[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test, performed according to OECD Guideline 471 and in accordance with GLP principles, the test substance was found to be not mutagenic with or without metabolic activation.

In addition, in a Mouse Lymphoma Assay, performed according to OECD Guideline 490 and in accordance with GLP principles, the test substance the test substance was found to be not mutagenic with or without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 August 2020 - 7 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Physical Description: Brown liquid
Storage Conditions: At room temperature

Target gene:

Histidine locus and tryptophan locus

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

Histidine mutations: TA1537 hisC3076; TA98 hisD3052/R-factor; TA1535 hisG46; TA100 hisG46/R-factor

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH, Giessen, Germany
- method of preparation of S9 mix: rat liver microsomal enzymes (S9 homogenate) 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 or 5.0 mL Milli-Q
water (first or second experiment respectively); 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.
- concentration of S9 mix: 5% (v/v) S9-fraction (first experiment); 10% (v/v) S9-fraction (second experiment)

Test concentrations with justification for top dose:

Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
First mutation experiment: 17, 52, 164, 512, 1600 and 5000 μg/plate
Second mutation experiment: 10, 50, 100, 500, 1000, and 5000 μg/plate

Vehicle / solvent:

- Vehicle used: DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9-mix

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 ± 4 h at 37.0 ± 1.0 °C
- Harvest time after the end of treatment: Not reported

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

Evaluation criteria:

No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

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

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up 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.

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Absence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Absence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Absence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Second mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Absence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

First mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

First mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

First mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With and without S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

First mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With and without S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

First experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With and without S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose-range Finding test/First Mutation Experiment
- Precipitation of the test item on the plates was observed in tester strains TA1535, TA1537 and TA98 in the absence of S9-mix.
- Cytotoxicity, as evidenced by a decrease in the number of revertants and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strains WP2uvrA and TA1537 (presence of S9-mix).
- No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Second mutation experiment
- In tester strain TA1537, the test item precipitated on the plates at the highest tested dose level in the presence of S9-mix.
- In the second mutation assay, cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in the absence of S9-mix in tester strains TA1535, TA98, TA100 and WP2uvrA, and in the presence of S9-mix in tester strains TA1537 and TA100.
- In the second mutation assay, no increase in the number of revertants was observed upon treatment with X-21415 under all conditions tested.

Table 1 Dose-Range Finding Test: Mutagenic Response of X-21415 in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
	TA100	WP2uvrA

 

Without S9-mix

 

Positive control	1668	±	62		428	±	5
Solvent control	97	±	6		21	±	3
1.7	109	±	15		23	±	3
5.4	105	±	20		19	±	1
17	98	±	9		24	±	4
52	100	±	6		20	±	5
164	125	±	14		15	±	1
512	117	±	19		16	±	10
1600	71	±	5		9	±	6
5000	6	±	4	^n NP	11	±	5	^n NP

 

With S9-mix¹

 

Positive control	830	±	24		1192	±	127
Solvent control	85	±	19		16	±	1
1.7	94	±	6		15	±	5
5.4	96	±	22		14	±	4
17	93	±	6		12	±	3
52	100	±	14		14	±	9
164	51	±	1	^n	13	±	2
512	12	±	5	^s	15	±	7
1600				^e MC	13	±	2
5000				^e NP  MC	15	±	1	^n NP

 

^1	Plate incorporation assay (5% S9)
MC	Microcolonies
NP	No precipitate
e	Bacterial background lawn extremely reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

 

Table 2. Experiment 1: Mutagenic Response of X-21415 in the Salmonella typhimurium Reverse Mutation Assay 

 

 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium.
	TA1535	TA1537	TA98

 

Without S9-mix

 

Positive control	767	±	5		1009	±	101		1290	±	138
Solvent control	10	±	2		3	±	2		15	±	5
17	7	±	3		5	±	2		16	±	7
52	8	±	3		2	±	2		16	±	3
164	7	±	3		0	±	1		13	±	7
512	3	±	1		1	±	0		11	±	3
1600	0	±	1	^NP	1	±	1	^NP	4	±	3	^NP
5000	0	±	1	^n SP	1	±	1	^n SP	0	±	0	^n SP

 

