Reaction mass of C12-14 tert-alkylamines and dimethyl hydrogen phosphate and methyl dihydrogen phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse Mutation Assay

Key study

In a study performed to the standardized guideline OECD 471, under GLP conditions, the test substance was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system (Bio Reliance, 2018).

Micronucleus Assay

Key study

In a study performed to the standardized guideline OECD 487, under GLP conditions, the test substance was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system (Bio Reliance, 2018).

Mammalian cell gene mutation test

Under the conditions of the assay described in this report, C12-14 tert-alkylamines, compounds with phosphoric acid, mono and dimethyl esters was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

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:

14 June 2017 to ****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Purity: >98% (nominal); This substance has a Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
- Description: Clear yellow highly viscous liquid

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity Assay: Conducted at dose levels of 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate in DMSO.
No precipitate was observed. Toxicity was observed beginning at 667, 1000 or 3333 µg per plate.

Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate for TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation, 5000 µg per plate for TA100 andTA1535 in the presence of S9 activation and 1500 µg per plate in the absence of S9 activation.

Mutagenicity Assay: Conducted at dose levels of 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate for TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation; 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate for TA100 and TA1535 in the presence of S9 activation; and 5.00, 15.0, 50.0, 150, 500 and 1500 µg per plate in the absence of S9 activation.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Preparation of Tester Strain
Overnight cultures were prepared from the appropriate frozen permanent stock. Following inoculation, each flask was placed in a shaker/incubator and incubated at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Exogenous Metabolic Activation
The S9 metabolic activation system was purchased commercially from MolTox (Boone, NC) and stored at 60°C or colder until use. It was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254. Each bulk preparation was assayed for its ability to metabolize benzo(a)pyrene and 2 aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.
The S9 mix was prepared on the day of use and contained: S9 (10%), sodium phosphate buffer (pH 7.4; 100 mM), MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM), and β-nicotinamide-adenine dinucleotide phosphate (4 mM). The Sham mix, containing 100 mM phosphate buffer at pH 7.4, was also prepared on the day of use.

Frequency and Route of Administration
The test system was exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983).

Preliminary Toxicity Assay to Select Dose Levels
The preliminary toxicity assay was used to establish the dose range over which the test substance would be assayed. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone and ten dose levels of the test substance, with a single plate/condition, on selective minimal agar in the presence and absence of S9 mix. Dose levels for the mutagenicity assay were based upon post-treatment toxicity.

Mutagenicity Assay
TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and at least five dose levels of test substance, in triplicate, in the presence and absence of S9 mix.
To confirm the sterility of the S9, Sham mixes, test substance and the vehicle, each was plated on selective agar with an aliquot volume equal to that used in the assay and incubated under the same conditions as the assay.
One half (0.5) milliliter of S9 or Sham mix, 100 µL of tester strain (cells seeded) and 50.0 µL of vehicle, positive control, or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C, vortexed, and overlaid onto minimal bottom agar. After the overlay solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2C. Plates that were not counted immediately following the incubation period were stored at 2 8C until colony counting could be conducted.

Scoring
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate. Colonies were enumerated either by hand or by machine.

Tester Strain Verification
On the day of use in each assay, all tester strain cultures were checked for the appropriate genetic markers.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Assay
The maximum dose of 5000 µg per plate was achieved using a concentration of 100 mg/mL and a 50.0 µL plating aliquot. No precipitate was observed. Toxicity was observed beginning at 667, 1000 or 3333 µg per plate.

Mutagenicity Assay
No precipitate was observed. Toxicity was observed beginning at 500, 1500 or at 5000 µg per plate.
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Conclusions:

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test material did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Executive summary:

In a study performed to the standardized guideline OECD 471, under GLP conditions, the test substance was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

 

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. No precipitate was observed. Toxicity was observed beginning at 667, 1000 or 3333 µg per plate. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate for TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation, 5000 µg per plate for TA100 andTA1535 in the presence of S9 activation and 1500 µg per plate in the absence of S9 activation.

