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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two well conducted in vitro studies (bacterial, and mammalian) on the test substance, and three well conducted in vitro supportive studies on a major component of the test substance are available. All resuts were negative for genetic toxicity.

Link to relevant study records

Referenceopen allclose all

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.
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
Target gene:
Thymidine kinase, TK +/- locus of the L5178Y mouse lymphoma cell line
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
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
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"
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'.

 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.

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.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 November 2014 to 02 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon for Salmonella.
Tryptophan operon for Escherichia
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg per plate

Experiment 2: 5, 15, 50, 150, 300, 1500 and 5000 µg per plate
Vehicle / solvent:
Acetone
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
-S9 (2 µg/plate for WP2uvrA, 3 µg/plate for TA100 and 5 µg/plate for TA1535)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
-S9 (80 µg/plate for TA1537)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
-S9 (0.2 µg/plate for TA98)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
+S9 (1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, and 10 µg/plate for WP2uvrA)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
+ S9 (5 µu/plate for TA98)
Details on test system and experimental conditions:
Test for Mutagenicity (Experiment 1) – Plate Incorporation Method
Dose selection
PP-R-001 (CAS#1613243-54-1) was tested using the following method. The maximum concentration was 5000 g/plate (the maximum recommended dose level). Eight concentrations (1.5, 5, 15, 50, 150, 500, 1500 and 5000 g/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
0.1 mL of the appropriate concentration of PP-R-001 (CAS#1613243-54-1), vehicle or appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously (see 4.5.1.2) except that following the addition of the PP-R-001 (CAS#1613243-54-1) formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 C± 3 C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required, predominantly due to interference in the base agar e.g. minor precipitation of salts or marks on the base of the plates slightly distorting the counts.


Test for Mutagenicity (Experiment 2) – Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the PP-R-001 (CAS#1613243-54-1) formulation or vehicle or 0.1 mL of appropriate positive control were incubated at 37 C± 3 C for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously (see 4.5.2.2) except that following the addition of the PP-R-001 (CAS#1613243-54-1) formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 C± 3 C for 20 minutes (with shaking) and addition of molten amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 C± 3 C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. A 100 mg/mL formulation of PP-R-001 (CAS#1613243-54-1) was also shown to be sterile. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for PP-R-001 (CAS#1613243-54-1), positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 3.

The maximum dose level of PP-R-001 (CAS#1613243-54-1) in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. PP-R-001 (CAS#1613243-54-1) induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains dosed in the absence of S9-mix and Salmonella strain TA100 dosed in the presence of S9-mix at 5000 µg/plate, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. The toxicity of PP-R-001 (CAS#1613243-54-1) to the bacterial cells was identical in the second experiment (pre-incubation method) with weakened background lawns again noted at 5000 µg/plate to all of the Salmonella strains dosed in the absence of S9-mix and Salmonella strain TA100 dosed in the presence of S9-mix. A precipitate (light and globular in appearance) was noted at 5000 g/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of PP-R-001 (CAS#1613243-54-1), either with or without S9 mix in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of PP-R-001 (CAS#1613243-54-1), either with or without S9-mix in Experiment 2 (pre incubation method).

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

PP-R-001 (CAS#1613243-54-1) was considered to be non-mutagenic under the conditions of the bacterial reverse mutation assay (Ames Test).
Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

 

Methods…….

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere treated with PP-R-001 (CAS#1613243-54-1) using both the Ames plate incorporation and pre‑incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co‑factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh formulations of PP-R-001 (CAS#1613243-54-1). The dose range was amended following the results of Experiment 1 and was 5 to 5000 µg/plate.

 

Seven dose levelsof PP-R-001 (CAS#1613243-54-1)were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit following the change in test methodology.

 

Results…….

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The maximum dose level of PP-R-001 (CAS#1613243-54-1) in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. PP-R-001 (CAS#1613243-54-1) induced a visible reduction in the growth of the bacterial background lawns of all of theSalmonellastrains dosed in the absence of S9-mix andSalmonellastrain TA100 dosed in the presence of S9-mix at 5000 µg/plate, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. The toxicity of PP-R-001 (CAS#1613243-54-1) to the bacterial cells was identical in the second experiment (pre-incubation method) with weakened background lawns again noted at 5000 µg/plate to all of theSalmonellastrains dosed in the absence of S9-mix andSalmonellastrain TA100 dosed in the presence of S9-mix. A precipitate (light and globular in appearance) was noted at 5000 mg/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of PP-R-001 (CAS#1613243-54-1), either with or without S9‑mix in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of PP-R-001 (CAS#1613243-54-1), either with or without S9-mix in Experiment 2 (pre‑incubation method). 

