Di-tert-butyl sec-butylidene diperoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All of the available in vitro studies (Ames, Mouse Lymphoma, Chrome Ab) are negative. No in vivo studies are necessary.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between May 1978 and August 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The procedures followed in the present protocol mimic as closely as possible those described by Ames and coworkers, in Mutation Research 31 (1975), 347-365.

GLP compliance:

no

Remarks:

Study pre-dates GLP

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The study report covers multiple substances. In this report the substance is referred to as Trigonox D-B50.

Target gene:

the histidine locus

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix using liver homogenate of Aroclor-induced rats

Test concentrations with justification for top dose:

0.08, 0.4, 2 and 10 µl test liquid/0.1ml acetone/plate
All dilutions/solutions were prepared immediately before use.

Vehicle / solvent:

acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

0.1, 0.25, 0.5 and 1.0 µg/0.1ml DMSO/plate for all strains

Positive control substance:

other: 2-aminoanthracene

Remarks:

with and without S9-mix

Details on test system and experimental conditions:

Bacterial strains
Origin
The Salmonella typhimurium mutants used viz. s. typh. TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were provided by Dr. B.N. Ames, Berkeley, California, USA. They are stored as frozen cultures at -80ºC.

Cultures for testing
To obtain cultures for mutagenesis testing, nutrient broth is inoculated with a thawed aliquot of the appropriate bacterial culture (0.1 ml per 10 ml nutrient bouillon) and grown up overnight with shaking at 37°C for 16 hours. The optical density is used as a measure for the total number of bacteria per ml. The bacterial cultures are stored in a refrigerator at 5ºC for up to four days.

MUTAGENESIS ASSAYS
Plate incorporation assay - Solids and non-volatile liiquids
The procedure used in this assay has been described in detail by Ames et al. (1975). Briefly the procedure is as follows: to 2 ml molten top agar (56°C) are added in this sequence 0.1 ml of a fully grown culture of one of the tester strains containing about 10^9 cells/ml , 0.1 ml of the appropriate dilution/suspension of the test product and 0.5 ml of the S-9 mix if indicated. The ingredients are thoroughly mixed and immediately poured onto minimal glucose agar plates.
After the top agar has been allowed to harden, the plates are incubated at 37°C for three days. Then the colonies (revertants which are histidine independent) are counted and the background lawn of bacterial growth is examined microscopically. Routinely, four to five different doses and a solvent control are tested with 5 different strains, viz. Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 99 and TA 100 with and without the liver microsome activation system. All determinations are made in triplicate.

S-9 mix and test product are checked for sterility. The dose range used in the mutagenesis assay is based on a preliminary test performed to assess the toxicity of the compound for the bacteria. If possible, the lowest toxic dose is taken as the highest dose for mutagenesis assay. In the case of questionable results the plate incorporation assay is partly repeated and/or, a number of revertant colonies is checked for histidine requirement and for other strain characteristics if appropriate.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

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:

The results obtained with the various peroxides did not provide evidence of a dose-related, reproducible increase in the numbers of his+ revertants with any of the five tester strains, either in the presence or in the absence of S-9 mix.
Incorporation of 1 µl Triqonox 48 and of 0.01 µl Trigonox HM per plate appeared slightly toxic to the bacteria as revealed by a less dense background lawn of bacterial growth. Incorporation of the other four peroxides did not reveal any indication of an interference of chemical toxicity with mutagenicity testing.
From the present results it can be concluded that none of the six peroxides examined revealed mutagenic activity in the plate incorporation assay with s. typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 in the presence or absence of the liver microsome activation system under the test conditions employed in this evaluation.

Conclusions:

From the present results it can be concluded that none of the six peroxides examined revealed mutagenic activity in the plate incorporation assay with s. typhimurium TA 1535, TA 1537, TA 1538, TA 98 or TA 100 in the presence or absence of the liver microsome activation system under the test conditions employed in this evaluation.

Executive summary:

1. The mutagenic activity of six organic peroxides: Trigonox B, Laurox, Trigonox 48, Trigonox HM, Trigonox D-B50 and Perkadox SE 10 was examined in the Salmonella/microsome mutagenicity test, using a set of five histidine requiring mutants of S. typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and liver homogenate of Aroclor-induced rats.

2. Incorporation of the peroxides up to non-inhibitory levels did not increase the number of his+ revertants in any of the five tester strains, either in the presence or in the absence of the liver microsome activation system.