 

In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate for TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate for TA100 and TA1535 in the presence of S9 activation and 5.00, 15.0, 50.0, 150, 500 and 1500 µg per plate in the absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 500, 1500 or at 5000 µg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

 

These results indicate that the test substance was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May 2017 to *****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Specific details on test material used for the study:

- Purity: >98% (nominal); This substance has a Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
- Description: Clear yellow highly viscous liquid

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Peripheral blood lymphocytes were obtained from a healthy non-smoking, 26 year old female and were cultured in complete medium (RPMI 1640 containing 15% fetal bovine serum, 2 mM L glutamine, 100 units penicillin, 100 µg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium with 2% phytohemagglutinin. The cultures were then incubated at 37 ± 1 °C in a humidified atmosphere of 5 ± 1% CO2 in air for 44 to 48 hours.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Micronucleus Assay
Based on the results of the preliminary toxicity test, the doses selected for testing in the micronucleus assay were as follows:
- Non-activated: 10, 20, 50, 75, 100, 125, 150 µg/mL, for 4 and 20 hrs,
- Non-activated: 5, 10, 50, 55, 65, 75, 80 µg/mL, for 24 and 0 hrs, and
- S9-activated: 10, 20, 50, 75, 100, 125, 150 µg/mL, for 4 and 20 hrs.

Precipitation of the test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose evaluated for the micronuclei was selected based on the following:
- 4-hour (-S9): 55 ± 5% cytotoxicity (CBPI relative to the vehicle control);
- 4-hour (+S9): 55 ± 5% cytotoxicity (CBPI relative to the vehicle control), and
- 24-hour (-S9): 55 ± 5% cytotoxicity (CBPI relative to the vehicle control).

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Vinblastine

Details on test system and experimental conditions:

Details on mammalian cell lines
Human peripheral blood lymphocytes were obtained from a healthy non-smoking individual (26 years of age) and were cultured in complete medium (RPMI 1640 containing 15% fetal bovine serum, 2 mM L glutamine, 100 units penicillin, 100 µg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing complete medium with 2% phytohemagglutinin. The cultures were incubated at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air for 44-48 hours.

Exogenous Metabolic Activation System
The S9 metabolic activation system was purchased commercially from MolTox (Boone, NC) and stored at 60°C or colder until use. It was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254. Each bulk preparation was assayed for its ability to metabolize benzo(a)pyrene and 2 aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

The S9 mix was prepared on the day of use and final concentration of its components in serum-free RPMI-1640 medium were: S9 (20 µL/mL), MgCl2 (2 mM), KCl (6 mM), glucose-6-phosphate (1 mM), and NADP (sodium salt; 1 mM).

Preliminary Toxicity Test for Selection of Dose Levels
Lymphocytes were exposed to vehicle alone and to nine concentrations of test substance for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9 using single cultures. Precipitation of test substance in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose, lowest precipitating dose, and the highest soluble dose was measured. The pH of the highest dose in the treatment medium was measured using test tape. Dose levels for the micronucleus assay were based upon post-treatment toxicity (cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) or visible precipitate in the treatment medium at the conclusion of the treatment period. .After the 4 hour treatment in the non-activated and the S9-activated studies, the cells were centrifuged, the treatment medium was aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium containing cytochalasin B at 6.0 µg/mL and returned to the incubator under standard conditions. For the 24 hour treatment in the non-activated study, cytochalasin B (6.0 µg/mL) was added at the beginning of the treatment.

Micronucleus Assay
Lymphocytes were exposed to vehicle alone and to a minimum of five concentrations of test substance for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9 using duplicate cultures. Precipitation of test substance in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment.

Collection of Cells (Preliminary Toxicity Test and Micronucleus Assay)
Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), and the suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange.

Cell Cycle Kinetics Scoring (Preliminary Toxicity Test and Micronucleus Assay)
For the preliminary toxicity test, at least 500 cells, if possible, were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture), if possible, were evaluated to determine the CBPI at each dose level and the control. The CBPI was determined using the following formula:

CBPI = 1X Mononucleated cells + 2 x Binucleated cells + 3 x Multinucleated cells / Total number of cells scored

% Cytostasis (cytotoxicity) = 100 -100 {(CBPItest-1) /(CBPIvehicle-1)}

Micronucleus Scoring
The slides from at least three test substance concentrations were coded and a minimum of 2000 binucleated cells from each concentration (if possible, 1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei. Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:
• the micronucleus should have the same staining characteristics as the main nucleus
• the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
• the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 ug/L

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Micronucleus Assay

The test substance was soluble in the treatment medium at all doses tested at the beginning and conclusion of the treatment period. The pH of the highest dose of test substance in treatment medium was 7.0. Cytotoxicity (CBPI relative to the vehicle control) was observed as follows:

 

Treatment Condition	Treatment Time	Highest Evaluated Dose (µg/mL)	Cytotoxicity (%)
Non-activated	4 hr	125	53
	24 hr	75	51
S9-activated	4 hr	125	56

 

No significant or dose‑dependent increases in micronuclei induction were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

The results for the positive and vehicle controls indicate that all criteria for a valid assay were met. 

Conclusions:

Under the conditions of the assay described in this report the test material was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.