With S9-mix¹

 

Positive control	320	±	24		251	±	14		1035	±	45
Solvent control	6	±	3		5	±	2		18	±	5
17	8	±	1		3	±	2		21	±	4
52	7	±	5		4	±	1		19	±	3
164	10	±	2		6	±	3		15	±	1
512	7	±	3		5	±	5		23	±	1
1000	6	±	5		2	±	2		16	±	7
5000	2	±	2	^n NP	11	±	1	^n NP	2	±	2	^n NP

 

^1	Plate incorporation assay (5% S9)
NP	No precipitate
SP	Slight Precipitate
n	Normal bacterial background lawn

 

Table 3. Experiment 2: Mutagenic Response of X-21415 in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

 

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

 

Without S9-mix

 

Positive control	913	±	42		956	±	80		1485	±	83		609	±	15		925	±	50
Solvent control	5	±	3		4	±	3		7	±	0		85	±	8		17	±	2
10	10	±	4		3	±	2		6	±	3		67	±	10		14	±	5
50	5	±	5		3	±	2		10	±	6		71	±	17		14	±	2
100	7	±	3		4	±	1		6	±	5		68	±	9		18	±	3
500	2	±	1		4	±	5		7	±	4		9	±	9		12	±	2
1000	3	±	2		3	±	4		4	±	1	^n	2	±	1	^n	16	±	7
5000	0	±	0	^n NP	4	±	3	^n NP				^e NP  MC				^e NP  MC	9	±	2	^n NP

 

 

With S9-mix¹

 

Positive control	246	±	12		254	±	46		666	±	105		1327	±	255		195	±	27
Solvent control	8	±	2		4	±	1		17	±	6		73	±	6		20	±	3
10	10	±	3		4	±	1		14	±	6		62	±	3		23	±	8
50	8	±	2		3	±	2		18	±	2		78	±	2		20	±	3
100	8	±	6		2	±	1		13	±	3		80	±	19		25	±	12
500	9	±	2		4	±	2		13	±	3		79	±	5		15	±	1
1000	8	±	3		2	±	2	^NP	19	±	5		81	±	15		19	±	6
5000	5	±	2	^n NP	0	±	0	^n SP	9	±	6	^n NP	45	±	7	^n NP	19	±	4	^n NP

 

 

^1	Plate incorporation assay (10% S9)
MC	Microcolonies
NP	No precipitate
SP	Slight Precipitate
e	Bacterial background lawn extremely reduced
n	Normal bacterial background lawn

Conclusions:

In an Ames test, performed according to OECD guideline 471 and in accordance with GLP principles, X-21415 was found to be not mutagenic with or without metabolic activation.

Executive summary:

An Ames test was performed according to OECD guideline 471 and in accordance with GLP principles. The test item did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate, and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that X-21415 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation. Since all acceptibility criteria were met, the study was considered valid.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 9th November 2021 Experimental completion date: 13th December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

Identification: X-21415

Chemical Name: Benzenesulfonic acid, C14-24-branched and linear alkyl derivs

CAS Number: 115829-36-2

Batch Number: 0921SA107S

Purity: UVCB* – 100% purity

Molecular Weight: N/A (UVCB)

Expiry Date: 1st September 2022

Appearance: Brown liquid

Storage Conditions: Room temperature in the dark

Target gene:

thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Source: cell line:
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time. Master stocks of cells were tested and found to be free of mycoplasma.

Cell culture:
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), were used during the course of the study. All donor horse serum was purchased heat inactivated from the supplier.

Metabolic activation:

with and without

Metabolic activation system:

Microsomal Enzyme Fraction
Rat S9 Microsomal fraction, adjusted to a final protein content of 20 mg/mL before use.
The S9 mix was prepared by mixing S9 with 100 mM phosphate buffer containing NADP (5 mM), G¬6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% S9-mix concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.

Test concentrations with justification for top dose:

Preliminary Toxicity Test:
The dose range used in the preliminary toxicity test was 19.53 to 5000 µg/mL. The maximum concentration was the maximum recommended concentration.

The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.

Mutagenicity Test:
The dose range of test item used in the main test was selected following the results of a preliminary toxicity test.

