tert-pentyl peroxypivalate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two Ames tests are available. In the first assay (key study), five strains were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. The test substance induced mutagenic activity in the bacterial reverse mutation test on the TA 102 strain of Salmonella typhimurium, with S9 mix. In the second assay the tester strains used were Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537, and Escherichia coli tester strain WP2uvrA. The substance was positive in tester strains WP2uvrA and TA100 in the presence of S9 mix. The substance did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells in an in vitro mammalian gene mutation assay.

An in vitro clastogenicity study is not available.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Experiments without S9 mix:
The selected treatment-levels were as follows:
156.25, 312.5, 625, 1250 and 2500 µg/plate: for the TA 1535 strain in the first experiment as
well as for the TA 102 strain in the second experiment,
312.5, 625, 1250, 2500 and 3125 µg/plate: for all strains (except for the TA 1535 strain) in the
first experiment,
78.125, 156.25, 312.5, 625 and 1250 µg/plate: for all strains (except for the TA 102 strain) in
the second experiment.

Experiments with S9 mix:
The selected treatment-levels were as follows:
156.25, 312.5, 625, 1250 and 2500 µg/plate: for the TA 1535 strain in the first experiment as
well as for all tester strains (except for the TA 98 strain) in the second experiment,
312.5, 625,1250, 2500 and 3125 µg/plate: for all strains (except for the TA 1535 strain) in the
first experiment,
78.125, 156.25, 312.5, 625 and 1250 µg/plate: for the T A 98 strain in the second experiment.

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

The five strains of Salmonella typhimurium (Ames et al., 1975; Maron and Ames, 1983): TA 1535, TA 1537, TA 98, TA 100 and TA 102 were supplied by B.N. Ames' Laboratory (University of California, Berkeley, USA). They are stored in a cryoprotective medium (1 ml nutrient broth and 0.09 ml dimethylsulfoxide) in a liquid nitrogen container. The day before treatment, cultures were inoculated from frozen permanents: a scrape was taken under sterile conditions and put into approximately 6 ml of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37°C for about 14 hours, to produce bacterial suspensions.
Each strain derived from Salmonella typhimurium L T 2 contains one mutation in the histidine operon, resulting in a requirement for histidine. In addition, to increase their sensitivity to mutagenic substances, further mutations have been added:
· the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules that do not penetrate the nonnal bacteria cell wall,
· the uvrB mutation is a deletion of a gene coding for the DNA excision repair system, which renders the bacteria unable to use this repair mechanism to remove the damaged DNA,
· the addition of the plasmid pKM 101 (conferring ampicillin resistance) to strains TA 98, TA 100 and TA 102 enhances their sensitivity of detection to some mutagens,
· in addition, the pAQ1 tetracycline resistant plasmidic factor has been added to the TA 102 strain.

Evaluation criteria:

Treatment of results
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtained in the presence of the vehicle), are presented in a table.

Acceptance criteria
This study would be considered valid since the following criteria are fully met:
The number of revertants in the vehicle controls is consistent with our historical data (appendix 2),
the number of revertants in the positive controls is higher than that of the vehicle controls and inconsistent with our historical data.

Evaluation criteria
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

None

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥1250 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥625 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥312.5 µg/plate, slight to moderate (in 2. experiment)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

only at 625 µg/plate, not at higher concentrations (1. experiment, not seen after preincubation in 2. experiment)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥625 and ≥1250 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥625 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight to moderate at high conc. (2. experiment)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight to moderate at high conc. (2. experiment)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
The test substance was freely soluble in the vehicle (ethanol) at 100 mg/mL. Consequently, with a maximum dose volume of 50 µg/plate, the dose-levels were: 10, 100, 500,1000, 2500 and 5000 µg/plate. A slight to moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels: ≥2500 µg/plate.

Without S9 mix, a slight toxicity was induced in the TA 98 and TA 102 strains at 5000 µg/plate. In the TA 100 strain, a slight to strong toxicity was noted at dose-levels greater than or equal to 2500 µg/plate.