 

Conclusion

PP-R-001 (CAS#1613243-54-1)was considered to be non-mutagenic under the conditions of the bacterial reverse mutation assay (Ames Test).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
April - May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Salmonella typhimurium strains TA98, 100, 1535, 1537 and Escherichia coli strain WP2uvrA-
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 mix.
Test concentrations with justification for top dose:
Concentration range in the main test (with and without metabolic activation): 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
Solvent: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ENNG, 9-AA, 4-NQO
Remarks:
without S9-Mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9-mix
Details on test system and experimental conditions:
Five concentrations of the test material were assayed in triplicate against
each tester strain, using the direct plate incorporation method in
accordance with the standard methods for mutagenicity tests using bacteria.

Known al iquots (0.1 ml) of one of the bacterial suspensions were
dispensed into sets of sterile test tubes followed by 2.0 ml of molten
trace histidine/tryptophan supplemented top agar at 45°C, 0.1 ml
of the appropriately diluted test material or vehicle control and
either 0.5 ml of the 59 liver microsome mix or phosphate buffer.
The contents of each test tube were mixed and equally distributed
onto the surface of Vogel-Bonner Minimal agar plates (one tube per
plate). This procedure was repeated, in triplicate, for each bacterial
strain and for each concentration of test material with and without
59-mix.

All of the plates were incubated at 37°C for approximately 48 hours
and the frequency of revertant colonies assessed using a Domino
colony counter. Due to the potentially volatile nature of the test
material, all of the plates were sealed in stainless steel containers
(one dose group per container) for the duration of the incubation
period.

The second experiment was performed using methodology as described for
experiment 1, using fresh bacterial cultures, test material and control
solutions in triplicate.
Evaluation criteria:
For a substance to be considered positive in this test system, it should have
induced a dose-related and statistically(S) significant increase in mutation rate
(of at least twice the spontaneous reversion rate) in one or more strains of
bacteria in the presence and/or absence of the 59 microsomal enzymes in both
experiments at sub-toxic dose levels. In the event of the two experiments
giving conflicting or equivocal results, then a third experiment may be
performed to confirm the correct response. To be considered negative the
number of induced revertants compared to spontaneous revertants should be
less than twofold at each dose level employed, the intervals of which should
be between two and five fold and extend to the limits imposed by toxicity,
solUbility or up to the maximum recommended dose of 5000 tJg/plate. In this
case the limiting factor was the maximum recommended dose.
Species / strain:
other: as specified above
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: as specified above
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
not valid
Additional information on results:
Observations:
None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: main test
Conclusions:
Interpretation of results (migrated information):
negative

The substance was considered to be non-mutagenic under the conditions of this test.
Executive summary:

This study was conducted to assess the mutagenic potential of the test material using a bacterial/microsome test system. The study was based on the in vitro technique described by Ames and his co-workers (1, 2, 3) and Garner et al (4) in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium to various concentrations of the test material. This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. This method also conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471, Method B14 in EC Commission Directive 92/69/EEC and the USA, EPA (TSCA) guidelines. A copy of the Certificate of Compliance with GLP, issued by the UK Department of Health, is included as Appendix IV.

The substance was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - December 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese Hamster Lung cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9-mix
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 156.25 -5000 µg/ml
Concentration range in the main test (without metabolic activation): 1.22 -40 µg/ml
Vehicle / solvent:
Acetone
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix
Details on test system and experimental conditions:
Expression time:
Experiment 1:
Without S9-mix: 12 hours continuous
With S9-mix: 4 exposure , a wash and a
further 8 hours treatment free.

Experiment 2:
Without S9-mix: 12, 24, 48 hours continuous,
6 hours and 18 hrs treatment free
With S9-mix: 6hrs treatment, 18 treatment free
4 hours treatment 8 hr treatmentfree

Selection time:
See above.

