Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are two in-vitro genetic toxicology studies which are high quality Klimisch 1 standard. These are An OECD 471 Ames bacterial reverse mutation test using S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 and an OECD 490 In-vitro mammalian Cell Gene Mutation test using the Thymidine Kinase Gene TK +/- in L5178Y cells (mouse lymphoma test). There is no in-vitro chromosomal aberration or in-vitro micronucleus test as there is an in-vivo mouse micronucleus test available. This was done for use registration with the EPA.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 17, 2009 to April 14, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidelines S2A and S2B (ICH, 1996, 1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate with and without S9
Vehicle / solvent:
The vehicle control article was deionized water.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
Sterility Controls
The most concentrated test substance dilution and the S9 mix (50 and 500 µL, respectively; the same volumes used in the assay) were checked for sterility by plating on selective agar.

Liver Homogenate
Liver homogenate (S9) was purchased from Molecular Toxicology, Inc. (Lot Nos. 2354 and 2376, containing 33.0 and 39.8 mg/mL protein, respectively). The homogenate was prepared from male Sprague-Dawley rats that had been injected (intraperitoneally) with Aroclor™ 1254 (200 mg/mL in corn oil) at 500 mg/kg, 5 days before sacrifice (Ames, et al., 1975).

S9 Mix
S9 mix was prepared on the day of use, maintained on ice, and contained the components indicated below (Table II; the mix and metabolic activation system are referred to as S9 interchangeably).

Tester Strains
The tester strains used were the Salmonella histidine auxotrophs TA98, TA100, TA1535, and TA1537 (Ames et al. 1975) and the E. coli tryptophan auxotroph WP2uvrA (Green and Muriel 1976). In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain additional mutations that enhance their sensitivity to some mutagenic compounds.

Source of Tester Strains
Salmonella tester strains were received from Dr. Bruce Ames, Department of Biochemistry, University of California (Berkeley, CA). E. coli tester strain WP2uvrA was received from The National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).

Preparation of Overnight Cultures
Inoculation
Overnight cultures were inoculated into flasks containing culturing broth and the flasks were placed in a shaker/incubator programmed to begin operation (shaking, 125 ± 25 rpm; incubation, 37 ± 2°C) so that overnight cultures were in late log phase when optical density (OD) monitoring began.

Harvest
To ensure cultures are harvested at the appropriate phase, the growth of the culture was monitored using a spectrophotometer. An aliquot of each culture was diluted 1:4 and its OD determined at 650 nm (OD650(1:4)). Cultures were harvested once a predetermined OD was reached which ensured that cultures had reached late exponential or early stationary phase (representative of cultures with ≥109 cells/mL) and had not overgrown. Overgrown (stationary) cultures may exhibit decreased sensitivity to some mutagens. Once the target OD was reached (OD650(1:4) = 0.4 to 0.6), the cultures were removed from incubation and held at >0 to 10°C until used in the assay.