With S9 mix, the test substance was totally toxic at 5000 µg/plate in the three tester strains. In addition, a slight toxicity was induced at 2500 µg/plate in both the TA 98 and TA 100 strains. In the TA 102 strain, a 3 and a 7 fold increase in the number of revertants was noted at dose levels of 1000 and 2500 µg/plate, respectively.

MUTAGENICITY EXPERIMENTS
The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test substance was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix:
The selected treatment-levels were as follows:
156.25, 312.5, 625, 1250 and 2500 µg/plate: for the TA 1535 strain in the first experiment as well as for the TA 102 strain in the second experiment, 312.5, 625, 1250, 2500 and 3125 µg/plate: for all strains (except for the TA 1535 strain) in the first experiment, 78.125, 156.25, 312.5, 625 and 1250 µg/plate: for all strains (except for the TA 102 strain) in the second experiment.
A slight emulsion was generally noted at dose-levels ≥1250 µg/plate.
In the first experiment, a slight toxicity was noted in the TA 1535 and TA 102 strains at dose levels ≥50 µg/plate and ≥2500 µg/plate, respectively. In the three remaining test strains, a slight to strong toxicity was noted at dose-levels ≥312.5 µg/plate.
In the second experiment, a slight to moderate toxicity was noted in the TA 102 and TA 100 strains (at the highest dose-level) as well as in the TA 1537 strain (at dose-levels ≥312.5 µg/plate). The test substance did not induce any significant increase in the number of revertants, in both experiments, in any of the five strains.

Experiments with S9 mix:
The selected treatment-levels were as follows:
156.25, 312.5, 625, 1250 and 2500 µg/plate: for the TA 1535 strain in the first experiment as well as for all tested strains (except for the TA 98 strain) in the second experiment,
312.5, 625,1250, 2500 and 3125 µg/plate: for all strains (except for the TA 1535 strain) in the first experiment.
78.125, 156.25, 312.5, 625 and 1250 µg/plate: for the TA 98 strain in the second experiment.
A slight emulsion was generally noted at dose-levels ≥1250 µg/plate.
In the first experiment, the test substance was slightly to totally toxic in the TA 1537, TA 100 and TA 102 strains (at doses ≥2500 µg/plate) as well as in the TA 98 strain (at doses ≥1250 µg/plate).

In the second experiment, the test substance was slightly to totally toxic at doses ≥625 µg/plate in the TA 1537 and TA 100 strains. A slight to moderate (for the TA 98 strain) and a slight to strong (for the TA 1535 strain) toxicity was noted at dose-levels ≥625 and ≥1250 µg/plate, respectively. In the TA 102 strain, a slight toxicity was noted at 2500 µg/plate. In the first experiment, a 2 fold increase in revertants was noted in the TA 98 strain at 625 µg/plate but not at higher dose-levels where toxicity was induced. The second experiment was performed using the preincubation method with this strain and no noteworthy increase in revertants was noted. Therefore the previous 2 fold increase in revertants was not considered as biologically relevant.

In the TA 102 strain in the first experiment, a 2.2-3.76 fold increase in the number of revertants was induced at dose-levels of between 312.5 and 1250 µg/plate. No increase was noted at higher dose-levels where toxicity could have masked the mutagenic effect. This result confirmed the increase previously noted in the preliminary test. In the second experiment, repeated under the same experimental conditions (direct plate incorporation method), a significant increase in revertants was reproduced, even if a lower effect was noted: up to 2.1 fold the vehicle control value at 1250 µg/plate.

Conclusions:

-Positive with metabolic activation:


Under our experimental conditions, the test substance induced mutagenic activity in the bacterial reverse mutation test on the TA 102 strain of Salmonella typhimurium, with S9 mix.

Executive summary:

The objective of this study was to evaluate the potential of the test substance to induce reverse mutation in Salmonella typhimurium.