3. It was concluded that the present results did not reveal mutagenic activity with any of the test products in the Salmonella/microsome mutagenicity test.

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: 21 August 2015 Experimental completion date: 15 September 2015

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 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/β-naphthoflavone

Test concentrations with justification for top dose:

Main test: 0, 0.63, 1.25, 2.5, 5, 10, 12, 14, 16 and 18 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

at 400 µg/mL and 150 µg/mL for 4-hour and 24-hour exposures respectively, was used as the positive control in the absence of metabolic activation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

at 1.5 µg/mL was used as the positive control in the presence of metabolic activation.

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 234.34 therefore the maximum proposed dose level in the solubility test was set at 2343 µg/mL, the maximum recommended 10mM dose level, and the purity of the test item was 49% which was not accounted for at the request of the sponsor. However the maximum dose level used was 1171.5 µg/mL, as acetone is dosed at 0.5% in this assay, due to the toxicity of acetone at higher concentrations. 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:

µg/mL 0 4.58 9.15 18.30 36.61 73.22 146.44 292.88 585.75 1171.5
pH 7.30 7.28 7.28 7.29 7.29 7.31 7.33 7.32 7.32 7.31
mOsm 382 381 385 384 384 384 380 374 369 353


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 (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch BCBN1209V (CAS No.62-50-0) at 400 µg/mL and 150 µg/mL for 4-hour and 24-hour exposures respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch MKBS0021V(CAS No. 6055-19-2) at 1.5 µg/mL was used as the positive control in the presence of metabolic activation.


Microsomal Enzyme Fraction
PB/BNF S9 was prepared in-house on 01 March 2015 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 Envigo 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 Envigo 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 the Main Experiment.

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 4.58 to 1171.5 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 using a Coulter counter and then serially diluted to 2 x 105 cells/mL.

The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post 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
Main Experiment
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. For the 4-hour exposure groups both with and without metabolic activation, the cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. In the 24-hour exposure in absence of metabolic activation the cells were processed to give 0.3 x 106 cells/mL in 10 mL cultures established in 25 cm2 tissue culture flasks The treatments were performed in duplicate (A + B), at eight dose levels of the test item (0.75 to 18 µg/mL in the 4-hour exposure in the absence of metabolic activation 0.63 to 36 µg/mL, in the presence of metabolic activation and 0.63 to 36 µg/mL in the 24 hour exposure), vehicle and positive controls. To each universal was added 2 mL of S9-mix (2%) if required, 0.1 mL of the treatment dilutions, (0.15 or 0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL (R10 is used for the 24-hour exposure group).

The treatment vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Evaluation criteria:

Please refer to "Any other information on materials and methods"

Statistics:

Please refer to "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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

 Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 4.58 to 1171.5 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

 

Dose (mg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
4.58	86	93	90
9.15	65	92	86
18.30	3	88	37
36.61	0	1	0
73.22	0	0	0
146.44	0	0	0
292.88	0	0	0
585.75	0	0	0
1171.5	0	0	0

 

In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was evidence of marked reductions of %RSG values of cells treated with test item. The sharp toxicity curve indicated that it would be difficult to achieve optimum toxicity in the main test. In the 4-hour exposures, a precipitate of the test item was observed at and above 73.22 µg/mL in absence of metabolic activation, and at and above 146.44 µg/mL in the presence of metabolic activation. In 24-hour exposure group, a precipitate of the test item was observed at and above 292.88 µg/mL. In the subsequent mutagenicity experiments the maximum dose was limited by test item-induced toxicity.

 

Mutagenicity Test

 4-hour exposure in the absence and presence of metabolic activation

There was evidence of toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values (Tables 3 and 6). There was evidence of reductions in viability (%V), therefore indicating that residual toxicity occurred, in both the absence and presence of metabolic activation. Optimum levels of toxicity were achieved in both exposure groups. The toxicity observed at 18 µg/mL in the absence of metabolic activation exceeded the upper acceptable limit of 90% and, therefore, this dose level was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances.

 

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10^-6viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

 

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6per viable cell, at any dose level, in either the absence or presence of metabolic activation (Tables 9 and 12). It should be noted that all mutant frequency values were within the acceptable range for a vehicle control culture, including those with excessive toxicity.

 

 24-hour exposure in the absence of metabolic activation

There was evidence of toxicity following exposure to the test item as indicated by the %RSG and RTG values. There was evidence of slight reductions in viability (%V), therefore indicating that residual toxicity occurred in this exposure group. Optimum levels of toxicity were achieved. The excessive toxicity observed at and above 30 µg/mL 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.