Test Design
The test article was evaluated in the initial mutagenicity assay at doses of 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 µg/plate with and without S9. Positive and vehicle controls were evaluated concurrently. All test and control articles were evaluated in duplicate plates. An independent confirmatory assay subsequently was performed at doses of 33.3, 100, 333, 1000, 2000, and 5000 μg/plate with and without S9. Positive and vehicle controls were evaluated concurrently and all test and control articles were evaluated in triplicate plates in the confirmatory assay. Tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and Maron and Ames (1983).
Rationale for test conditions:
The bacterial reverse mutation assay has been shown to be a sensitive, rapid and accurate indicator of the mutagenic activity of many materials including a wide range of chemical classes. By using several different tester strains, both base pair substitution and frameshift mutations can be detected. Salmonella and E. coli strains used in this assay are histidine and tryptophan auxotrophs, respectively, by virtue of conditionally lethal mutations in the appropriate operons. When these histidine (his–) or tryptophan (trp–) dependent cells are exposed to the test article and grown under selective conditions (minimal media with a trace amount of histidine or tryptophan), only those cells which revert to histidine (his+) or tryptophan (trp+) independence are able to form colonies. Trace amounts of histidine or tryptophan added to the media allow all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. his+ or trp+ revertants are readily discernable as colonies against the limited background growth of his– or trp– cells.
Evaluation criteria:
Criteria for a Positive Response
A test substance is considered to have produced a positive response if it induces a dose dependent increase in revertant frequency that is ≥2.0-fold vehicle control values for tester strains TA98, TA100, and WP2uvrA, or ≥3.0-fold vehicle control values for tester strains TA1535 and TA1537. In addition, any response should be reproducible.
Criteria for a Negative Response
A test substance is considered to have produced a negative response if no dose-dependent, ≥2.0-fold or ≥3.0-fold increases are observed in tester strains TA98, TA100, and WP2uvrA, or TA1535 and TA1537, respectively.
Criteria for an Equivocal Response
Even after repeated trials, a test substance may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the dose-dependency or fold increase requirements but are reproducible). In those rare instances, the test article may be considered to have produced an equivocal response.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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:
no 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:
no 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Test Article Handling
Deionized water was the vehicle for this study. In solubility testing, the test substance was observed to form an opaque, white, homogeneous suspension at approximately 100, 50, and 25 mg/mL, and a translucent, white, homogeneous suspension at approximately 12.5 mg/mL, in deionized water. At 100 mg/mL, which was the most concentrated dose formulation prepared for treatment; the test substance was observed to form an off-white, opaque, non-viscous, homogeneous suspension. The test substance diluted to a solution at 3.2 mg/mL and remained freely soluble at all succeeding lower dilutions prepared.

Mutagenicity Assay
The test substance was evaluated in the initial mutagenicity assay, in all five tester strains, at doses of 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate in the presence and absence of S9 mix. All doses of the test substance, as well as the concurrent positive and vehicle controls, were evaluated in duplicate plates. No positive increases in the mean number of revertants/plate were observed with any of the tester strains in the presence or absence of S9 mix. No dose-related decreases in revertant frequency were observed with any of the tester strains in the presence or absence of S9. Reduced bacterial background lawns were observed at ≥1600 μg/plate in the absence of S9 mix with tester strains TA100, TA1535, and TA1537.

Based on the results of the initial assay, the test substance was re-evaluated in the confirmatory mutagenicity assay at doses of 33.3, 100, 333, 1000, 2000, and 5000 μg/plate with and without S9. All doses of the test substance, as well as the concurrent positive and vehicle controls, were evaluated in triplicate plates. No positive increases in the mean number of revertants/plate were observed with any of the tester strains in the presence or absence of S9 mix. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.
Conclusions:
Under the study conditions, the test substance was negative in the Bacterial Reverse Mutation Assay with a confirmatory assay.
Executive summary:

A study was conducted to determine the potential of the test substance and its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and at the tryptophan locus of Escherichia coli strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9) according to OECD Guideline 471, EPA OPPTS Method 870.5100 and ICH Guidelines S2A and S2B, in compliance with GLP. The test substance was evaluated in the initial mutagenicity assay, in all five tester strains, at doses of 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate with and without S9. No positive increases were observed with any of the tester strains in the presence or absence of S9 mix. Based on the results of the initial assay, the test substance was re-evaluated in the confirmatory mutagenicity assay at doses of 33.3, 100, 333, 1000, 2000, and 5000 μg/plate with and without S9. No positive increases were observed with any of the tester strains in the presence or absence of S9 mix. All positive and vehicle control values were within acceptable ranges and all criteria for a valid study were met. Under the study conditions, the test substance was negative in the Bacterial Reverse Mutation Assay with a confirmatory assay (Farabaugh, 2009).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 24 July 2018 Experimental Completion Date: 21 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cell Line
The L5178Y TK +/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit in Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time. The L5178Y cell line has successfully been used in in vitro experiments for many years. L5178Y cells are characterized by a high proliferation rate (doubling time 10 - 12 h in stock cultures) and cloning efficiency of untreated cells of usually more than 50 %, both characteristics are necessary for the appropriate performance of the study. The stock of cells is stored in liquid nitrogen at approximately -196 °C. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Culture
The culture medium used was RPMI 1640 with Glutamax-l and 20mM HEPES buffer supplemented with Penicillin (100 units/ml), Streptomycin (100 μg/mL), Sodium Pyruvate (1mM) and Amphotericin B (2.5 μg/mL) giving R0 media.
For use, a sample of cells were removed from cryogenic storage before the start of the study and grown in R0 media supplemented with 10% horse serum (known as R10 media) at approximately 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and are sub-cultured accordingly. R10 media was used for general maintenance of cells and cell washing at the end of the test item exposure period. R20 media, (R0 with 20% horse serum) was used for the seeding of viability and mutant frequency plates and for maintenance of cultures during the expression period. The serum concentration during the exposure period was 5% for the 4-hour groups and 10% for the 24 hour group.