 

A preliminary toxicity test was performed to define the dose-levels of the substance to be used for the mutagenicity study. The test substance was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except the second with S9 mix (excluding the TA 102 strain), which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test substance was dissolved in ethanol.

 

Experiments without S9 mix: The selected treatment-levels were as follows: 156.25, 312.5, 625, 1250 and 2500 µg/plate: for the TA 1535 strain in the first experiment as well as for the TA 102 strain in the second experiment. 312.5, 625, 1250, 2500 and 3125 µg/plate: for all strains (except for the TA 1535 strain) in the first experiment, 78.125, 156.25,312.5,625 and 1250 µg/plate: for all strains (except for the TA 102 strain) in the second experiment. A slight emulsion was generally noted at dose-levels ≥1250 ug/plate. In the first experiment, a slight toxicity was noted in the TA 1535 and TA 102 strains at dose levels ≥50 µg/plate and ≥2500 µg/plate, respectively. In the three remaining tester strains, a slight to strong toxicity was noted at dose-levels ≥312.5 µg/plate.

In the second experiment, a slight to moderate toxicity was noted in the TA 102 and TA 100 strains (at the highest dose-level) as well as in the TA 1537 strain (at dose-levels ≥312.5 µg/plate). The test substance did not induce any significant increase in the number of revertants, in both experiments, in any of the five strains. Experiments with S9 mix: The selected treatment-levels were as follows: 156.25, 312.5, 625, 1250 and 2500 µg/plate: for the TA 1535 strain in the first experiment as well as for all tester strains (except for the TA 98 strain) in the second experiment, 312.5, 625,1250, 2500 and 3125 µg/plate: for all strains (except for the TA 1535 strain) in the first experiment, 78.125, 156.25, 312.5, 625 and 1250 µg/plate: for the TA 98 strain in the second experiment. A slight emulsion was generally noted at dose-levels ≥1250 µg/plate. In the first experiment, the test substance was slightly to totally toxic in the TA 1537, TA 100 and TA 102 strains (at doses ≥2500 µg/plate) as well as in the TA 98 strain (at doses ≥1250 µg/plate). In the second experiment, the test substance was slightly to totally toxic at doses ≥625 µg/plate in the TA 1537 and TA 100 strains. A slight to moderate (for the TA 98 strain) and a slight to strong (for the TA 1535 strain) toxicity was noted at dose-levels ≥625 and ≥1250 µg/plate, respectively. In the TA 102 strain, a slight toxicity was noted at 2500 µg/plate. In the first experiment, a 2 fold increase in revertants was noted in the T A 98 strain at 625 µg/plate but not at higher dose-levels where toxicity was induced. The second experiment was performed using the preincubation method with this strain and no noteworthy increase in revertants was noted. Therefore the previous 2 fold increase in revertants was not considered as biologically relevant. In the TA 102 strain in the first experiment, a 2.2-3.76 fold increase in the number of revertants was induced at dose-levels of between 312.5 and 1250 µg/plate. No increase was noted at higher dose-levels where toxicity could have masked the mutagenic effect. This result confirmed the increase previously noted in the preliminary test. In the second experiment, repeated under the same experimental conditions (direct plate incorporation method), a significant increase in revertants was reproduced, even if a lower effect was noted: up to 2.1 fold the vehicle control value at 1250 µg/plate.

 

Under our experimental conditions, the test substance induced mutagenic activity in the bacterial reverse mutation test on the TA 102 strain of Salmonella typhimurium, with S9 mix.