4-hour without S9: 0, 5, 10, 20, 40, 50, 60, 70, 80 Concentration of X-21415 (µg/mL)
4-hour with S9: 0, 5, 10, 20, 40, 50, 60, 70, 80 Concentration of X-21415 (µg/mL)
24-hour without S9: 0, 2.5, 5, 10, 20, 25, 30, 35, 40 Concentration of X-21415 (µg/mL)

Due to optimum levels of toxicity not being achieved in any of the three exposure groups, this experiment was abandoned prior to plating and repeated using a revised concentration range

Mutagenicity Test Repeat I:
4-hour without S9: 0, 10, 20, 40, 50, 55, 60, 65, 70, 75, 80 Concentration of X-21415 (µg/mL)
4-hour with S9: 0, 10, 20, 40, 50, 55, 60, 65, 70, 75, 80 Concentration of X-21415 (µg/mL)
24-hour without S9: 0, 5, 10, 20, 30, 35, 40, 45, 50 Concentration of X-21415 (µg/mL)

Due to optimum levels of toxicity not being achieved in the 24-hour exposure group, this exposure group was abandoned prior to plating and repeated using a revised concentration range.

Mutagenicity Test Repeat II:
24-hour with S9: 0, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 Concentration of X-21415 (µg/mL)

Vehicle / solvent:

Identity: Dimethyl sulfoxide (DMSO)

Supplier: Fisher

Batch number: 206994

Purity: 99.9%

Expiry date: 1st November 2025

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Absence of S9-mix 24 hours exposure

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Presence of S9-mix 4 hours exposure

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Absence of S9-mix 4 hours exposure

Details on test system and experimental conditions:

Cell Cleansing:
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen, they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24-hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24-hours the cells were cultured in THG medium (i.e., THMG without Methotrexate) before being returned to R10 medium.

Test Item Preparation:
The test item was a UVCB (Substances of Unknown or Variable composition, Complex reaction products or Biological materials) and, therefore, no correction for the purity of the test item was applied to the formulations. Consequently, the maximum concentration was initially set at 5000 µg/mL, the maximum recommended concentration. Following solubility checks performed in-house on the same test item (Labcorp Study No. 8483321), the test item was accurately weighed and formulated in dimethyl sulfoxide (DMSO) prior to serial dilutions being prepared.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.
The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration, or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test Procedure:
Preliminary Toxicity Test:
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 19.53 to 5000 µg/mL. The maximum concentration was the maximum recommended concentration.
Following the exposure periods, the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL.
The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL. After a further 24 hours, the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent solvent control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item concentrations for the mutagenicity experiments. Maximum concentrations were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB*) the upper concentration may need to be higher, and the maximum concentration will be 5 mg/mL.
ii) Precipitating concentrations will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 %. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA .

Mutagenicity Test:
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) using eight concentrations of the test item and solvent and positive controls. To each universal was added 2 mL of S9 mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24-hour exposure group).
Three exposure groups were used for the Mutagenicity Test:
i) 4-hour exposure to the test item without S9-mix. The concentrations of test item used were 5 to 80 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%). The concentrations of test item used were 5 to 80 µg/mL.
iii) 24-hour exposure to the test item without S9-mix. The concentrations of test item used were 2.5 to 40 µg/mL.
The exposure vessels were incubated for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Mutagenicity Test Repeat I:
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) using up to ten concentrations of the test item and solvent and positive controls. To each universal was added 2 mL of S9 mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24-hour exposure group).
Three exposure groups were used for the Mutagenicity Test Repeat I:
i) 4-hour exposure to the test item without S9-mix. The concentrations of test item used were 10 to 80 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%). The concentrations of test item used were 10 to 80 µg/mL.
iii) 24-hour exposure to the test item without S9-mix. The concentrations of test item used were 5 to 50 µg/mL.
The exposure vessels were incubated for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Mutagenicity Repeat II:
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 0.3 x 106 cells/mL in 10 mL cultures in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), using ten concentrations of the test item and solvent and positive controls. To each universal was added 0.2 mL of the exposure dilutions, ,0.2 mL of the positive control, and sufficient R10 medium to bring the total volume to 20 mL. The concentrations of test item used were 20 to 65 µg/mL.