Cell Cleansing
TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before a stock of cells is frozen these homozygous mutants (TK -/-) must be removed. The cells are cleansed of mutants by culturing in THMG medium for 24 hours. This medium contains 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 are cultured in THG medium (THMG medium without Methotrexate) before being returned to R10 media. The "cleansed" cells are frozen in 1 ml aliquots at between 1 to 3 x 106 cells/mL and stored in liquid nitrogen freezers at approximately –196 °C. A fresh ampoule is removed from the frozen stock and cultured to provide adequate numbers of cells for testing.
Metabolic activation:
with and without
Metabolic activation system:
liver S9 from male Sprague-Dawley rats treated with Phenobarbital / B-Naphtha flavone
Test concentrations with justification for top dose:
Preliminary Toxicity Test
0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 µg/mL
The dose levels were limited by an increase in pH observed above 2500 μg/mL

Mutagenicity Test
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/mL or 10 mM. (limited by pH)
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce approximately 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).

Dose range
4-hour without S9: 25, 50, 100, 200, 300, 400, 500, 600
4-hour with S9 (2%): 25, 50, 100, 200, 300, 400, 500, 600
24-hour without S9: 1.25, 2.5, 5, 10, 20, 40, 60, 80
The maximum dose level was limited by test item induced toxicity.
Vehicle / solvent:
Following solubility checks performed in-house, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
400 μg/mL and 150 μg/mL for 4-hour and 24-hour exposures respectively
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
1.5 μg/mL
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
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 10^5 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 9.77 to 2500 μg/mL for all three of the exposure groups. The dose levels were limited by an increase in pH observed above 2500 μg/mL. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 10^5 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 10^5 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 Concentration toxicity, and a comparison of each Concentration SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

Main Experiment
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. For the 4-hour exposure groups both with and without metabolic activation, the cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. In the 24-hour exposure in absence of metabolic activation the cells were processed to give 0.3 x 10^6 cells/mL in 10 mL cultures established in 25 cm2 tissue culture flasks. The Concentrations were performed in duplicate (A + B), at 8 dose levels of the test item (25 to 600 μg/mL in both 4-hour exposure groups and 1.25 to 80 μg/mL in the 24-hour exposure group), vehicle and positive controls. To each universal was added 2 mL of S9-mix (2%) if required, 0.2 mL of the concentration dilutions, (0.15 or 0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL (R10 is used for the 24-hour exposure group). The Concentration vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Measurement of Survival, Viability and Mutant Frequency
At the end of the Concentration period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%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 Concentration toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate Scoring
Microtiter plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37 °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 mutation plates were also recorded (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgement. 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 mutation plates. The plates were incubated for approximately two to three hours. MTT is a vital stain that is taken up by viable cells and metabolized to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.
Evaluation criteria:
The normal range for mutant frequency per survivor is 50-170 x 10^-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should ideally be within this range. Experiments where the vehicle control values are markedly greater than 200 x10^-6 mutant frequency per survivor are not normally acceptable and will be repeated.
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
Optimum toxicity is approximately 20% survival (80% toxicity), but no less than approximately 10% survival (90% toxicity). RTG values are usually the primary factor to designate the level of toxicity. However, under certain circumstances, %RSG values may also be taken into account. Dose levels that have RTG survival values markedly less than 10% are excluded from any statistical analysis.
For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. The Global Evaluation Factor (GEF) value was set at 126 x 10^-6 for the microwell method. Any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
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
Positive controls validity:
valid
Additional information on results:
Preliminary Cytotoxicity Test
In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was evidence of marked reductions of %RSG values of cells treated with test item. In the 4-hours +S9 and 24-hour –S9 exposure groups precipitate of the test item was observed at 2500 μg/mL. In the subsequent mutagenicity experiments the maximum dose was limited by test item induced toxicity.