Reference 2

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: 13th February 2015 Experimental completion date: 8th April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

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 not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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/ Beta-naphthoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 3.68 to 941.5 µg/mL

Experiment 1: 5, 10, 20, 30, 40, 50, 60, 120 µg/mL
Experiment 2: 1.25, 2.5, 5, 10, 15, 20, 25, 30 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:

-S9 (400 µg/mL and 150 µg/mL for Experiment's 1 and 2 respectively

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

+ S9

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 had a molecular weight of 188.3. Therefore, the maximum proposed dose level in the solubility test was set at 1883 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 188.3 mg/ml and dosed at 0.5% to give a maximum achievable dose level of 941.5 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). The pH and osmolality readings are in the following table:

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 batches BCBK5968V and BCBN1209 at 400 µg/mL and 150 µg/mL for Experiment 1 and Experiment 2, respectively, were used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) (CAS No. 6055-19-2) Sigma-Aldrich batch MKBS0021V at 1.5 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 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 Experiments 1 and 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 3.68 to 941.5 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:

i) Maximum recommended dose level, 5000 µg/mL or 10 mM.
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 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 (5 to 120 µg/mL in the absence of metabolic activation, and 5 to 120 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 or 0.15 mL for the positive controls) 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 (2% S9 final concentration) at eight dose levels of the test item (1.25 to 30 µg/mL in the absence of metabolic activation, and 30 to 100 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 or 0.15 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

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 sharp in all three of the exposure groups. A greasy/oily precipitate of the test item was observed at and above 235.38 µg/mL in all three exposure groups immediately upon dosing. 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 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, very near to optimum levels of toxicity were considered to have been achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at 120 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. The toxicity observed at 60 µg/mL in the absence of metabolic activation and 100 µg/mL in the presence of metabolic activation markedly exceeded the upper acceptable limit of 90%. Therefore, these dose levels were 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.

 

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 either the absence and presence of metabolic activation, including the dose levels that achieved very near optimum toxicity (Tables 3 and 6). 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.

 

 

Experiment 2

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. 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. Based on the %RSG values observed, optimum levels of toxicity were considered to have been achieved in both the absence and presence metabolic activation. The excessive toxicity observed at and above 25 µg/mL in the absence of metabolic activation and at and above 90 µg/mL in the presence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. In the presence of metabolic activation, a dose level (80 µg/mL), 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 RTG value observed. Acceptable levels of toxicity were seen with both positive control substances.

 

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.

 

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.

 

The test item induced very small but statistically significant and dose related (linear-trend) increases in the mutant frequency x 10^-6per viable cell in both the absence and presence of metabolic activation (Tables 9 and 12). The GEF value was not exceeded at any test item dose in either the absence or presence of metabolic activation. 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 Vitro Mammalian 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 eight dose levels using a 4‑hour exposure group in the presence of metabolic activation (2% 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 Tert-amyl peroxypivalate (CAS# 29240-17-3) (µg/mL) plated for viability and mutant frequency
4-hour without S9	10, 20, 30, 40, 50, 60
4-hour with S9 (2%)	10, 20, 40, 60, 80, 100

 

Experiment 2

Group	Concentration of Tert-amyl peroxypivalate (CAS# 29240-17-3) (µg/mL) plated for viability and mutant frequency
24-hour without S9	1.25, 2.5, 5, 10, 15, 20
4-hour with S9 (2%)	37.5, 40, 50, 70, 75, 80

 

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.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Genetic toxicity in vivo

Description of key information

There are two in vivo micronucleus studies. In the first oral study in mice the test substance does not induce damage to the chromosomes or the mitotic apparatus of bone marrow cells after two oral administrations, with a 24-hour interval, at the dose-levels of 62.5, 125 or 250 mg/kg/day (expressed as active material). At doses higher than 250 mg/kg bw/day significant toxicity was seen and therefore these doses were chosen in the main study, a depression in the PCE:NCE was not observed.

In the second study substance induced mortality and numerous signs of severe clinical toxicity in the treated animals. There was a depression in the PCE:NCE ratio and although this was not statistically significant it does suggest, along with the clinical observations, that the test article or a metabolite reached the target tissue. Also in this study the test substance did not induce a statistically significant increase in micronuclei in bone marrow PCEs and is considered negative in the mouse bone marrow micronucleus test under the conditions of exposure in this assay.