Fixation time:
Two hours before harvest time
Evaluation criteria:
Many experiments with the CHL cell line have established a range of aberration
frequencies acceptable for control cultures, these are commonly in the range of
o to 3 % cells with aberrations (lshidate, 1987), Data Book of Chromosomal
Aberration Test In Vitro, (Revised Edition).
A positive response was recorded for a particular treatment if the % cells with
aberratipns (gaps included) was equal to or exceeded 10%, an equivocal
response was recorded for values between 5 and 10% and a negative response
for values less than 5%. For polyploid cells, an incidence greater than 10% is
generally recorded as positive.
However, consideration is given to a number of factors, such as the frequency
of chromosome exchange events which are comparatively rare in control
cultures, and the ultimate designation must rely upon experience and sound
scientific judgement (UKEMS Guidelines for Mutagenicity Testing, 1983).
Species / strain:
other: CHL cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
( 312.5 µg/ml)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: CHL cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 9.77 µg/ml)
Vehicle controls validity:
valid
Positive controls validity:
not valid

Chromosome Aberration Test - Experiment 1: The test material showed the expected level of toxicity similar to that seen in the preliminary toxicity study. These data show a dose-related increase in toxicity in the with-59 exposure group and approximately 50% mitotic inhibition was achieved at 1250 ug/ml. In the exposure group without-59 the toxicity response curve was very steep, there being little evidence of toxicity at 19.53 ug/ml and total mitotic inhibition at 29.3 ug/ml. The vehicle control cultures gave values of chromosome aberrations within the expected range. The positive control cultures gave significant increases in the frequency of aberrations indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected. The test material was seen to induce no statistically significant increases in the frequency of cells with aberrations. The test material did not induce a significant increase in the number of polyploid cells at any dose level in either of the treatment cases.

Chromosome Aberration Test - Experiment 2: The test material showed the expected level of toxicity similar to that seen in the preliminary cytotoxicity study and Experi ment 1. In most cases the maximum evaluated dose level achieved an approximate 50% inhibition of mitotic index. The vehicle control cultures gave values of chromosome aberrations within the expected range. The positive control cultures, except cyclophosphamide without S9 treatment, gave significant increases in the frequency of cells with aberrations indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected. Cyclophosphamide is used in the absence of activation as a control for the with-activation treatment group. The test material was seen to induce no statistically significant or dose-related increases in the frequency of cells with aberrations. The test material did not induce a significant increase In the number of polyploid cells at any dose level in any of the six treatment cases.

Conclusions:
Interpretation of results (migrated information):
negative

The test material did not induce any statistically significant or dose-related increases in the frequency of cells with aberrations. The test material was shown to be toxic to CHL cells in vitro in all six treatment cases, with a very steep dose response curve. The test material, INITIATOR D-129, was shown to be non-clastogenic to CHL cells in vitro.
Executive summary:

This study was designed to assess the potential chromosomal mutagenicity of a test material, on the metaphase chromosomes of the Chinese Hamster lung (CHl) cell line according to the requirements of the japanese New Chemical Substance law (MITI) and the updated Annex V B 10 Method.

Duplicate cultures of Chinese hamster lung (CHL) cells were treated with the test material at a minimum of four dose levels, in each treatment case together with vehicle and positive controls in each of two experiments. In Experiment 1 a single cell-harvest time-point at 12 hours, both with and without metabolic activation, was used. In Experiment 2 six treatment regimes were used: 6 hours exposure both with and without the addition of metabolic activation (followed by 18 hours treatment-free incubation); 4 hours exposure with the addition of metabolic activation (followed by 8 hours treatment-free incubation); 12 hours continuous exposure, 24 hours continuous exposure and 48 hours continuous exposure. The dose range for metaphase analysis was selected from a series of at least four dose levels chosen on the basis of the results of a preliminary toxicity test. The test material was evaluated at doubling dose levels between 2.5 and 2500 ug/ml depending on the particular treatment regime used; the continuous exposures were more toxic than the pulse exposures.

The vehicle (solvent) controls gave frequencies of aberrations within the range expected for the CHL cell line.