Mutagenicity Test
4-hour exposure in the absence and presence of metabolic activation
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 Optimum toxicity was considered to have been achieved in the absence of metabolic activation with very near to optimum toxicity achieved in the presence of metabolic activation. There was no evidence of marked reductions in viability (%V); therefore indicating that residual toxicity had not occurred. No precipitate was observed. The excessive toxicity observed at 500 and 600 μg/mL in both the absence and presence of metabolic activation resulted in these dose level not being plated for viability or 5-TFT resistance.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
The test item did not induce any toxicologically significant increases in the mutant frequency in either of the exposure groups. The GEF value was not exceeded at any dose level.

24-hour exposure in the absence of metabolic activation
There was evidence of marked toxicity following exposure to the test item as indicated by the %RSG and RTG values. There was evidence of moderate reductions in viability (%V), therefore indicating that residual toxicity had occurred in this exposure group. Optimum levels of toxicity were achieved. The excessive toxicity observed at and above 60 μg/mL resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with the positive control substance. Precipitate was not observed in the 24-hour exposure group.
The 24-hour exposure without metabolic activation (S9) Concentration, demonstrated that the extended time point had a marked effect on the toxicity of the test item. The vehicle control mutant frequency was within the normal range of 50 to 170 x 10^-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.
The test item did not induce any toxicologically significant increases in the mutant frequency in the 24-hour exposure in the absence of metabolic activation. The GEF value was not exceeded at any test item dose level, and all of the mutant frequency values were within the acceptable range for a vehicle control.
Conclusions:
The test item, Amides, C5-9, N-[3-(dimethylamino)propyl] (CASRN 1044764-00-2) did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, 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 Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods

One main experiment was performed. In the main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at up 8 dose levels, in duplicate, together with vehicle (DMSO) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9) and a 24-hour exposure 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 dose levels were as follows:

Main Experiment

Group

Concentration of Amides, C5-9, N-[3-(dimethylamino)propyl] (CASRN 1044764-00-2) (µg/mL)

 plated for mutant frequency

4-hour without S9

25, 50, 100, 200, 300, 400, 500, 600

4-hour with S9 (2%)

24-hour without S9

1.25, 2.5, 5, 10, 20, 40, 60, 80

Results

The maximum dose level was limited by test item induced toxicity. No precipitate was observed. The vehicle (DMSO) controls had acceptable mutant frequency values that were within the acceptable range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion

The test item Amides, C5-9, N-[3-(dimethylamino)propyl] (CASRN 1044764-00-2), did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There is a Klimisch 1 in-vivo OECD 474 Mammalian erythrocyte micronucleus test in bone marrow of CD -1 mice.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 16, 2009 to May 15, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
mouse
Strain:
CD-1
Details on species / strain selection:
Young adult male and female ICR(CD-1) mice. This is an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test substances. The mouse has been routinely utilized as an animal model of choice for the mammalian bone marrow erythrocyte micronucleus assay.
Sex:
male/female
Details on test animals and environmental conditions:
Dose range-finding study: young adult male and female ICR(CD-1) mice were received on 26 March 2009 from Harlan, Frederick, Maryland.
Micronucleus Assay: young adult male ICR(CD-1) mice were received on 14 April 2009 from Harlan, Indianapolis, IN.

Identification and Acclimation
Animals were randomized into groups using a computer program. Following randomization, each study animal was uniquely identified by ear tag. Animals were acclimated to laboratory conditions for at least 5 days.

Housing
The animals were housed in sanitary polycarbonate cages containing Sani-Chips® Hardwood Chip Laboratory bedding. The animals were housed, separated by gender, up to five animals per cage during acclimation, and by full dose group/harvest timepoint after randomization. Each batch of wood chips was analyzed by the manufacturer for specific microorganisms and contaminants.

Environmental Conditions
Environmental controls were set to maintain the following animal room conditions: temperature range of 18 to 26°C, relative humidity range of 30 to 70%, 10 or greater air changes/hour, and a 12-hour light/12-hour dark cycle. Actual temperature and humidity readings were monitored continuously and averaged twice daily. Any variations to these conditions are maintained in the raw data and had no effect on the outcome of the study.

