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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start Date: 24 May 2021
Experimental Completion Date: 29 June 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl 4-(dimethylamino)benzoate
EC Number:
244-289-3
EC Name:
2-ethylhexyl 4-(dimethylamino)benzoate
Cas Number:
21245-02-3
Molecular formula:
C17H27NO2
IUPAC Name:
2-ethylhexyl 4-(dimethylamino)benzoate

Method

Target gene:
Thymidine kinase locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Labcorp were stored as frozen stocks in liquid nitrogen. Full details of the supplier are documented in central records. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and placed in an incubator set to 37°C. When the cells were growing well, subcultures were established in an appropriate number of flasks.
Metabolic activation:
with and without
Metabolic activation system:
S-9 post-mitochondrial fraction from rat liver induced with ß-Naphthoflavone/Phenobarbital
Test concentrations with justification for top dose:
Range-Finder Experiment- 3 Hour Treatments in the Absence and Presence of S-9:
0, 0.7813, 1.563, 3.125, 6.25, 12.5, 25, 50, 100, 200 µg/mL.
(limited by solubility in the culture medium)

Mutation Experiment - (3 hour treatment)
In the absence of S-9: 0, 10, 20, 30, 40, 45, 50, 55, 60, 80,100 µg/mL
In the presence of S-9: 0, 20, 40, 80, 100, 120, 130, 140, 150, 175, 200 µg/mL
Vehicle / solvent:
(DMSO) diluted 100 fold in the treatment medium.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
Cytotoxicity Range-Finder Experiment
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment culture volume was 20 mL.
Following 3 hour treatment, cells were centrifuged (200 g) for 5 minutes, washed with tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 50 mL RPMI 10.
All cultures were placed in an incubator set to 37°C for 1 day, counted and where possible, diluted to 2 x 10^5 cells/mL. Cultures were incubated for a further day and counted.

Mutation Experiment
Treatment of Cell Cultures
At least 10^7 cells in a volume of 17.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.

After 3 hours in an incubator set to 37°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and finally resuspended in 50 mL RPMI 10 medium.

Cells were transferred to tissue culture flasks for growth throughout the expression period. The solubility of the test article in culture was assessed, by eye, at the beginning and end of treatment

Expression Period
Cultures were maintained in flasks for a period of 2 days during which the tk-/- mutation would be expressed. During the expression period, subculturing was performed as required with the aim of retaining an appropriate number of cells/flask.

In the Range-Finder Experiment, at the end of the expression period, toxicity was assessed by measuring suspension growth (and hence relative suspension growth (RSG)), compared to the concurrent vehicle control values. Cultures were not plated for viability assessment.

Plating for Viability
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10^4 cells/mL in readiness for plating for TFT resistance. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were placed in a humidified incubator set to 37°C, gassed with 5% v/v CO2 in air until scoreable (8 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for TFT Resistance
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10^4 cells/mL. TFT (300 µg/mL) was diluted approximately 100 fold into these suspensions to give a final concentration of 3 µg/mL. Using an eight channel pipette, 0.2 mL of each suspension was placed into each well of four 96 well microlitre plates (384 wells at 2 x 10^3 cells/well). Plates were placed in a humidified incubator set to 37°C, gassed with 5% v/v CO2 in air until scoreable (12 days). Wells containing viable clones were identified by eye using background illumination and counted.

In addition, the number of wells containing large colonies and the number containing small colonies were scored for the vehicle and positive controls.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF
2. The linear trend test was statistically significant.
The test article was considered as positive in this assay if both of the above criteria were met.
The test article was considered as negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Toxicity
In the cytotoxicity Range-Finder Experiment, nine concentrations were tested in the absence and presence of S-9 ranging from 0.7813 to 200 µg/mL (limited by solubility in the culture medium). Upon addition of the test article to the cultures, precipitate was observed at the highest four concentrations tested in the absence and presence of S-9 (25 to 200 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration in the absence of S-9 only (200 µg/mL). The highest concentrations to provide >10% RSG were 50 µg/mL in the absence of S 9 and 100 µg/mL in the presence of S-9, which gave 14% and 32% RSG, respectively (see table 1 in Any other information on results incl. tables).

No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations analysed (50, 100 and 200 µg/mL) as compared to the concurrent vehicle controls (measured data not reported).
In the Mutation Experiment, ten concentrations were tested, ranging from 10 to 100 µg/mL in the absence of S-9 and from 20 to 200 µg/mL in the presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the highest nine concentrations tested in the absence of S-9 (20 to 100 µg/mL) and at all concentrations tested in the presence of S-9 (20 to 200 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration in the presence of S-9 only (200 µg/mL). Two days after treatment, the highest four concentrations in the absence of S-9 (55 to 100 µg/mL) and the highest three concentrations in the presence of S-9 (150 to 200 µg/mL) were considered too toxic for selection to determine viability and TFT resistance. All other concentrations were selected in the absence and presence of S-9. However, the highest two concentrations selected in the absence of S-9 (45 and 50 µg/mL) and in the presence of S-9 (130 and 140 µg/mL) were later rejected from analysis due to extreme toxicity (<10% RTG). The highest concentrations analysed were 40 µg/mL in the absence of S-9 and 120 µg/mL in the presence of S-9, which gave 16% and 6% RTG, respectively.

