choice of solvent/vehicle: solubility based on visual assessment. The substance proved to be insoluble in Milli-Q water, DMSO, ethanol and acetone, but formed a clear light yellow solution in hexane.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

hexane

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments
2: Test 1 plate incorporation; Test 2 pre-incubation

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
1E09 cells/mL (0.1 mL,plate)
- Test substance added in medium; Test 1 in agar (plate incorporation); Test 2 preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period (Test 2):
30 min by 70 rpm at 37 ± 1°C
- Exposure duration/duration of treatment:
48 ± 4 h at 35.8 - 38.1°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition, reduction of the number of revertant colonies

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- automatically with a Sorcerer Colony Counter

Evaluation criteria:

A substance is considered negative (not mutagenic) in the test if:
a)The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b)The negative response should be reproducible in at least one follow up experiment.
A substance is considered positive (mutagenic) in the test if:
a)The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b)In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

NA

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Test 1: precipitate at 512 µg/plate and above; Test 2 precipitate at 52 µg/plate

Vehicle controls validity:

valid

Remarks:

within historical control ranges

Positive controls validity:

valid

Remarks:

within historical control ranges

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Test 1 and 2 precipitate at 164 µg/plate

Vehicle controls validity:

valid

Remarks:

within historical control ranges

Positive controls validity:

valid

Remarks:

within historical control ranges

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Test 1: precipitate at 512 µg/plate; Test 2: precipitate at 52 µg/plate

Vehicle controls validity:

valid

Remarks:

within historical control ranges

Positive controls validity:

valid

Remarks:

within historical control ranges

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Test 1 precipitate at 164 µg/plate; Test 2: precipitate at 52 µg/plate

Vehicle controls validity:

valid

Remarks:

within historical control ranges

Positive controls validity:

valid

Remarks:

within historical control ranges

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Test 1: precipitate at 164 µg/plate; Test 2: precipitate at 52 µg/plate

Vehicle controls validity:

valid

Remarks:

within historical control ranges

Positive controls validity:

valid

Remarks:

within historical control ranges

Additional information on results:

Cytotoxicity:
In Test 1 in TA1537 without metabolic activation a decrease of number of revertants slightly below historical control values was seen at the highest tested concentrations
In Test 2 in individual replicates fluctuations in the number of revertant colonies below the laboratory historical control data range were observed in TA98 and TA 1537 without metabolic acyivation and WP2uvrA with metabolic activation

Plate incubation Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia colistrain.
	TA100	WP2uvrA	TA1535	TA1537	TA98

Without S9-mix

 

Positive control	944	±	26		1593	±	136		886	±	47		864	±	56		794	±	31
Hexane	111	±	9		19	±	1		8	±	2		6	±	3		15	±	7
1.7	114	±	7		25	±	2		3	±	1		4	±	1		7	±	4
5.4	109	±	10		21	±	5		4	±	1		2	±	1		5	±	3
17	110	±	17		24	±	6		6	±	3		3	±	2		7	±	4
52	111	±	13	^NP	32	±	2	^NP	5	±	1		4	±	2	^NP	6	±	2
164	105	±	15	^SP	20	±	6	^SP	4	±	3	^NP	2	±	2	^SP	8	±	2	^NP
512	101	±	10	^MP	23	±	2	^SP	6	±	1	^n MP	2	±	2	^n MP	6	±	4	^n MP
1600	97	±	13	^MP	18	±	3	^MP
5000	84	±	4	^n MP	18	±	7	^n MP

With S9-mix¹

 

Positive control	1538	±	88		355	±	26		282	±	19		370	±	40		1160	±	74
Hexane	89	±	7		21	±	5		8	±	5		4	±	4		13	±	2
1.7	87	±	1		17	±	3		4	±	2		2	±	3		6	±	2
5.4	84	±	8		27	±	9		5	±	2		4	±	6		8	±	7
17	99	±	20		24	±	7		7	±	2		1	±	1		8	±	2
52	97	±	20	^NP	23	±	3	^NP	8	±	3		3	±	2		5	±	1
164	101	±	9	^SP	28	±	5	^SP	6	±	2	^NP	3	±	1	^NP	8	±	2	^NP
512	99	±	15	^SP	22	±	2	^MP	9	±	3	^SP n	3	±	1	^n MP	13	±	1	^n MP
1600	80	±	16	^MP	22	±	6	^MP
5000	83	±	3	^n MP	18	±	1	^n MP

 

MP	Moderate Precipitate
NP	No precipitate
SP	Slight Precipitate
n	Normal bacterial background lawn

Pre-incubation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain.
	TA1535	TA1537	TA98	TA100	WP2uvrA

Without S9-mix

 

Positive control	926	±	107		177	±	8		1556	±	70		884	±	7		1276		±	5
Hexane	10	±	5		3	±	2		9	±	1		92	±	13			19	±	5
1.7	5	±	2		4	±	1		9	±	3		93	±	19			15	±	1
5.4	7	±	2		2	±	1		13	±	3		104	±	13			16	±	6
17	5	±	0		2	±	2		8	±	5	^NP	108	±	13			14	±	8	^NP
52	7	±	3	^NP	3	±	2	^NP	7	±	6	^SP	108	±	6	^NP		17	±	2	^SP
164	10	±	8	^SP	3	±	4	^SP	13	±	6	^SP	113	±	6	^SP		19	±	8	^SP
512	9	±	4	^n MP	3	±	3	^n MP	9	±	1	^n MP	88	±	8	^n MP		12	±	5	^n MP