Kirkland et al. (Mutation Research 775-776 (2014) 69-80) conclude that “Thus, in the case of an Ames-positive chemical, negative results in 2 in vitro mammalian cell tests covering both mutation and clastogenicity/aneugenicity endpoints should be considered as indicative of absence of in vivo genotoxic or carcinogenic potential. ”This therefore implies for the mutagenicity endpoint a negative “In Vitro Mammalian Cell Gene Mutation Test” supported by in silico data is sufficient evidence to waive the need for in vivo gene mutation testing.”

QSAR models (TOPKAT, VEGA, DEREK) are negative for (Ames) mutagenicity (See section 13 of IUCLID for the read across justification document for the output).

 

In this situation the Ames is positive, the MLA is negative and the in vivo micronucleus is negative. In silico methods indicate that the mutagenicity endpoint is negative. Based on the available information further in vivo mutagenicity testing is not required.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

The relative humidity recorded in the animal room was sometimes outside the target range specified in the protocol. This minor deviation was not considered to have compromised the validity or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

Number: 12 male and 12 female mice for the preliminary toxicity test
56 mice: 28 males and 28 females for the cytogenetic study.
Strain: Swiss leo: OFI (lOPS Caw). Reason for this choice: rodent species generally accepted by regulatory authorities for this type of study.
Breeder: Iffa Credo, I'Arbresle, France.
Age: on the day of treatment, the animals were approximately 6 weeks old.
Veterinary care at CIT: upon their arrival at CIT, the animals were given a complete examination to ensure that they were in good clinical conditions.
Acclimatisation: at least 5 days before the day of treatment.
Constitution of groups: upon arrival, the animals were randomly allocated to the groups by sex' Subsequently, each group was assigned to a different treatment group.
Identification: individual tail marking upon treatment.
Environmental conditions
Upon their arrival at CIT, the animals were housed in an animal room, with the following
. environmental conditions:
· temperature: 21 ± 2°C,
· relative humidity: 30 to 70%,
· light/dark cycle: 12 hl12 h (07:00 - 19:00),
· ventilation: about 12 cycles/hour of filtered non-recycled fresh air.
The housing conditions (temperature, relative humidity, light/dark cycle and ventilation) werechecked regularly. The animals were housed by groups in polycarbonate cages. Each cage contained autoclaved sawdust (SICSA, 94142 Alfortville, France).
Bacteriological and chemical analysis of the sawdust, including the detection of possible contaminants (pesticides, heavy metals) of the sawdust are performed by the suppliers.

Food and water
All animals had free access to A04 C pelleted maintenance diet (UAR, 91360 ViIlemoisson-surOrge, France) and tap water (filtered using a 0.22 micron filter). Each batch of food was analysed (composition and contaminants) by the supplier. Bacteriological and chemical analysis of water, including the detection of possible contaminants (pesticides, heavy metals and nitrosarnines) are performed regularly by extemallaboratories. The results of these analyses are archived at CIT.
No contaminants are known to be present in the diet, drinking water or bedding material at levels which may be expected to interfere with or prejudice the outcome of the study.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Administration
· Route for the vehicle and the test substance: oral, since it is a possible route of exposure in man
· Frequency: two treatments separated by 24 hours,
· Volume: 10 mL/kg,
· CPA: oral route, one treatment.
The quantity of each substance administered to each animal was adjusted according to the most
recently recorded body weight.

Frequency of treatment:

Two treatments separated by 24 hours

Post exposure period:

The animals of the treated and vehicle control groups were killed 24 hours after the last
treatment and the animals of the positive control group were killed 24 hours after the single
treatment.

Remarks:

Doses / Concentrations:
62.5, 125 or 250 mg/kg/day (expressed as active material), at a 24-hour interval
Basis:
other: percent active

No. of animals per sex per dose:

In the main study, three groups of five male and five female mice.

Control animals:

yes, concurrent vehicle

Positive control(s):

One group of five males and five females received the positive control test substance
(cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.

Tissues and cell types examined:

Bone marrow smears were then prepared. For each animal, the number of the micronuc1eated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE)
erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Details of tissue and slide preparation:

At the time of sacrifice, all the animals were killed by CO2 inhalation in excess. The femurs of the 'animals were removed and the bone marrow was eluted out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. All the slides were coded for scoring:

For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochrornatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Evaluation criteria:

For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.