Assessments:
Measurement of Survival, Cloning Efficiency and Mutant Frequency:
At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/mL. The cultures were incubated at 37±2 °C with 5% CO2 in air and sub-cultured daily for the expression period of two days, by counting and dilution to 2 x 105 cells/mL.
On day 2 of the experiment, the cells were counted, diluted to 1x104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for cloning efficiency (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the solvent control, and when combined with the cloning efficiency (%V) data, a Relative Total Growth (RTG) value.

Plate Scoring:
Microtitre plates were scored using a magnifying mirror box after eleven to thirteen days incubation at 37±2 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutant plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item. Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutant plates. The plates were incubated for approximately two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

Calculation of Percentage Relative Suspension Growth (%RSG):
The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48-hours cell count/2)
Day 0 Factor = dose 0-hour cell count/solvent control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/solvent control SG] x 100

Rationale for test conditions:

Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual concentration. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Concentrations that have RTG survival values markedly less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e., increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, i.e., the mutant frequency of the concurrent solvent control plus 126, which is based on the analysis of the distribution of the solvent control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is

Evaluation criteria:

Acceptability of the Assay:
This mutation assay is considered acceptable as it meets the following acceptability criteria, the current recommendations of the IWGT will be considered. Please go to section: Any other information on materials and methods incl. tables.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and 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 examined

True negative controls validity:

not applicable

Positive controls validity:

valid

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 19.53 to 5000 µg/mL.  The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (mg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
19.53	90	74	94
39.06	34	46	6
78.13	5	7	0
156.25	1	0 P	-
312.5	-	1 P	-
625	-	- P	-
1250	-	- P	-
2500	- P	- P	-
5000	- P	- P	- P

P = A precipitate of the Test Item observed at the end of the exposure period
- = No data generated

treated with the test item when compared to the concurrent solvent controls in all three of the exposure groups.  The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult.

At the end of the exposure periods a precipitate of the test item was observed at and above 2500 µg/mL in the 4-hour exposure group in the absence of metabolic activation, at and above 156.25 µg/mL in the 4-hour exposure group in the presence of metabolic activation, and at 5000 µg/mL in the 24-hour exposure group.  In the subsequent mutagenicity test, the maximum concentration was limited by test item-induced toxicity as recommended by the OECD 490 guideline.

Mutagenicity Test – Main Experiments

Mutagenicity Test

No reportable data was generated.

Mutagenicity Test Repeat I

No reportable data was generated from the 24-hour exposure group

Summary of Results

Concentration (µg/mL)	4-hours-S9 Repeat I			Concentration (µg/mL)	4-hours+S9 Repeat I
	%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1.00	107.51	0	100	1.00	107.60
10	104	-	-	10	91	-	-
20	101	-	-	20	87	-	-
40	95	0.80	224.33	40	96	-	-
50	99	0.97	146.22	50	86	-	-
55	48	0.34	195.97	55	67	0.59	146.51
60	31	0.26	229.82	60	63	0.67	119.02
65	21	0.17	219.84	65	59	0.54	115.75
70	X	X	X	70	40	0.40	137.65
75	X	-	-	75	31	0.30	125.12
80	-	-	-	80	15	0.14	109.32
MF threshold for a positive response=			233.51	MF threshold for a positive response=			233.60
Positive control				Positive control
EMS				CP
400	73	0.48	1014.68	1.5	117	1.16	399.62
Concentration (µg/mL)	24-hours-S9 Repeat II
	%RSG	RTG	MF§
0	100	1.00	148.86
20	86	-	-
25	68	-	-
30	85	0.88	134.78
35	67	0.51	171.45
40	61	0.71	121.07
45	40	0.39	123.52
50	37	0.36	139.22
55	28	0.29	129.27
60	11	0.10	110.75
65	5	-	-
MF threshold for a positive response=			274.86
Positive control
EMS
150	56	0.40	1144.08

		Solvent Control Mutant Frequencies per survivor x 10^-6
		Without S9	With S9
Vehicle Control 95% control limits		66.59 – 188.91	78.27 – 176.07
Positive Control 95% control limits		861.41 – 1462.99	310.76 – 1491.16

As was seen previously, there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any marked reductions in cloning efficiency (%V), therefore indicating that no residual toxicity occurred in either of the exposure groups

Based on the RTG and %RSG values, it was considered that optimum levels of toxicity were achieved in both the absence and presence of metabolic activation.  The excessive toxicity observed at and above 75 µg/mL in the absence of metabolic activation resulted in these concentrations not being plated for cloning efficiency or 5-TFT resistance.  Acceptable levels of toxicity were seen with both positive control substances.