 

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a marked effect on the toxicity of the test item. Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10^-6viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

 

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6per viable cell, at any dose level, in either the absence or presence of metabolic activation. It should be noted that all mutant frequency values were within the acceptable range for a vehicle control culture.

 

Conclusions:

Interpretation of results (migrated information):
negative

The test item, 2,2-di(tert-butylperoxy) butane (CAS # 2167-23-9) 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", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

 

 

Methods…….

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

 

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

 

Main Experiment

Group	Concentration of 2,2-di(tert-butylperoxy) butane (CAS # 2167-23-9) (µg/mL) plated for mutant frequency
4-hour without S9	5, 10, 12, 14, 16, 18
4-hour with S9 (2%)	10, 12, 18, 24, 30, 36
24-hour without S9	2.5, 5, 10, 12, 18, 24

 

 

Results……..

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

 

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels, either with or without metabolic activation.

 

Conclusion

The test item, 2,2-di(tert-butylperoxy) butane (CAS # 2167-23-9) did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental staring date: 7 February 2001 Experimental completion date: 18 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

other: In vitro mammalian chromosome aberration test

Specific details on test material used for the study:

The substance is referred to as LUPEROX 220M50 in this report
chemical name: 2,2-DI-(t-BUTYLPEROXY) BUTANE]
batch number: ATE 512001 1001 MXL
description: colourless liquid
date of receipt: 11 January 2001
storage conditions: at room temperature and protected from light
purity: 50.5% in isododecane
expiry date: November 2001

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human lymphocytes are primary cell cultures recommended by international regulations for the mammalian chromosome aberration test; they have a stable karyotype with 46 chromosomes and an average cell cycle time of 12-14 hours.
Human lymphocytes were prepared from whole blood samples obtained from two healthy donors and collected into heparinised sterile tubes.

Cytokinesis block (if used):

colcemid solution (10 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix consists of induced enzymatic systems contained in rat liver microsomal fraction (S9 fraction) and the cofactors necessary for their function.

Test concentrations with justification for top dose:

All the dose-levels were expressed as active substance, taking into account a purity of 50.5%. Dose-levels were selected on the basis of pH, osmolality and solubility.

Experiments without S9 mix:
With a treatment volume of 1 5 μL/5.5 mL culture medium, the treatment-levels were as follows:
17.43, 34.86, 69.72, 139.4, 278.9, 557.8, 836.7 and 1115.6 μg/mL, for the first experiment,
34.86, 69.72, 139.4, 278.9, 557.8 and 836.7 μg/mL, for the second experiment.

Experiments with S9 mix:
With a treatment volume of 15μL/5.5 mL culture medium, the treatment-levels were as follows:
17.43, 34.86, 69.72, 139.4, 278.9, 557.8, 836.7 and 1115.6 μg/mL, for the first experiment,
34.86, 69.72, 139.45, 278.9, 418.35 and 557.8 μg/mL, for the second experiment.

Vehicle / solvent:

The vehicle was ethanol, batch No. V8M084248M 5 (Carlo Erba, Val de Reuil, France).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

3 µg/mL or 0.2 µg/mL in the three hour and contunous treatments respectively

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

50 µg/mL and 25 mg/mL (the treatment which gave satisfactory response in terms of quality and quantity of metaphases and extent of chromosomal damage was analysed)

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

Treatment
A range of dose-levels of LUPEROX 220M50 (two cultures/dose-level) were tested, with and without metabolic activation. In case of treatment with S9 mix, the final concentration of S9 fraction in the culture medium was 1.5% (i.e. 15% S9 mix).
In each experiment, the following controls were included using at least duplicate cultures:
vehicle controls: cultures treated with the vehicle,
positive controls: cultures treated (only for the first harvest time) with:
- MMC, in the absence of S9 mix,
- CPA, in the presence of S9 mix.
To prepare each culture, 0.5 mL of heparinised whole blood was added to 5 mL of RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and phytohaemagglutinin (PHA: a mitogen to stimulate the lymphocytes to divide). The cultures were then placed at 37°C for 48 hours.

First experiment
Lymphocyte cultures were then exposed to the test or control substances, both in the absence and presence of S9 mix, for 3 hours then rinsed. One and a half hours before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. Harvest time was 20 hours from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.
As this experiment gave positive results, no other experiments were undertaken.
Second experiment
- without S9 mix, cells were exposed continuously to the test or control substances.
- with S9 mix, cells were exposed to the test or control substances for 3 hours and then rinsed.
One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. Harvest times were 20 hours and 44 hours from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later.

