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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 12the November 2014 Experimental completion date: 22nd December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
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:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Identification: PP-R-001 (also known as Trigonox 501-CS40)
Chemical Name: 1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives
IUPAC Name: Reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propy l-1,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimeth yl-3,6,9-tri-n-propyl-1,2,4,5,7,8-hexoxonane
CAS Number: 1613243-54-1
Batch Number: BYK004856-98
Purity: Not applicable
Physical state / Appearance: Clear, colorless liquid
Expiry / Retest Date: 03 April 2017
Storage Conditions: Approximately 4 ºC in the dark

Method

Target gene:
Thymidine kinase, TK +/- locus of the L5178Y mouse lymphoma cell line
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Mutagenicity Test
Experiment 1: 0, 1.25m 2.5, 3, 10, 20, 30, 35 and 40 µg/ml

Experiment 2: 0, 1.25, 2.5, 5, 10, 15, 20, 25, 30, 35 and 40 µg/ml

Vehicle / solvent:
Acetone
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Solvent treatment groups were used as the vehicle control)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
400 µg/ml and 150 µg/ml for Experiments 1 and 2 respectively
Details on test system and experimental conditions:
Test Item Preparation
Following solubility checks performed in-house, the test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared. The test item was considered to be a multi-constituent substance (UVCB). Therefore, the maximum proposed dose level in the solubility test was set at 5000 µg/mL, the maximum recommended dose level, and no correction for the purity of the test item was applied. Acetone is toxic to L5178Y cells at dose volumes greater than 0.5% of the total culture volume. Therefore, the test item was formulated at 500 mg/ml and dosed at 0.5% to give a maximum achievable dose level of 2500 µg/mL. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991).

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Control Preparation
Vehicle and positive controls were used in parallel with the test item. Solvent (acetone) (CAS No. 67-64-1) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) (CAS No. 62-50-0) Sigma batch BCBK5968V at 400 µg/mL and 150 µg/mL for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) (CAS No. 6055-19-2) Sigma-Aldrich batch SLBG4216V at 2 µg/mL was used as the positive control in the presence of metabolic activation. The positive controls were formulated in dimethyl sulfoxide (DMSO) (CAS No. 67-68-5).

Microsomal Enzyme Fraction
PB/BNF S9 was prepared in-house on 12 October 2014 and 23 November 2014 from the livers of male Sprague-Dawley rats weighing approximately 250g. These had each received, orally, three consecutive daily doses of phenobarbital/β-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day. This procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012. The conduct of the procedure may be reviewed, as part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process. The S9 was stored at approximately 196 °C in a liquid nitrogen freezer.

S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.

20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1. 10% S9-mix (i.e. 1% final concentration of S9) was added to the cultures for Experiment 2.

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 9.77 to 2500 µg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.

The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained. 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 treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

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:

Maximum recommended dose level, 5000 µg/mL or 10 mM.

The presence of excessive precipitate where no test item-induced toxicity was observed.

Test item-induced toxicity, where the maximum dose level used should produce 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).

Mutagenicity Test
Experiment 1
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (1.25 to 40 µg/mL in the absence of metabolic activation, and 2.5 to 60 µg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.1 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL.

The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

Experiment 2
As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL cultures in R10 medium for the 4 hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours (Moore et al, 2007) therefore 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (1% S9 final concentration) at ten dose levels of the test item (1.25 to 40 µg/mL in the absence of metabolic activation, and 5 to 50 µg/mL in the presence of metabolic activation), vehicle and positive controls. To each culture vessel was added 2 mL of S9 mix if required, 0.1 mL of the treatment dilutions, (0.2 mL for the positive controls) and sufficient R0 medium to give a final volume of 20 mL (R10 was used for the 24 hour exposure group).

The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.
Evaluation criteria:
Please see "Any other information on materials and methods"
Statistics:
Please see "Any other information on materials and methods"

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary cytotoxicity test
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 all three of the exposure groups. The onset of test item-induced toxicity was extremely sharp in all three of the exposure groups. Precipitate of the test item was observed at and above 156.25 µg/mL in all three of the exposure groups immediately upon dosing, and appeared greasy / oily at and above 312.5 µg/ml. Based on the %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item induced toxicity.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

 Experiment 1

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 RTG and %RSG values (Tables 3 and 6). There was also evidence of modest reductions in viability (%V), therefore indicating that residual toxicity had occurred in both of the exposure groups (Tables 3 and 6). Based on the RTG and %RSG values observed, optimum levels of toxicity were considered to have been achieved in both the absence and presence of metabolic activation (Tables 3 and 6). The excessive toxicity observed at and above 30 µg/mL in the absence of metabolic activation, and at 60 µg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 50 µg/mL in the presence of metabolic activation markedly exceeded the upper acceptable limit of 90%. Therefore, this dose level was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances .

 

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. 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 (Tables 3 and 6).

 

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6per viable cell, at any of the dose levels in the absence of metabolic activation, including the dose level that achieved optimum toxicity (Table 3). A very modest, but statistically significant, dose related (linear-trend) increase in mutant frequency was observed in the presence of metabolic activation (Table 6). However, the GEF was not exceeded at any of the dose levels, including the dose level that achieved optimum toxicity, and the mutant frequency values observed were within the acceptable range for vehicle controls. The response was therefore considered artefactual and of no toxicological significance. With no evidence of any toxicologically significant increases in mutant frequency, in either the absence or presence of metabolic activation, the test item was considered to have been adequately tested. Precipitate of the test item was not observed at any of the dose levels during the course of the experiment.

 

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

 

Experiment 2

The results of the microtitre plate counts and their analysis are presented in Tables 8 to 13.

 

As was seen previously, there was evidence of marked toxicity in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values (Tables 9 and 12). There was also evidence of a modest reduction in viability (%V) in the presence of metabolic activation, therefore indicating that residual toxicity had occurred in this exposure group (Table 12). Based on the %RSG values observed, optimum levels of toxicity were considered to have been achieved in both the absence and presence metabolic activation prior to plating for viability and 5-TFT resistance (Tables 9 and 12). However, following the incubation period, the RTG value at 30 µg/mL in the presence of metabolic activation exceeded the upper acceptable toxic limit of 90%, therefore, this dose level was excluded from the statistical analysis. In the absence of metabolic activation, a dose level (30 µg/mL) that exceeded the upper limit of acceptable toxic limit of 90%, based on the %RSG value, was plated for viability and 5-TFT resistance as sufficient cells were available at the time of plating. This dose level was also later excluded from the statistical analysis based on the %RSG and RTG values observed. The excessive toxicity observed at and above 35 µg/mL, in both the absence and presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).

 

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had no marked effect on the toxicity of the test item. However, it should be noted that the lowering of the S9 concentration to 1% in this second experiment resulted in greater levels of toxicity being observed when compared to 4-hour exposure groups in the presence of 2% metabolic activation in the Preliminary Toxicity Test and Experiment 1.

 

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. 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 (Tables 9 and 12).

 

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6per viable cell at any of the dose levels, including the dose levels that achieved optimum toxicity based on the %RSG values in both the absence and presence of metabolic activation, but marginally exceeded the upper limit of acceptable toxicity based on the RTG value in the presence of metabolic activation (Tables 9 and 12). It should also be noted that there was also no evidence of an increase in mutant frequency at the dose level that exceeded the upper limit of acceptable toxicity based on %RSG and RTG values in the absence of metabolic activation (Table 9). Therefore, with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, in either the absence or presence of metabolic activation, the test item was once again considered to have been adequately tested. Precipitate of the test item was not observed at any of the dose levels during the course of the experiment.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.
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.476 "In VitroMammalian Cell Gene Mutation Tests" adopted 21 July 1997, 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…….

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (acetone), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at ten dose levels using a 4‑hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

 

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for viability and expression of mutant colonies were as follows:

 

Experiment 1

Group

Concentration of PP-R-001 (CAS# 1613243-54-1) (µg/mL) plated for viability and mutant frequency

4-hour without S9

1.25, 2.5, 5, 10, 20

4-hour with S9 (2%)

5, 10, 20, 30 ,40, 50

 

Experiment 2

Group

Concentration of PP-R-001 (CAS# 1613243-54-1) (µg/mL) plated for viability and mutant frequency

24-hour without S9

5, 10, 15, 20, 25, 30

4-hour with S9 (1%)

5, 10, 15, 20, 25, 30

 

Results………

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of the test item was not observed at any of the dose levels during the course of the Mutagenicity Test. The vehicle controls (acetone) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment 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 dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

 

Conclusion

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.