Executive summary:

In a study performed to the standardized guideline OECD 487, under GLP conditions, the test substance was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 µg/mL, which was the limit dose for this assay. Cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses ≥ 150 µg/mL in all three treatment conditions. At the conclusion of the treatment period, visible precipitate was observed at 5000 µg/mL in all three treatment conditions. Based upon these results, the doses chosen for the micronucleus assay ranged from 10 to 150 µg/mL for the non activated and S9-activated 4-hour exposure groups; and from 5 to 80 µg/mL for the non-activated 24 hour exposure group.

In the micronucleus assay, cytotoxicity (≥ 50% CBPI relative to the vehicle control) was observed at doses ≥ 125 µg/mL in the non activated and S9 activated 4-hour exposure groups; and at doses ≥ 75 µg/mL in the non activated 24-hour exposure group. The doses selected for evaluation of micronuclei were 50, 100, and 125 µg/mL for the non-activated and S9-activated 4-hour exposure groups; and 10, 55, and 75 µg/mL for the non-activated 24 hour exposure group.

No significant or dose dependent increases in micronuclei induction were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). The results indicate that the test substance was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.  

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 August 2017 to ****

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

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

Specific details on test material used for the study:

- Purity: >98% (nominal); this substance has a Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
- Description: Clear yellow highly viscous liquid

Target gene:

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

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/- cells, clone 3.7.2C, obtained from the American Type Culture Collection (repository number CRL-9518), Manassas, VA. Each batch of frozen cells was tested and found to be free of mycoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the concentrations tested were 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL.
The maximum concentration evaluated approximated the limit dose for this assay. No visible precipitate was observed at the beginning or end of treatment. Relative suspension growth (RSG) was 65, 13 and 57% at concentrations of 39.1 µg/mL (4-hour treatment with S9), 39.1 µg/mL (4-hour treatment without S9) and 19.5 µg/mL (24-hour treatment without S9), respectively. RSG was 0% at all higher concentrations using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 10, 15, 30, 40, 50, 60 and 80 µg/mL (4-hour treatment with S9), 1, 5, 10, 20, 30, 40 and 50 µg/mL (4-hour treatment without S9) and 1, 5, 10, 20, 30 and 40 µg/mL (24-hour treatment without S9).

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

Solubility Determination
Dimethyl sulfoxide (DMSO)was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL with sonication at 36.9ºC for 10 minutes in the solubility test conducted.

Preparation of Cells
L5178Y/TK+/- cells, clone 3.7.2C, were obtained from the American Type Culture Collection (repository number CRL-9518), Manassas, VA. Each batch of frozen cells was tested and found to be free of mycoplasma contamination. Prior to use in the assay, L5178Y/TK+/- cells were cultured for 24 hours in the presence of thymidine, hypoxanthine, methotrexate and glycine to poison the TK-/- cells. L5178Y/TK+/- cells were prepared in 50% conditioned F0P supplemented with 10% horse serum and 2 mM L-glutamine (F10P) and 50% Fischer's Media for Leukemic Cells of Mice with 0.1% Pluronics F 68 (F0P). All media contained antibiotics.

Exogenous Metabolic Activation
The S9 metabolic activation system was purchased commercially from Moltox (Boone, NC) and stored at 60°C or colder until use. It was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254. Each lot of S9 was assayed for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.
The S9 mix was prepared on the day of use and final concentrations of its components in the test system were: DL-isocitric acid (17.4 mM), NADP (sodium salt; 3.0 mM), and S9 (10 µL/mL).

Preliminary Toxicity Test for Selection of Dose Levels
L5178Y/TK+/- cells were exposed to the vehicle alone in duplicate cultures and nine concentrations of test substance using single cultures for 4 hours in the presence and absence of S9, and for 24 hours in the absence of S9. The maximum concentration evaluated was based on solubility limitations of the test substance in the vehicle; precipitation of the test substance in the treatment medium was determined with the unaided eye at the beginning and end of treatment. The pH of the treatment medium was measured and adjusted if necessary to maintain neutral pH. Osmolality in treatment medium of the vehicle control, the highest concentration, the lowest precipitating concentration and the highest soluble concentration also was measured. Dose levels for the definitive assay were based on post-treatment cytotoxicity (growth inhibition relative to the vehicle control) or solubility limitations of the test substance in the treatment medium.

For the preliminary toxicity assay only, after a 4-hour treatment in the presence and absence of S9, cells were washed with culture medium and cultured in suspension for two days post treatment, with cell concentration adjustment on the first day. After a 24 hour treatment in the absence of S9, cells were washed with culture medium and immediately readjusted to 3 x 10^5 cells/mL. Cells were then cultured in suspension for an additional two days post-treatment with cell concentration adjustment on the first day.