Preparation of microscope slides
After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

Microscopic evaluation
The cytotoxicity of the test substance was evaluated using the mitotic index (number of cells in mitosis/1000 cells examined), which indicates whether a substance induces mitotic inhibition.
Mitotic index was determined without blind scoring.
Analysis of 200 metaphases/dose-level (with 44 to 46 chromosomes) was made, with 100 metaphases/culture whenever possible. Only 50 metaphases/culture were analysed when at least 10% cells with structural chromosome aberrations were observed. All analyses were performed with blind scoring.
The following structural aberrations were recorded for each metaphase: gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverizations) and the following numerical aberrations: polyploidy and endoreduplication.

Treatment of results
A cell having one or more of the above-mentioned structural chromosome aberrations was recorded as a single cell with structural chromosome aberrations. Therefore the total frequency of cells with structural chromosome aberrations was not necessarily equivalent to the total number of aberrations.

Evaluation criteria:

A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.

Statistics:

For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using the X2 test, in which p = 0.05 was used as the lowest level of significance.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test substance was tested as supplied or diluted in the vehicle (ethanol).
In the culture medium, the dose-level of 1115.6 μg/mL (obtained with a treatment with 15 μL of the test substance as supplied) showed a marked emulsion. At this dose-level, the pH was about 7.1 (as for the vehicle control) and the osmolality equal to 293 mOsm/kg H20 (340 for the vehicle control).

Experiments without S9 mix:

A slight to moderate emulsion was observed at the end of the treatment period at dose-levels ≥ 557.8 μg/mL.

Cytotoxicity:
A marked decrease in the mitotic index (-69%) was noted in the first experiment at 1115.6 μg/mL.
In the second experiment, no noteworthy decrease in the mitotic index was induced at the 20-hour harvest time. At the 44-hour harvest time, a 49% decrease in the mitotic index was noted at the highest dose-level test of 836.7 μg/mL and this was related to the female donor.

Chromosomal aberration analysis:
The dose-levels selected for metaphase analysis were as follows:
- 1115.6, 836.7 and 557.8 μg/mL, for the first experiment, the dose-level of 1115.6 μg/mL being the highest dose-level used for treatment and inducing 69% decrease in the mitotic index.
- 836.7, 557.8 and 278.9 μg/mL, for the second experiment at the 20-hour harvest time, the dose-level of 836.7 μg/mL being the highest dose-level used for treatment
- 836.7 μg/mL, for the second experiment at the 44-hour harvest time, this dose-level being the highest dose-level used for treatment. At this dose, the metaphases were analysed only in the male donor, due the strong decrease in the mitotic index induced in the female donor.
In the first experiment, after a 3-hour treatment, no significant increase in the frequency of aberrant cells was noted at the dose-levels tested.
In the second experiment, a slight but a statistically significant (p < 0.05) increase in the frequency of aberrant cells was noted at 278.9 μg/mL. However, this increase was not considered as biologically relevant since the frequency of aberrant cells (3%) was very consistent with the vehicle control historical range (0-2.5%) and no noteworthy increase was induced at higher dose-levels. The statistically significant difference was attributed to the low vehicle control frequency of aberrant cells (0%).
No noteworthy increase in the frequency of aberrant cells was induced at the 44-hour harvest time.

Experiments with S9 mix:

A slight to moderate emulsion was observed at the end of the treatment period at dose-levels ≥ 557.8 μg/mL.

Cytotoxicity:
At the 20-hour harvest time, a marked decrease in the mitotic index (68-80%) was noted at dose-levels ≥ 418.35 μg/mL.
At the 44-hour harvest time, a slight to marked (30-82%) decrease in the mitotic index was induced at dose-levels ≥ 418.35 μg/mL.

Chromosomal aberration analysis:
The dose-levels selected for metaphase analysis were as follows:
- 557.8, 278.9 and 139.4 μg/mL, for the first experiment, the dose-level of 557.8 μg/mL inducing 68% decrease in the mitotic index.
- 418.35, 278.9 and 139.45 μg/mL, for the second experiment at the 20-hour harvest time, the dose-level of 418.35 μg/mL inducing 75% decrease in the mitotic index.
- 418.35 μg/mL, for the second experiment at the 44-hour harvest time, this dose-level being the highest readable dose-level and showed 30% decrease in the mitotic index.

In the first experiment, a slight but a statistically significant (p < 0.05) increase in the frequency of aberrant cells was noted at 278.9 μg/mL. However, this increase was not considered as biologically relevant since the frequency of aberrant cells (4%) was very consistent with the vehicle control historical range (0-3%) and no noteworthy increase was induced at the higher dose-level of 557.8 μg/mL. In addition, in the second experiment, the increase in the frequency of aberrant cells was not reproduced.
No noteworthy increase in the frequency of aberrant cells was induced at the 44-hour harvest time.
The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.

Conclusions:

Under the experimental conditions, the test substance LUPEROX 220M50 (batch No. ATE 512-0011-001) does not induce chromosome aberrations in cultured human lymphocytes.

Executive summary:

The objective of this study was to evaluate the potential of the test substance LUPEROX 220M50 to induce chromosome aberrations in cultured human lymphocytes.

Methods

The test substance was tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. No preliminary cytotoxicity test was performed. Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used for the first experiment and dose-levels for scoring of chromosomal aberrations were selected on the basis of cytotoxicity indicated by reduction of mitotic index (MI). For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemagglutinin) and incubated at 37°C in a humidified atmosphere of 5% C02 I 95% air, for 48 hours.

First experiment

Lymphocyte cultures were exposed to the test or control substances, with or without S9 mix, for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis.

As this first experiment was negative, the study was continued with a second experiment.

Second experiment

- without S9 mix, cells were exposed continuously to the test or control substances.

- with S9 mix, cells were exposed to the test or control substances for 3 hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively. One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis.

For both experiments, after hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

The test substance LUPEROX 220M50 was dissolved in ethanol.

The dose-levels of the positive controls were as follows:

without S9 mix, mitomycin C: 3 μg/mL (3 hours of treatment) or 0.2 μg/mL (continuous treatment),

with S9 mix, cyclophosphamide: 25 or 50 μ g/mL.

Results

All the dose-levels were expressed as active substance, taking into account a purity of 50.5%.

In the culture medium, the dose-level of 1115.6 μg/mL (obtained with a treatment with 15 μL of the test substance as supplied) showed a marked emulsion. At this dose-level, the pH and the

osmolality were equivalent to the vehicle control culture values.

Experiments without S9 mix:

With a treatment volume of 15 μL/5.5 mL culture medium, the treatment-levels were as follows:

- 17.43, 34.86, 69.72, 139.4, 278.9, 557.8, 836.7 and 1115.6 μg/mL, for the first experiment,

- 34.86, 69.72, 139.4, 278.9, 557.8 and 836.7 μg/mL, for the second experiment.

Cytotoxicity:

A slight to marked decrease in the mitotic index was noted mainly at dose-levels ≥ 836.7 μg/mL, depending on the experiment.

Chromosomal aberration analysis:

The dose-levels selected for metaphase analysis were as follows:

- 1115.6, 836.7 and 557.8 μg/mL, for the first experiment,

- 836.7, 557.8 and 278.9 μg/mL, for the second experiment at the 20-hour harvest time,

- 836.7 μg/mL, for the second experiment at the 44-hour harvest time.

No increase in the frequency of aberrant cells which could be considered as biologically relevant was noted.

Experiments with S9 mix:

With a treatment volume of 15 μL/5.5 mL culture medium, the treatment-levels were as follows:

- 17.43, 34.86, 69.72, 139.4, 278.9, 557.8, 836.7 and 1115.6 μg/mL, for the first experiment,

- 34.86, 69.72, 139.45, 278.9, 418.35 and 557.8 μg/mL, for the second experiment.

Cytotoxicity:

A slight to marked decrease in the mitotic index was noted at dose-levels ≥ 418.35 μg/mL, depending on the harvest time.

Chromosomal aberration analysis:

The dose-levels selected for metaphase analysis were as follows:

- 557.8, 278.9 and 139.4 μg/mL, for the first experiment,

- 418.35, 278.9 and 139.45 μg/mL, for the second experiment at the 20-hour harvest time,

- 418.35 μg/mL, for the second experiment at the 44-hour harvest time.

No increase in the frequency of aberrant cells which could be considered as biologically relevant was noted.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.

Conclusion

Under our experimental conditions, the test substance LUPEROX 220M50 does not induce chromosome aberrations in cultured human lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo studies are necessary.

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:

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

All of the available in vitro studies (Ames, Mouse Lymphoma, Chrome Ab) are negative. Available data are conclusive, and no in vivo genotoxicity studies are necessary.