Although 120 µg/mL in the presence of S-9 gave less than 10% RTG, the second highest concentration (100 µg/mL) gave 24% RTG and therefore both concentrations were analysed (see table 2 in Any other information incl. tables)

Mutation.
The acceptance criteria were met and the study was accepted as valid.
No increases in mutant frequency (MF), which exceeded the Global Evaluation Factor (GEF) of 126 mutants per 10^6 viable cells (compared to concurrent controls), were observed in any treated cultures and no significant linear trends were observed in either the absence or presence of S-9. The maximum concentration analysed in the presence of S-9 gave <10% RTG, however, no increases in MF were observed at this concentration and therefore it was considered to be a suitable maximum dose.

In addition, for the vehicle and positive controls, the number of wells containing small colonies and the number containing large colonies were scored. Thus the small and large colony MF could be estimated and the proportion of small mutant colonies could be calculated. For the vehicle controls, the proportion of small colony mutants in the absence and presence of S-9 ranged from 24% to 27%. Marked increases in the number of both small and large colony mutants were observed following treatment with the positive control chemicals MMS and B[a]P.

Any other information on results incl. tables

Table 1: RSG Values - Range-Finder Experiment - 3 Hour Treatments in the Absence and Presence of S-9


































































Concentration



-S-9



+S-9



µg/mL



%RSG



%RSG



0



100



100



0.7813



96



102



1.563



101



106



3.125



99



103



6.25



106



104



12.5



112



111



25 P



99



96



50 P



14



90



100 P



2



32



200 P



1 PP



4



%RSG                  Percent relative suspension growth


P                           Precipitation noted at time of treatment
PP                        Precipitation noted following treatment incubation period


 


 


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


























































































3 Hour Treatment –S-9



3 Hour Treatment +S-9



Concentration



%RTG



MF §



Concentration



%RTG



MF §



µg /mL



 



 



µg/mL



 



 



0



100



81.69



0



100



60.37



10



106



81.27



20 P



78



114.24



20 P



103



87.93



40 P



80



91.51



30 P



65



90.54



80 P



56



102.17



40 P



16



85.56



100 P



24



90.33



 



 



 



120 P



6



83.86



MMS 15



62



345.29



B[a]P 4



49



615.08



MMS 20



39



521.15



B[a]P 6



39



713.97



-S-9 Linear trend test on mutant frequency: Not Significant
+S-9 Linear trend test on mutant frequency: Not Significant (negative slope)
§                           5-TFT -resistant mutants/106 viable cells 2 days after treatment
%RTG                 Percent Relative Total Growth
P                           Precipitation noted at time of treatment

Applicant's summary and conclusion

Conclusions:
It is concluded that SpeedCure EHA did not induce mutation at the tk locus of mouse lymphoma L5178Y cells when tested in the absence and presence of a rat liver metabolic activation system (S-9). The highest concentration analysed was limited by toxicity in both the absence and presence of S-9.
Executive summary:

Summary


Introduction

SpeedCure EHA was assayed for the ability to induce mutation at the tk locus (5-trifluorothymidine [TFT] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by a β-Naphthoflavone/Phenobarbital-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).


Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges, and clear increases in mutation were induced by the positive control chemicals Methyl methane sulphonate (without S-9) and Benzo[a]pyrene (with S-9). Therefore, the study was accepted as valid.


Method

A 3 hour treatment incubation period was used for each experiment.
In the cytotoxicity Range-Finder Experiment, nine concentrations were tested in the absence and presence of S-9 ranging from 0.7813 to 200 μg/mL (limited by expected solubility in the culture medium). The highest concentrations to provide >10% relative suspension growth (RSG) were 50 μg/mL in the absence of S-9 and
100 μg/mL in the presence of S-9, which gave 14% and 32% RSG, respectively.
In the Mutation Experiment, ten concentrations were tested, ranging from 10 to
100 μg/mL, in the absence of S-9 and ranging from 20 to 200 μg/mL in the presence of S-9. Two days after treatment, the highest concentrations selected to determine viability and TFT resistance were 40 μg/mL in the absence of S-9 and 120 μg/mL in the presence of S-9, which gave 16% and 6% RTG, respectively. Although 120 μg/mL in the presence of S-9 gave less than 10% RTG, the second highest concentration (100 μg/mL) gave 24% RTG and therefore both concentrations were analysed.
Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges, and clear increases in mutation were induced by the positive control chemicals Methyl methane sulphonate (without S-9) and Benzo[a]pyrene (with S-9). Therefore, the study was accepted as valid.


Results

No increases in mutant frequency (MF), which exceeded the Global Evaluation Factor (GEF) of 126 mutants per 106 viable cells (compared to concurrent controls), were observed in any treated cultures and no significant linear trends were observed in either the absence or presence of S-9. The maximum concentration analysed in the presence of S-9 gave <10% RTG, however, no increases in MF were observed at this concentration and therefore it was considered to be a suitable maximum concentration.


Conclusion

It is concluded that SpeedCure EHA did not induce mutation at the tk locus of mouse lymphoma L5178Y cells when tested in the absence and presence of a rat liver metabolic activation system (S-9). The highest concentration analysed was limited by toxicity in both the absence and presence of S-9.