Mouse Lymphoma Assays
L5178Y/TK+/- cells were exposed to the vehicle alone and five to seven concentrations of test substance at appropriate dose intervals using duplicate cultures for 4 hours in the presence and absence of S9, and for 24 hours in the absence of S9. Precipitation was determined with the unaided eye at the beginning and end of treatment.

Treatment of Target Cells
The preparation and addition of the test substance to the test system was carried out under filtered lighting. A mixture containing 50 µL of test substance or vehicle control formulation or 100 µL of positive control formulation were added to treatment medium. All pH adjustments were performed prior to adding S9 or target cells. Either medium or S9 mix (as appropriate) and 6 x 106 L5178Y/TK+/- cells were added, cultures were capped tightly, and incubated with mechanical mixing at 37 ± 1°C for 4 or 24 hours.
For the definitive assay only, at the end of the exposure period, the cells were washed with culture medium, resuspended in F10P, and incubated at 37 ± 1°C for two days following treatment. Cell population adjustments to 3 x 10^5 cells/mL were made as follows:
• 4 hour treatment – 1 and 2 days after treatment.
• 24 hour treatment – immediately after test substance removal; 2 and 3 days after treatment.

Selection of Mutant Phenotype
Cells from selected dose levels were cultured in triplicate with 2-4 μg TFT/mL at a density of 1 x 10^6 cells/100 mm plate in cloning medium containing 0.22 to 0.24% agar. For estimation of cloning efficiency at the time of selection, 200 cells/100 mm plate were cultured in triplicate in cloning medium without TFT (viable cell (VC) plate). Cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 10 or 11 days. The plates were stored under refrigerated conditions (2-8°C) for 8 days prior to scoring.

The total number of colonies per culture was determined for the VC plates and the total relative growth calculated. The total number of colonies per TFT plate was then determined for those cultures with ≥10% total growth (including at least one concentration between 10 and 20% total growth, if possible). Colonies were counted and the diameter of the TFT colonies from the positive control and vehicle control cultures were determined over a range from 0.2 to 1.1 mm.

Extended Treatment and/or Confirmatory Assay
Verification of a clear positive response was not required (OECD Guideline 490). For negative results without activation, an extended treatment assay was performed in which cultures were continuously exposed to the test substance for 24 hours without S9 activation. The extended treatment assay was performed concurrently with the initial assay. For negative results with S9 activation, a confirmatory assay was not required unless the test substance was known to have specific requirements of metabolism.

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:

valid

Positive controls validity:

valid

Conclusions:

Under the conditions of the assay described in this report, the test material was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

Executive summary:

In a study performed to the standardized guideline OECD 490, under GLP conditions, the test substance was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

 

In the preliminary toxicity assay, the concentrations tested were 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL. The maximum concentration evaluated approximated the limit dose for this assay. No visible precipitate was observed at the beginning or end of treatment. Relative suspension growth (RSG) was 65, 13 and 57% at concentrations of 39.1 µg/mL (4-hour treatment with S9), 39.1 µg/mL (4-hour treatment without S9) and 19.5 µg/mL (24-hour treatment without S9), respectively. RSG was 0% at all higher concentrations using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 10, 15, 30, 40, 50, 60 and 80 µg/mL (4-hour treatment with S9), 1, 5, 10, 20, 30, 40 and 50 µg/mL (4-hour treatment without S9) and 1, 5, 10, 20, 30 and 40 µg/mL (24-hour treatment without S9).

 

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. 4-hour treatment with S9 was retested as the mean solvent mutant frequency was higher than the protocol specified acceptance criteria. Cultures treated at concentrations of 1, 5, 10, 20 and 30 µg/mL (4-hour treatment without S9) and 1, 5, 10 and 20 µg/mL (24-hour treatment without S9) exhibited 19 to 97% and 16 to 87% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 23 to 146% (4-hour treatment without S9) and 14 to 79% (24 hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/10^6 clonable cells were observed under any treatment condition. 

 

In the retest of definitive mutagenicity assay, the concentrations tested were the same as definitive mutagenicity assay for 4-hour treatment with S9. No visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 10, 15, 30, 40 and 50 µg/mL (4-hour treatment with S9) exhibited 20 to 90% RSG, and were cloned. Relative total growth of the cloned cultures ranged from 20 to 100% (4 hour treatment with S9). No increases in induced mutant frequency ≥90 mutants/10^6 clonable cells were observed.

 

The results indicate the test substance was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available