With S9 -mix

 

Positive control	266	±	7		179	±	26		897	±	56		1446	±	84		575	±	36
Hexane	8	±	4		5	±	2		18	±	7		82	±	10		16	±	3
1.7	14	±	6		3	±	2		9	±	6		76	±	6		6	±	2
5.4	5	±	5		6	±	2		20	±	3		85	±	8		16	±	6
17	10	±	4	^NP	4	±	4	^NP	16	±	8	^NP	85	±	6	^NP	18	±	3	^NP
52	12	±	2	^SP	3	±	3	^SP	14	±	3	^SP	113	±	9	^SP	19	±	1	^SP
164	11	±	1	^SP	11	±	6	^SP	18	±	2	^SP	108	±	4	^SP	20	±	7	^SP
512	5	±	2	^n MP	6	±	4	^n MP	10	±	3	^n MP	70	±	16	^n MP	19	±	5	^n MP

 

MP	Moderate Precipitate
NP	No precipitate
SP	Slight Precipitate
n	Normal bacterial background lawn

  Historical controls

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 – 29	3 – 27	3 – 20	3 – 23	8 - 61	8 – 60	61 – 176	60 - 176	10 – 61	9 - 68
Mean	10	11	6	6	16	22	112	108	27	33
SD	3	3	2	3	5	7	18	21	8	9
n	3303	3265	3232	3212	3251	3326	3336	3246	3021	2993

Historical positive controls

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	-	+	-	+	+	-	+
Range	128 – 1530	73 – 1481	58 – 1422	439 – 1993	408 - 2379	93 – 1999	109 - 1968	54 – 1239	365 – 1978	250 – 2018
Mean	919	256	802	907	1308	1073	437	328	1305	910
SD	172	122	362	167	386	537	158	154	236	355
n	3215	3122	2777	3231	3179	2923	2987	3187	3202	3216

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Apr 2016 and Apr 2019. 

  

 

 

 

Conclusions:

The substance is not mutagenic in bacteria with and without metbolic activation

Executive summary:

In an Ames test the substance was tested for induction of gene mutations in S. typhimurium; TA98, TA100, TA1535, and TA1537, and E. coli strain WP₂uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). Incidental cytotoxicity was observed characterized as a reduction of the number of revertants below the historical control range. Precipitation was observed at concentrations between 52 and 512 µg/plate. In two independent tests (test 1 plate incorporation; test 2 pre-incubation) no increase of the number of revertants compared to the vehicle (hexane) controls was observed at any of the concentrations evaluated. Therefore it is concluded that the substance is not mutagenic in bacteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In an Ames test the substance was tested for induction of gene mutations in S. typhimurium; TA98, TA100, TA1535, and TA1537, and E. coli strain WP₂uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). Incidental cytotoxicity was observed characterized as a reduction of the number of revertants below the historical control range. Precipitation was observed at concentrations between 52 and 512 µg/plate. In two independent tests (test 1 plate incorporation; test 2 pre-incubation) no increase of the number of revertants compared to the vehicle (hexane) controls was observed at any of the concentrations evaluated. Therefore it is concluded that the substance is not mutagenic in bacteria.

In a chromosome aberration test with human lymphocytes, cultures treated with the substance at the following concentrations were selected for chromosomal aberration analysis along with the appropriate solvent and positive control cultures: 1000, 500, 100 µg/mL. In Experiment 1 cultures were treated for a period of 3 hours both in the presence and absence of S9-mix. In Experiment 2 cultures were treated for a period of 3 hours in the presence of S9 -mix and 20 hours in the absence of S9-mix. Cultures were harvested 68 hours after culture initiation. Concentrations above 1000 µg/ml were considered not to be suitable for analysis due to precipitated test substance present on the slides obscuring the metaphases or too few metaphases for analysis. No statistically or biologically significant increases in the percentage of aberrant cells, compared to the solvent control values, were recorded in cultures from either experiment treated in either the presence or absence of S9-mix. The sensitivity of the test system, and the metabolic activity of the S9-mix employed, were clearly demonstrated by the increases in the percentage of aberrant cells induced by the positive control agents, mitomycin C and cyclophosphamide. It is concluded that, under the conditions of this assay, the substance is not clastogenic to cultured human lymphocytes treated in vitro in either the presence or absence of S9-mix.

The substance was tested for its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix) according to the procedures as described in OECD 490. Two experiments were performed, 3 hours in presence and absence of S9 and 24 hours in absence of metabolic activation. The highest concentrations tested were based on solubility in the culture medium.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

In the absence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. 
In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency. 
In conclusion, the substance is not mutagenic in the mouse lymphoma L5178Y test system.

 

 

Justification for classification or non-classification

Based on the in vitro studies on genetic toxicity there is no indication of mutagenic effects of the substance. The substance is not classified as mutagenic according to EC Regulation 1272/2008 (CLP).