Statistics:

When there was no significant within-group heterogeneity, using the heterogeneity chi-square test value (Lovell and colI., 1989), the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the x2 value (Lovell and colI., 1989). When there was Significant within-group heterogeneity, then that group was compared with the control group using a non-parametric analysis, the Mann-Whitney test (Schwartz, 1969).
The student "t" test was used for the PE/NE ratio comparison. Probability values of p < 0.05 was considered as significant.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In order to select the top dose-level for the cytogenetic study, 2000, 1000, 500 and 250 mg/kg/day were administered, to three males and three females. The interval between each administration was 24 hours.
At 2000 and 1000 mg/kg/day, all animals were found dead 24 hours after the first treatment. At 500 mglkg/day, hypoactivity and/or piloerection were noted in 113 males and 1/3 females, 2 and 6 hours after the second treatment. These two animals were found dead 24 hours after the second treatment.
At 250 mglkg/day, piloerection was observed in all animals 6 hours after the second treatment and persisted 18 hours later. The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since observable toxic effects were noted, the top dose-level was based on the toxicity level, such that a higher dose-level was expected to induce lethality.
Consequently, 250 mg/kg/day was selected as the top dose-level. The two other dose-levels were 125 and 62.5 mg/kg/day.

No clinical signs and no mortality were observed in the animals of both sexes given 125 and 62.5 mg/kg/day. At 250 mg/kg/day, piloerection was noted in 3/5 males as well as in 1/5 females from the main group. For both males and females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test substance, were equivalent to those of the vehicle group. The mean values of MPE as wen as the PE/NE ratio for the vehicle control groups were consistent with our historical data. For one female from the positive control group (corresponding to slide 26) no significant increase in the frequency of MPE was noted. However since for the remaining nine animals cyclophosphamide induced a clear increase in the frequency of MPE and since a very significant increase (p < 0.001) in the mean frequency was noted, this was considered as indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

 

Under our experimental conditions, the test substance does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 62.5, 125 or 250 mg/kg/day (expressed as active material).

Conclusions:

Interpretation of results: negative
Under our experimental conditions, the test substance does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hourinterval, at the dose-levels of 62.5, 125 or 250 mg/kg/day (expressed as active material).

Executive summary:

The objective of this study was to evaluate the potential of the test substance to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of mice.

 

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study. In the main study, three groups of five male and five female Swiss leo: OFI (lOPS Caw) mice received two oral treatments of the substance at dose-levels of 62.5, 125 or 250 mg/kg/day (expressed as active material), at a 24-hour interval. One group of five males and five females received the vehicle (com oil) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test substance (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

 

The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since observable toxic effects were noted in the preliminary test, the top dose-level was based on the toxicity level, such that a higher dose-level was expected to induce lethality. Consequently, 250 mg/kg/day was selected as the top dose-level. The two other dose-levels were 125 and 62.5 mg/kg/day. For both males and females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test substance, were equivalent to those of the vehicle group. The mean values of MPE as well as the PE/NE ratio for the vehicle control groups were consistent with our historical data. For one female from the positive control group, no significant increase in the frequency of MPE was noted. However since for the remaining nine animals cyclophosphamide induced a clear increase in the frequency of MPE and since a very significant increase (p<0.001) in the mean frequency was noted, this was· considered as indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

 

Under our experimental conditions, the test substance does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, with a 24-hour interval, at the dose-levels of 62.5, 125 or 250 mg/kg/day (expressed as active material).