The positive control treatments gave significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

Conclusion: The test material did not induce any statistically significant or dose-related increases in the frequency of cells with aberrations in either Experiment 1 or in Experiment 2. The test material was shown to be toxic to CHL cells in vitro in all six treatment cases. The test material was shown to be non-clastogenic to CHL cells in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
August-October 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y/TK+/-3.7.2C mouse lymphoma cells
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphtoflavone induced rat liver S9-mix
Test concentrations with justification for top dose:
with metabolic acitivation:
test 1: 1-387 µg/mL, test 2: 1-350 µg/mL
without metabolic activation:
test 1: 1-66 µg/mL, test 2: 5-53 µg/mL, test 3: 10-56 µg/mL
Vehicle / solvent:
acetone
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9-mix
Negative solvent / vehicle controls:
yes
Positive controls:
no
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix
Details on test system and experimental conditions:
Exposure duration:
with S9-mix: 3 h
without S9-mix: 3 and 24 h

Expression time: 2 days

Selection time: 11 or 12 days

Seletive medium: trifluorthymidine (TFT)

Evaluation criteria:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated
for its biological relevance including a comparison of the results with the historical control data range.
A test substance is considered positive (mutagenic) in the mutation assay if:
a) It induced at least a three-fold increase in the mutation frequency compared to the solvent control
in a dose-dependent manner; or
b) In case a positive result was repeated, the positive response should be reproducible in at least
one repeated experiment.
c) In addition, the observed increase should be biologically relevant and was compared with the
historical control data range.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations showed a mutation frequency of at least three-fold compared
to the solvent controL
b) The results were confirmed in an independently repeated test.
A test substance is considered equivocal (questionable) in the mutation assay if:
- No clear conclusion for positive or negative result could be made after an additional confirmation
study.
Statistics:
The experimental results were not subjected to statistical analysis.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 44-50 µg/mL
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(at 333 µg/mL in a preliminary test)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation occurred at 40 µg/mL

The test article precipitated in the exposure medium at concentrations of 100 ug/ml and above. The test article was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 ug/ml.

First mutagenicity test

Evaluation of toxicity In the absence of S9-mix, the dose levels of 77 to 111 ug/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix, no toxicity was observed and all dose levels were evaluated. The dose levels selected to measure mutation frequencies at the TK-Iocus were: Without S9-mix: 1, 5, 11, 22, 33, 44, 55 and 66 ug/ml exposure medium. With S9-mix: 1, 11, 28, 55, 83, 111, 276 and 387 ug/ml exposure medium. In the absence of S9-mix, the relative total growth of the highest test sUbstance concentration was reduced by 93% compared to the total growth of the solvent controls. In the presence of S9-mix, no reduction of the relative total growth was observed up to the highest dose level tested. Evaluation of the mutagenicity No significant increase in the mutation frequency at the TK locus was observed after treatment with Trigonox 301 either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the Trigonox 301 treated cultures were comparable to the numbers of small and large colonies of the solvent controls. 8.3.2. Second mutagenicity test To obtain more information about the possible mutagenicity of Trigonox 301, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period and in the presence of 12% (v/v) S9-mix with a 3 hour treatment period. Based on the results of the dose range finding test and experiment 1, the following dose levels were selected for mutagenicity testing. Without S9-mix: 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 ug/ml exposure medium. With 12% (v/v) S9-mix: 1, 10, 25, 50, 75, 100, 250 and 350 ug/ml exposure medium. Evaluation of toxicity In the absence of S9-mix, no dose level was reached with a survival of 10 to 20%, this part of the experiment was repeated with the following concentrations: 5, 10, 20, 30, 40, 50, 53, 56, 60, 63, 66 and 70 ug/mL The dose levels of 56 to 70 ug/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix, no toxicity was observed and all dose levels were evaluated. The dose levels selected to measure mutation frequencies at the TK-Iocus were: Without S9-mix: 5, 10, 20, 30, 40, 50 and 53 ug/ml exposure medium. With S9-mix: 1, 10, 25, 50, 75, 100, 250 and 350 ug/ml exposure medium. In the absence of S9-mix, the relative total growth of the highest test SUbstance concentration was reduced by 93% compared to the total growth of the solvent controls. In the presence of S9-mix, no reduction of the relative total growth was observed up to the highest dose level tested. Evaluation of mutagenicity No significant increase in the mutation frequency at the TK locus was observed after treatment with Trigonox 301 either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the Trigonox 301 treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Third mutagenicity test Since no dose level was reached with a survival of 10-20% and no dose level with intermediate toxicity in the absence of S9-mix in the second experiment, an additional mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. The following dose levels were selected for the third mutagenicity test: 5, 10, 20, 30, 35, 40, 43, 46, 50, 53, 56, 60, 63, 65 and 70 ug/ml exposure medium. Evaluation of toxicity Since the dose levels of 5 to 35 ug/ml showed no cytotoxicity, the lowest dose of 5 ug/ml was regarded not relevant for mutation frequency measurement. The dose levels of 43 to 53 ug/ml showed no remarkable difference in the cell growth. Therefore, the concentrations of 43 and 53 ug/ml were not selected for mutation frequency measurement. The concentrations of 60 to 70 ug/ml were not selected for mutation frequency measurement, since these concentrations were too toxic. The dose levels selected to measure mutation frequencies at the TK-Iocus were: 10, 20, 30, 35, 40, 46, 50 and 56 uglml exposure medium. The relative total growth of the highest test substance concentration was reduced by 75% compared to the total growth of the solvent controls.