Diet, Water, and Contaminants
Harlan Teklad Certified Rodent Diet® #2016C was available ad libitum. The manufacturer analyzed the diet for nutritional components and environmental contaminants. Tap water was available ad libitum. Water samples are routinely analyzed for specified microorganisms and environmental contaminants. Results of the diet and water analyses are reviewed for acceptability and are on file at Covance-Vienna.
Route of administration:
oral: gavage
Vehicle:
Prior to dosing, the top stock of the test substance was prepared by adding the appropriate volume of the vehicle, RO/DI water, to a pre-weighed quantity of the test substance and mixed, forming a colorless, opaque, non-viscous, homogeneous suspension. Lower concentrations were obtained by dilution with the vehicle. The formulations were held at room temperature prior to dosing and stirred during the dosing procedure.
Details on exposure:
Dose Range-finding Study

In the dose range-finding study, the test substance was formulated in reverse osmosis / deionized (RO/DI) water and administered once, by oral gavage to 3 males and 3 females per dose level. The animals were dosed at 500, 1000, or 2000 mg/kg. At initiation of treatment, the animals were approximately 8 weeks old, and their body weights ranged from 34.7 to 39.3 g and 23.4 to 28.8 g, for the males and females, respectively.
The daily observations of toxic signs and/or mortality data were used to estimate the highest appropriate dose level (maximum tolerated dose) for the micronucleus assay.

Micronucleus Assay

Since no relevant differences in toxicity between the sexes were observed in the dose range-finding study, only males were used in the micronucleus assay.
In the dose range-finding study, the maximum tolerated dose was estimated to be 800 mg/kg. In the micronucleus assay, the test substance was formulated in RO/DI water and administered once at 200, 400 and 800 mg/kg to 5 males per dose level.
The high dose, unless non-toxic, should have produced some indication of toxicity, e.g., toxic signs, death, or depression of the ratio of PCEs to NCEs. The use of a high dose, as defined above, increased the likelihood that a weak clastogen could be detected.
At initiation of treatment, the animals were approximately 8 weeks old, and their body weights ranged from 32.4 to 39.3 g.
Duration of treatment / exposure:
Observation period: 2 days after dosing
Frequency of treatment:
All animals were examined immediately after dosing, approximately 1 hour after dosing, and at least daily for the duration of this assay for signs of clinical toxicity and/or mortality.
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males per dose
Control animals:
yes, concurrent no treatment
Tissues and cell types examined:
Extraction of Bone Marrow
The hind limb bones (tibias) were removed for marrow extraction from up to five surviving animals in each treatment and control group. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal).
Details of tissue and slide preparation:
Preparation of Slides
Following centrifugation to pellet the marrow, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. The slides were fixed in methanol, stained in May-Grünwald solution and Giemsa, and protected by mounting with coverslips. For control of bias, all slides were coded prior to analysis.

Slide Analysis
Slides prepared from the bone marrow collected from up to five animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least 500 erythrocytes per animal.

The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond- and ring-shaped micronuclei occasionally occurred. Micronuclei were sharp bordered and generally between one-twentieth and one-fifth the size of the PCEs. The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluishgray and red, respectively).
The historical background frequency of micronucleated cells was expressed as percentage micronucleated cells based on the number of PCEs analyzed. The historical background frequency of micronuclei in the mouse strains at this laboratory is about 0.0 to 0.4%, which is within the range of the published data (Salamone and Mavournin, 1994).
Evaluation criteria:
The criteria for a positive response are the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response. A test substance that does not induce both of these responses is considered negative. Statistical significance is not the only determinant of a positive response; the Study Director also considers the biological relevance of the results in the final evaluation.
Statistics:
- Assay data analysis was performed using an analysis of variance (Winer, 1971) on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances.
- If the analysis of variance was statistically significant (p ≤ 0.05), Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were statistically significantly different from the vehicle control.
The 200, 400, and 800 mg/kg dose groups, as well as the positive control group, were compared with the vehicle control group at the 5% probability level.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Dose Range-finding Study

Survival and Clinical Observations
Mortality was observed in 1 of 3 females at 1000 mg/kg, and in 3 of 3 males and 3 of 3 females at 2000 mg/kg. Based on these results, the maximum tolerated dose was estimated to be 800 mg/kg.