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Apparently well conducted GLP study. However, the test article was only identified by the sponsor's trade name. While the study is reliable with restrictions, it was conducted by the IP route, a non-physiologic route of human occupational expsosure. Therefore, this study was not selected as the key study for hazard assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

For the second dose range finding study, the daily animal observations were inadvertently not performed on the 1250 mg/kg dose level males. This had no impact on the integrity of the study

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Young adult male and female mice of the Crl:CD-1 ®(ICR) BR strain were purchased from Charles River Laboratories, Raleigh, NC. This is an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test articles. The protocol for this study was approved by the Covance-IACUC prior to the initiation of dosing.
The animals were acclimated for at least 7 days before being placed on study. The animals were housed up to 5 per cage during quarantine and by sex and dose group after randomization in sanitary polycarbonate cages containing Sani-Chips® Hardwood Chip Laboratory bedding. Each batch of wood chips was analysed by the manufacturer for specific microorganisms and contaminants. The animals were housed under the following climatic conditions: temperature, 64 degrees F - 79°F; humidity, 30% - 70%; light cycle, 12 hours light/dark; at least 10 air changes per hour. A commercial diet, PMI® Feeds, Inc. Certified Rodent Diet® # 5002 (pellets), and tap water were available ad libitum. The feed was analysed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water was analysed biannually on a retrospective basis for specified microorganisms, pesticides, heavy metals, alkalinity, and halogens.
The animals were randomly assigned to study groups and uniquely identified by ear tag. Treatment groups were identified by cage label/card. The animals were weighed prior to dosing and dosed based upon the individual animal weights. The weight variation of the animals did not exceed ±20% of the mean weight of each sex. All animals were dosed on an acute (one-time only) basis.
Personnel handling the animals or working within the animal facilities were required to wear suitable protective garments and equipment.

Route of administration:

intraperitoneal

Vehicle:

mineral oil

Details on exposure:

See materials and methods discussion.

Frequency of treatment:

Dosed by single intraperitoneal injection to six males per dose level/harvest timepoint

Post exposure period:

24 or 48 hours post-dosing.

Remarks:

Doses / Concentrations:
312.5, 625 and 1250 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

6 males per dose group per harvest time

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Tissues and cell types examined:

Bone marrow . Polychromatic erythrocytes (PCEs), were analyzed for the presence of micronuclei

Details of tissue and slide preparation:

The criteria for the identification of micronuclei were those of Schmid (1976).

Evaluation criteria:

The criteria for a positive response was the detection of a statistically significant
positive response for at least one dose level and a statistically significant dose related
response. A test article that did not induce both of these responses was
considered negative. Statistical significance was not the only determinant of a
positive response, the study director also considered 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), a Dunnett's t-test (Dunnett, 1955; 1964)
was used to determine which dose groups, if any, were statistically significantly
different from the vehicle control. Analyses were performed separately for each
sampling time. Additionally, parametric or nonparametric tests for trend may
have been employed to identify any dose-related response.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All animals were examined immediately after dosing, about 1 hour after dosing, and at least daily for the duration of this studies for toxic signs and/or mortalities. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest timepoints.
The test article, Trigonox 125 C-75, induced mortality and numerous signs of severe clinical toxicity in the treated animals. There was a depression in the PCE:NCE ratio at the 312.5 mg/kg and 625 mg/kg dose levels and although this was not statistically significant it does suggest, along with the clinical observations, that the test article or a metabolite reached the target tissue. Trigonox 125 C-75 induced no statistically significant increases in micronucleated PCEs over the levels observed in the vehicle controls at any of the harvest timepoints. The positive control, cyclophosphamide, induced statistically significant increases in micronucleated PCEs as compared to the vehicle controls, with a mean and standard error of 2.80% ± 0.57%.
The vehicle control group had less than approximately 0.4% micronucleated PCEs, for both studies. The positive control group was significantly higher (p < 0.01) than the vehicle control group.

Conclusions:

Interpretation of results: negative
The test article, Trigonox 125 C-75, induced mortality and numerous signs of
severe clinical toxicity in the treated animals. There was a depression in the
PCE:NCE ratio in the two surviving animals at the 1250 mg/kg dose level at the
48 hour time point in the first micronucleus assay. There was also a depression in
the PCE:NCE ratio at the 312.5 mg/kg and 625 mg/kg dose levels at the 24 hour
time point in the first micronucleus assay. Although these were not statistically
significant it does suggest, along,with the clinical observations, that the test article
or a metabolite reached the target tissue. Trigonox 125 C-75 did not induce a
statistically significant increase in micronuclei in bone marrow PCEs and is
considered negative in the mouse bone marrow micronucleus test under the conditions
of exposure in this assay.