Conclusions:
Interpretation of results (migrated information):
negative

The test substance is not mutagenic in the TK mutation test ystem under the experimental conditions
Executive summary:

Evaluation of the mutagenic activity of Trigonox 301 in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat), This report describes the effects of Trigonox 301 on the induction of forward mutations at the thymidine-kinase locus (TK-Iocus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9 .. mix (rat liver S9-mix induced by a combination of phenobarbital and l1-naphthoflavone). The study procedures described in this report were based on the most recent OEeD and EEC guidelines. Batch DVT 0706516079 of Trigonox 301 was a clear colourless liquid with a purity of 41 %. The test substance was dissolved in acetone. In the first experiment, Trigonox 301 was tested up to concentrations of 66 and 387 ug/ml in the absence and presence of 8% (v/v) S9-mix, respectively, The incubation time was 3 hours. Trigonox 301 was tested up to the cytotoxic level of 93% in the absence of S9-mix. In the presence of S9-mix, no toxicity was observed, however Trigonox 301 was tested beyond the precipitating dose level of 44 ug/ml. In the second experiment, Trigonox 301 was tested up to concentrations of 53 and 350 ug/ml in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Trigonox 301 was tested up to a cytotoxic level of 93% in the absence of S9-mix. In the presence of S9-mix, no toxicity was observed, however Trigonox 301 was tested beyond the preCipitating dose level of 40 iJg/ml. Since no dose level with a survival of 10-20% or with intermediate toxicity was reached in the absence of S9-mix in the second experiment, an additional mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. Trigonox 301 was tested up to a concentration of 56 ug/ml in the absence S9-mix. The incubation time was 24 hours, Trigonox 301 was tested up to the cytotoxic level of 75%. Mutation frequencies in cultures treated with positive control chemicals were increased by 12-, 11- and 5A-fold for MMS in the absence of S9-mix, and by 16- and 13-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, Trigonox 301 did not induce a significant increase in the mutation frequency in the first experiment This result was confirmed in independent repeat experiments with modifications in the duration of treatment time. In the presence of S9-mix, Trigonox 301 did not induce a significant increase in the mutation frequency in the first experiment This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

It is concluded that Trigonox 301 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

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

Genetic toxicity in vivo

Description of key information

No in vivo testing is required as all in vitro endpoints are negative

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:

Additional information

Additional information from genetic toxicity in vitro:

A bacterial gene mutation study (Ames bacterial mutagenicity test) and a mammalian gene mutation study (MLA) were performed. Both in vitro tests did not reveal genotoxic effects of the test substance.

Additionally, read-across supportive data are available from three in vitro genotoxicity studies: the Ames test (including the testing of the E coli strain), a mouse lymphoma assay, and most importantly, a chromosome aberration test. All 3 tests, which did not show genotoxic effects, were done with CAS# 24748 -23 -0, a major component of the test subtance, CAS 1613243 -54 -1 ( 1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives).

Justification for selection of genetic toxicity endpoint

A well conducted GLP study on genetic toxicity in mammalian cells.

Justification for classification or non-classification

Based on the absence of genotoxicity effect in all vitro tests, it was concluded that the data are conclusive and that the test substance does not meet the criteria for classification.