Micronucleus Assay

Survival and Clinical Observations
Mortality was observed in 4 of 13 males at 800 mg/kg. Clinical observations were noted in only one male at 800 mg/kg, and consisted of squinted eyes and labored respiration. All other animals appeared normal immediately after dosing and remained normal until the appropriate harvest timepoint. All animals in the vehicle and positive control groups appeared normal after dosing and remained normal until the appropriate harvest timepoint.


- The test substance, induced signs of clinical toxicity in the treated animals at 800 mg/kg, which included squinted eyes and labored respiration.
- The test substance did not induce statistically significant increases in micronucleated PCEs at any test substance dose examined (200, 400, and 800 mg/kg).
- The test substance was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose.
- The vehicle control group had approximately ≤0.3% micronucleated PCEs and the group mean was within the historical control range.
- The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with a mean and standard deviation of 1.11 ± 0.39%.
Conclusions:
Under the study conditions, the test substance was evaluated as negative in the mouse bone marrow micronucleus assay.
Executive summary:

A study was conducted to determine the test substance for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei inpolychromatic erythrocytes (PCE) in CD®-1 mouse bone marrow according to OECD Guideline 474 and EPA OPPTS Method 870.5395, in compliance with GLP. In the dose range-finding study, the test substance was formulated in reverse osmosis /deionized (RO/DI) water and administered once, by oral gavage to 3 males and 3 femalesper dose level. The animals were dosed at 500, 1000, or 2000 mg/kg bw and observed for up to 2 d after dosing for toxic signs and/or mortality. Based on the results of the dose range-finding study, the maximum tolerated dose was estimated to be 800 mg/kg bw. In the micronucleus assay, the test substance was formulated in RO/DI water and administered once at 200, 400 and 800 mg/kg bw. Bone marrow was extracted and at least 2000 PCEs per animal were analyzed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 total erythrocytes for each animal. Mortality was observed in 4 of 13 males at 800 mg/kg bw. Clinical observations were noted in only one male at 800 mg/kg bw and consisted of squinted eyes and labored respiration. The test substance did not induce statistically significant increases in micronucleated PCEs at any dose examined. In addition, the test substance was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose. Under the study conditions, the substance was evaluated as negative in the mouse bone marrow micronucleus assay (Xu, 2009).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The test substance was evaluated in the bacterial reverse mutation assay, in the initial mutagenicity assay, in all five tester strains, at doses of 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate with and without S9. No positive increases were observed with any of the tester strains in the presence or absence of S9 mix. Based on the results of the initial assay, the test substance was re-evaluated in the confirmatory mutagenicity assay at doses of 33.3, 100, 333, 1000, 2000, and 5000 μg/plate with and without S9. No positive increases were observed with any of the tester strains in the presence or absence of S9 mix. All positive and vehicle control values were within acceptable ranges and all criteria for a valid study were met. Under the study conditions, the test substance was negative in the Bacterial Reverse Mutation Assay with a confirmatory assay

The test item Amides, C5-9, N-[3-(dimethylamino)propyl] (CASRN 1044764-00-2), did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic in this assay.

The test substance did not induce statistically significant increases in micronucleated PCEs at any dose examined. In addition, the test substance was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose. Under the study conditions, the substance was evaluated as negative in the mouse bone marrow micronucleus assay

Justification for classification or non-classification

The test substance has been identified in the USA as Amides, C5-9, N-[3-(dimethylamino)propyl], however for RAECH there is not sufficient C5 to justify the name so it is call N-[3-(dimethylamino)propyl] C6-9 alkyl amides. It was negative in all three available genotoxicity tests. The in-vitro tests the Bacterial reverse mutation (AMES) test and the mouse lymphoma mammalian cell gene mutation assay TK +/- in L5178Y. It was also negative in the mouse bone marrow erythrocyte micronucleus test. This confirmed that it is not mutagenic in bacteria and mammalian cells and did not cause chromosome damage in-vivo as indicated by a lack of induction of micronuclei in polychromatic erthyrocytes in mouse bone marrow. Based on these consistent negative results there is not requirement for any additional in-vivo testing or for classification due to mutagenic, clastogenic or anugenic adverse effects, by EU CLP (GHS) criteria.