Executive summary:

The objective of this study was to evaluate the test article, Trigonox 125 C-75, for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in Crl:CD-1 ®(ICR) BR mouse bone marrow.

In the dose rangefinding and micronucleus studies, the animals dosed with the vehicle control were shared with assay 19396-0-4550ECD. In the micronucleus studies, the animals dosed with the positive control were shared with assay 19396-0-4550ECD. In the first dose range finding study, the test article was solubilized in mineral oil and dosed by intraperitoneal injection to three males and three females per dose level. The animals were dosed at 0, 200, 500, 800, 1500 and 2000 mg/kg and observed for 2 days after dosing for toxic signs and/or mortality. Mortality was observed in all of the animals at the 1500 mg/kg dose level and three males and two females at the 2000 mg/kg dose level. The dosing interval between 800 mg/kg and 1500 mg/kg was explored in a second dose range finding study.

 

In the second dose range finding study, the test article was solubilized in mineral oil and dosed by intraperitoneal injection to three males and three females per dose level. The animals were dosed at 1000 and 1250 mg/kg and observed for 2 days after dosing for toxic signs and/or mortality. The PCE:NCE ratio was evaluated in males and females at the 800, 1000 and 1250 mg/kg (both trial 1 and trial 2 animals). The PCE:NCE ratio was also evaluated in the surviving female at the 2000 mg/kg dose level. No depression in the PCE:NCE ratio was observed in the dose range finding studies. The clinical observations were of similar severity in both sexes therefore only males were selected for testing in the definitive micronucleus assay.

 

Based on the results of the dose range finding studies, the maximum tolerated dose was estimated to be 1250 mg/kg. In the initial micronucleus assay, the test article was solubilized in mineral oil and dosed by intraperitoneal injection to six males per dose level/harvest timepoint. The animals were dosed at 312.5,625 and 1250 mg/kg. The first five surviving animals dosed at the 312.5 and 625 mg/kg dose levels and with the positive control were euthanized approximately 24 hours after dosing for extraction of the bone marrow. The first five surviving animals dosed at the 1250 mg/kg dose level and with the vehicle control were euthanized approximately 24 hours after dosing for extraction of the bone marrow. There was an insufficient number of surviving animals at the 48 hour 1250 mg/kg dose level. This portion of the definitive assay was repeated in a second definitive micronucleus assay. At least 2000 PCEs per animal were analyzed for the frequency of micronuclei.

 

In the second definitive micronucleus assay, the test article was solubilized in mineral oil and dosed by intraperitoneal injection to six males per dose level/harvest timepoint. The animals were dosed at 1000 mg/kg. The first five surviving animals dosed at the 1000 mg/kg dose level and with the vehicle control were euthanized approximately 48 hours after dosing for extraction of the bone marrow. At least 2000 PCEs per animal were analyzed for the frequency of micronuclei. In both micronucleus assays, cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 200 erythrocytes for each animal.

 

The test article, Trigonox 125 C-75, induced mortality and numerous signs of severe clinical toxicity in the treated animals. There was a depression in the PCE:NCE ratio at the 312.5 mg/kg and 625 mg/kg dose levels and although this was not statistically significant it does suggest, along with the clinical observations, that the test article or a metabolite reached the target tissue. Trigonox 125 C-75 did not induce a statistically significant increase in micronuclei in bone marrow PCEs and is considered negative in the mouse bone marrow micronucleus test under the conditions of exposure in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vitro and in vivo study data, the test item is considered to be not mutagenic according to Regulation (EC) No 1272/2008 (CLP), as amended for the 17th time in Regulation (EU) 2021/849.