The solvent controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus.  The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

The test item did not induce any increases in the mutant frequency at any of the concentrations in the main test that exceeded the Global Evaluation Factor (GEF). Although the concentration range used did not achieve optimum levels of toxicity in both the absence and presence of metabolic activation, optimum levels of toxicity were achieved in the presence of metabolic activation, and there were at least four analysable concentrations in each exposure group, as recommended by the OECD 490 guideline.  The results observed in both of the 4‑hour exposure groups were considered to fulfil the criteria for a clearly negative outcome.

 

Conclusions:

The test item, X-21415, did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non mutagenic in this assay

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.  The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016.

Methods

Initially one main Mutagenicity Test was performed.  In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight concentrations in duplicate, together with solvent (dimethyl sulfoxide (DMSO)), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test.  The concentrations selected for the Main Experiment for the 4-hour exposures and 24-hour exposure were as follows:

Mutagenicity Test

Group	Concentration of X-21415 (µg/mL)
4-hour without S9	0, 5, 10, 20, 40, 50, 60, 70, 80
4-hour with S9	0, 5, 10, 20, 40, 50, 60, 70, 80
24-hour without S9	0, 2.5, 5, 10, 20, 25, 30, 35, 40

Due to optimum levels of toxicity not being achieved in any of the three exposure groups, this experiment was abandoned prior to plating and repeated using a revised concentration range.

Mutagenicity Test Repeat I

Group	Concentration of X-21415 (µg/mL)
4-hour without S9	0, 10, 20, 40, 50, 55, 60, 65, 70, 75, 80
4-hour with S9	0, 10, 20, 40, 50, 55, 60, 65, 70, 75, 80
24-hour without S9	0, 5, 10, 20, 30, 35, 40, 45, 50

Due to optimum levels of toxicity not being achieved in the 24-hour exposure group, this exposure group was abandoned prior to plating and repeated using a revised concentration range.

Mutagenicity Test Repeat II

Group	Concentration of X-21415 (µg/mL)
24-hour with S9	0, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65

Results

Mutagenicity Test

No reportable data was generated.

Mutagenicity Test Repeat I

No reportable data was generated for the 24-hour exposure group.

The maximum concentration used in the 4-hour exposure groups was limited by test item‑induced toxicity.  No precipitate of test item was observed at any of the concentrations at the end of the exposure period.

The solvent control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus.  The positive controls produced marked increases in the mutant frequency per viable cell, achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

The test item did not induce any increases in the mutant frequency at any of the concentrations in the main test that exceeded the Global Evaluation Factor (GEF), using a concentration range that achieved optimum levels of toxicity in both the absence and presence of metabolic activation, and at least four analysable concentrations in each exposure group, as recommended by the OECD 490 guideline.  The results observed in both of the 4‑hour exposure groups were considered to fulfil the criteria for a clearly negative outcome.

Mutagenicity Test Repeat II

The maximum concentration used in the 24-hour exposure group was limited by test item‑induced toxicity.  No precipitate of test item was observed at any of the concentrations at the end of the exposure period.

The solvent control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus.  The positive control produced marked increases in the mutant frequency per viable cell, achieving the acceptability criterion recommended by the OECD guideline, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

The test item did not induce any increases in the mutant frequency at any of the concentrations in the main test that exceeded the Global Evaluation Factor (GEF), using a concentration range that achieved optimum levels of toxicity, and at least four analysable concentrations, as recommended by the OECD 490 guideline.  The results observed in the 24‑hour exposure group were considered to fulfil the criteria for a clearly negative outcome

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Due to the lack of observed mutagenic potential, classification for germ cell mutagenicity is not required under Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures.