2,2'-(2-methylpropylidene)bis[4,6-xylenol]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro Ames Assay

Key value determined in a GLP accredited laboratory study in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay, performed in accordance with OECD Guideline 471 and EU Method B.13/14.

In Vitro Chromosome Aberration

Key value determined in a GLP accredited laboratory study to induce chromosome aberrations in cultured peripheral human lymphocytes, perfomed in accordance with OECD Guideline 473.

In vitro mammalian cell gene mutation test

Key value determined in a GLP accreidted laboratory study to induce the forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells, performed in accordance with OECD Guideline 490.

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:

28 April 2016 to 12 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study performed in accordance with OECD, MITI & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium (uvrB-)Escherichia coli (WP2uvrA)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mixture

Test concentrations with justification for top dose:

Lowinox® 22IB46 was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. Lowinox® 22IB46 precipitated on the plates at the dose levels of 1600 and 5000 μg/plate.

Vehicle / solvent:

The vehicle of the test article, being ethanol (Extra pure, Merck, Darmstadt, Germany).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

Cell culture
Preparation of bacterial cultures: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for testing.
Agar plates: Agar plates (ø 9 cm) containing 25 ml glucose agar medium.
Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Sigma).
Top agar: Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions: All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 35.0 – 38.0°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Metabolic activation system: Rat liver microsomal enzymes (S9 homogenate) was obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that had been injected intraperitoneal with Aroclor 1254 (500 mg/kg body weight). Each S9 batch is characterised with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Preparation of S9-mix: S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck).
The above solution was filter (0.22 μm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Study design
First experiment: Seven concentrations of the test item, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Second experiment: Based on the results of the first mutation assay, five doses (increasing with approximately half-log steps) of the test item were selected and tested in triplicate in each strain in the second experiment. The highest concentration of Lowinox® 22IB46 used in the second mutation assay was 5000 μg/plate.

Experimental procedure: The test item was tested both in the absence and presence of S9-mix in each strain, in two independent experiments. The vehicle control and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in ethanol, and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.

Colony counting: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD, EC and MITI).Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames) (TA1535: 2006, TA1537: 2009, TA98: 2015, TA100: 2015) and (Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK) (WP2uvrA, 2008)

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

First mutation experiment: Lowinox® 22IB46 was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix.
Precipitate: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 1600 and 5000 μg/plate at the end of the incubation period.
Toxicity: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutagenicity: No increase in the number of revertants was observed upon treatment with Lowinox® 22IB46 under all conditions tested.

Second mutation assay: To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based on the results of the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate.
Precipitate: Precipitation of Lowinox® 22IB46 on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 1600 and 5000 μg/plate at the end of the incubation period.
Toxicity: With the exception of tester strain TA98 (absence of S9-mix) and TA1535 (presence of S9-mix) where an extreme reduction of the revertant colonies was observed at the highest tested concentration), there was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Mutagenicity: No increase in the number of revertants was observed upon treatment with Lowinox® 22IB46 under all conditions tested.

Experiment 1: Mutagenic response of Lowinox®22IB46 in theSalmonella typhimuriumreverse mutation assay

and in theEscherichia colireverse mutation assay

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain
	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
Without S9-mix
Positive control Solvent control 5.4 17 52 164 512 1600 5000	1158 ± 6 ± 10 ± 10 ± 10 ± 10 ± 14 ± 9 ± 19 ±	16 2 5 6 5 2 5^NP 2^MP 2^n HP	844 ± 4 ± 5 ± 5 ± 4 ± 4 ± 7 ± 6 ± 5 ±	54 3 1 2 3 1 4^NP 5^MP 1^n HP	1972 ± 16 ± 21 ± 23 ± 24 ± 25 ± 22 ± 19 ± 20 ±	153 5 3 1 5 2 1^NP 3^MP 4^n HP	1076 ± 102 ± 96 ± 108 ± 111 ± 111 ± 84 ± 119 ± 69 ±	37 14 14 8 5 5 5^NP 20^MP 7^n HP	1811 ± 19 ± 20 ± 21 ± 20 ± 22 ± 27 ± 22 ± 18 ±	55 5 9 2 8 7 6^NP 1^MP 10^n HP
With S9-mix^1
Positive control Solvent control 5.4 17 52 164 512 1600 5000	411 ± 8 ± 7 ± 8 ± 4 ± 13 ± 9 ± 6 ± 8 ±	8 3 4 2 3 4 4^NP 2^MP 3^n HP	370 ± 4 ± 7 ± 6 ± 6 ± 4 ± 5 ± 6 ± 6 ±	96 5 3 1 2 2 2^NP 1^MP 3^n HP	1297 ± 25 ± 28 ± 23 ± 22 ± 29 ± 28 ± 19 ± 26 ±	66 6 2 1 7 2 3^NP 2^MP 1^n HP	1731 ± 99 ± 119 ± 115 ± 96 ± 83 ± 110 ± 122 ± 91 ±	113 2 10 11 6 16 19^NP 4^MP 7^n HP	624 ± 25 ± 26 ± 26 ± 28 ± 25 ± 28 ± 21 ± 25 ±	17 8 3 6 5 1 6^NP 6^MP 2^n HP

¹Plate incorporation assay (5% S9)

^HPHeave Precipitate

^MPModerate Precipitate

^NPNo precipitate

ⁿNormal bacterial background lawn

 

Experiment 2: Mutagenic response of Lowinox®22IB46 in theSalmonella typhimuriumreverse mutation assay

and in theEscherichia colireverse mutation assay

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain
	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
Without S9-mix
Positive control Solvent control 52 164 512 1600 5000	229± 6 ± 7 ± 10 ± 7 ± 5 ± 5 ±	13 2 2 4 2^NP 1^MP 1^n MP	686 ± 5 ± 7 ± 7 ± 7 ± 6 ± 4 ±	56 5 3 6 3^NP 2^MP 2^n MP	1407 ± 17 ± 20 ± 22 ± 23 ± 16 ± 6 ±	74 8 3 3 7^NP 7^MP 2^n MP	1139 ± 119 ± 133 ± 119 ± 128 ± 108 ± 114 ±	51 11 9 16 16^NP 12^MP 22^n MP	1190 ± 24 ± 24 ± 23 ± 22 ± 17 ± 18 ±	40 5 5 6 11^NP 3^MP 7^n MP
With S9-mix^1
Positive control Solvent control 52 164 512 1600 5000	861 ± 10 ± 7 ± 9 ± 11 ± 5 ± 3 ±	57 2 5 2 4^NP 2^MP 1^n MP	545 ± 8 ± 7 ± 7 ± 10 ± 7 ± 8 ±	44 2 3 3 4^NP 3^MP 2^n MP	683 ± 25 ± 26 ± 27 ± 25 ± 14 ± 19 ±	69 6 6 7 6^NP 5^MP 7^n MP	1453 ± 110 ± 92 ± 98 ± 71 ± 77 ± 81 ±	90 6 11 15 6^NP 3^MP 10^n MP	502 ± 34 ± 31 ± 34 ± 23 ± 24 ± 24 ±	13 12 10 11 4^NP 8^MP 6^n MP

¹Plate incorporation assay (10% S9)

^MPModerate Precipitate

^NPNo precipitate

ⁿNormal bacterial background lawn

 

Conclusions:

Based on the results of this study it is concluded that Lowinox® 22IB46 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of Lowinox® 22IB46 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

 

Lowinox® 22IB46 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by induced by Aroclor 1254).

 

The study procedures described in this report are in compliance with the following guidelines:

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test". (adopted July 21, 1997).

- European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.

- Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised March 31st, 2011).

 

Batch WB44L0016 of Lowinox® 22IB46 was a white to cream coloured powder with a purity of 99.7%. The test item was dissolved in ethanol.

 

In the first mutation assay, Lowinox® 22IB46 was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. Lowinox® 22IB46 precipitated on the plates at the dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

 

In the second mutation assay, Lowinox® 22IB46 was tested up to concentrations of 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix. Lowinox® 22IB46 precipitated on the plates at the dose levels of 1600 and 5000 μg/plate. Cytotoxicity, as evidenced by a biologically decrease in the number of revertants, was observed in tester strain TA98 in the absence of S9-mix and TA1535 in the presence of S9-mix at the highest tested concentration.

 

Lowinox® 22IB46 did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Based on the results of this study it is concluded that Lowinox® 22IB46 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 March 2016 to 15 July 2016

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)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted September 26, 2014).

Deviations:

yes

Remarks:

see "Any other information"

GLP compliance:

yes

Type of assay:

other: chromosome aberrations

Specific details on test material used for the study:

Test item: 207366/A
Identification: Lowinox® 22IB46
Appearance: White to cream coloured powder
Batch: WB44L0016
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 18 November 2018 (retest date)
Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: 2,2’-(2-methylpropylidene)bis[4,6-xylenol]
CAS Number: 33145-10-7

Target gene:

Structural chromosome aberrations.

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (e.g. OECD).
Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2015) are presented below:
Dose range finding study: age 27, AGT = 13.5 h
First cytogenetic assay: age 34, AGT = 12.9 h
Cytogenetic assay 1A: age 33, AGT = 14.2 h
Second cytogenetic assay: age 30, AGT = 12.9 h
Cytogenetic assay 2A: age 35, AGT = 12.9 h

Cell culture
Blood samples: Blood samples were collected by venepuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.
Culture medium: Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) (Life technologies) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).
Lymphocyte cultures: Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Remel, Europe Ltd., Dartford, United Kingdom) was added.
Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 55 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.7 - 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First cytogenetic assay: up to 50 and 70 μg/ml.
Second cytogenetic assay: up to 45 μg/ml

Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay.

Vehicle / solvent:

The vehicle for the test item was dimethyl sulfoxide.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Metabolic activation system
Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per ml physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life technologies).
The above solution was filter (0.22 μm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Study design

Dose range finding test
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Lowinox® 22IB46 was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.
Lymphocytes (0.4 ml blood of a healthy donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of Lowinox® 22IB46 for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time.
The highest tested concentration was determined by the solubility of Lowinox® 22IB46 in the culture medium.
After 3 h exposure to Lowinox® 22IB46 in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 ml culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time).
Cytotoxicity of Lowinox® 22IB46 in the lymphocyte cultures was determined using the mitotic index.
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

Cytogenetic assay
The cytogenetic assay was carried out as described by Evans, 1984 (2) with minor modifications. Lowinox® 22IB46 was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments. To be able to select appropriate dose levels for scoring of chromosome aberrations several repeat assays had to be performed.
First cytogenetic assay
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of Lowinox® 22IB46 for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 ml culture medium and incubated for another 20 - 22 h (24 h fixation time). Appropriate negative and positive controls were included in the first cytogenetic assay.
Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay. As clear negative results were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary. The follow up experiment was performed with the following modifications of experimental conditions.
Second cytogenetic assay
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of Lowinox® 22IB46 for 24 h and 48 h in the absence of S9-mix.
The cells were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time). Appropriate negative and positive controls were included in the second cytogenetic assay.

Chromosome preparation
During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/ml medium) (Acros Organics, Geel, Belgium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v).

Preparation of slides
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Histolab, Gothenburg, Sweden) and mounted with a coverslip in an automated cover slipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

Mitotic index/dose selection for scoring of the cytogenetic assay
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of 55 ± 5%, whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

Analysis of slides for chromosome aberrations
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with Charles River Den Bosch study identification number and code was placed over the marked slide. One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated. Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for chromosome aberrations.

Rationale for test conditions:

Not specified

Evaluation criteria:

Acceptability of the assay
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analysed by the Fisher’s exact test (one-sided, p < 0.05).

Statistics:

Data evaluation and statistical procedures
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

In case the Fisher’s exact test shows that there are statistically significant differences between one or more of the test item groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

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:

Dose range finding test
At a concentration of 200 μg/ml Lowinox® 22IB46 precipitated in the culture medium at the 24 h and 48 h exposure time. At the 3 h exposure time the test item precipitated in the culture medium at a concentration of 400 μg/ml. In the dose range finding study, blood cultures were treated with 5.4, 17, 52, 200 and 400 μg Lowinox® 22IB46/ml culture medium with and without S9-mix.

First cytogenetic assay
Further investigation showed that a concentration of 150 μg/ml already precipitated in the culture medium.
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
Without S9-mix: 5, 15, 25, 50, 75 and 100 μg/ml culture medium (3 h exposure time, 24 h fixation time).
With S9-mix: 5, 15, 25, 50, 75, 100, 125 and 150 μg/ml culture medium (3 h exposure time, 24 h fixation time).
In the presence of S9-mix no appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 50 μg/ml not enough cytotoxicity was observed (32%), whereas the next higher concentration of 75 μg/ml was too toxic for scoring (72%).
The experiment was repeated in cytogenetic assay 1A.
With S9-mix: 5, 30, 40, 50, 60, 70 and 80 μg/ml culture medium (3 h exposure time, 24 h fixation time).
The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix: 5, 25 and 50 μg/ml culture medium (3 h exposure time, 24 h fixation time).
With S9-mix: 5, 40 and 70 μg/ml culture medium (3 h exposure time, 24 h fixation time).
Both in the absence and presence of S9-mix, Lowinox® 22IB46 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
Both in the absence and presence of S9-mix, Lowinox® 22IB46 did not increase the number of polyploid cells and cells with endo-reduplicated chromosomes.

Second cytogenetic assay
To obtain more information about the possible clastogenicity of Lowinox® 22IB46, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Lowinox® 22IB46 in the absence of S9-mix for 24 or 48 hours. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 5, 15, 25, 35, 45 and 55 μg/ml culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time).
At the 48 h exposure time no appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 35 μg/ml not enough cytotoxicity was observed (36%), whereas the next higher concentration of 45 μg/ml was too toxic for scoring (84%).
The experiment was repeated in cytogenetic assay 2A.
Without S9-mix: 5, 15, 25, 30, 35, 40, 45 and 50 μg/ml culture medium (48 h exposure time, 48 h fixation time).
Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 5, 35 and 45 μg/ml culture medium (24 h exposure time, 24 h fixation time). 5, 40 and 45 μg/ml culture medium (48 h exposure time, 48 h fixation time).
At the 24 h continuous exposure time, Lowinox® 22IB46 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
At the 48 h continuous exposure time, Lowinox® 22IB46 induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested concentration of 45 μg/ml only. Although a significant trend was observed, the increase was within the 95% control limits of the negative control historical data range. Therefore this increase is not considered biologically relevant.
Lowinox® 22IB46 did not induce a biologically relevant increase in the number of polyploid cells and cells with endo-reduplicated chromosomes.

Mitotic index of human lymphocyte cultures treated with Lowinox® 22IB46

in the dose range finding test

Lowinox® 22IB46 concentration (μg/ml)	Number of metaphases
	Absolute	Number of cells scored	Percentage of control
Without metabolic activation (-S9-mix)
3 h exposure time, 24 h fixation time
Control^a1) Control^a2) 5.4 17 52 200 400^b)	98 86 84 81 29 ^_c) ^_c)	1008 1000 1002 1003 1000 ^_c) ^_c)	100 100 86 83 30 ^_c) ^_c)
24 h exposure time, 24 h fixation time
Control^a1) Control^a2) 5.4 17 52 200^b) 400^b)	92 102 102 82 11 ^_c) ^_c)	1002 1006 1002 1000 1004 ^_c) ^_c)	100 100 111 89 12 ^_c) ^_c)
48 h exposure time, 48 h fixation time
Control^a1) Control^a2) 5.4 17 52 200^b) 400^b)	95 55 87 83 2 ^_c) ^_c)	1000 1000 1005 1000 1000 ^_c) ^_c)	100 100 92 87 2 ^_c) ^_c)

a) Dimethyl sulfoxide 1% (1) and 2% (2) respectively.

b) Lowinox® 22IB46 precipitated in the culture medium

c) Cell lysis

 

Mitotic index of human lymphocyte cultures treated with Lowinox® 22IB46

in the dose range finding test

Lowinox® 22IB46 concentration (μg/ml)	Number of metaphases
	Absolute	Number of cells scored	Percentage of control
With metabolic activation (+S9-mix)
3 h exposure time, 24 h fixation time
Control^a1) Control^a2) 5.4 17 52 200 400^b)	120 110 114 96 82 ^_c) ^_c)	1006 1004 1000 1006 1006 ^_c) ^_c)	100 100 95 80 68 ^_c) ^_c)

a) Dimethyl sulfoxide 1% (1) and 2% (2) respectively.

b) Lowinox® 22IB46 precipitated in the culture medium

c) Cell lysis

 

Mitotic index of human lymphocyte cultures treated with Lowinox® 22IB46

in the first cytogenetic assay

Lowinox® 22IB46 concentration (μg/ml)	Number of metaphases^a)
	Absolute	Number of cells scored	Percentage of control
Without metabolic activation (-S9-mix)
3 h exposure time, 24 h fixation time
Control^b) 5 15 25 50 75 100 MMC-C; 0.5μg/ml MMC-C; 0.75μg/ml	73 – 71 62 – 66 54 – 50 45 – 48 28 – 25 0 – 0 0 – 0 48 – 46 35 – 37	1000 – 1000 1006 – 1003 1003 – 1004 1005 – 1004 1000 – 1001 1000 – 1000 1000 – 1000 1000 – 1003 1002 – 1002	100 89 72 65 37 0 0 65 50
With metabolic activation (+S9-mix)
3 h exposure time, 24 h fixation time
Control^b) 5 15 25 50 75 100 125 150^c) CP; 10μg/ml	53 – 56 45 – 43 39 – 44 38 – 43 35 – 39 11 – 19 6 – 5 1 -^_d) ^_d)-^_d) 34 – 38	1002 – 1000 1006 – 1000 1003 – 1008 1000 – 1000 1000 – 1000 1000 – 1002 1000 – 1001 1000  ^_d) ^_d)  ^_d) 1000 -^_e)	100 81 76 74 68 28 10 1 ^_d) 66

a) Duplicate cultures

b) Dimethyl sulfoxide

c) Lowinox® 22IB46 precipitated in the culture medium

d) Cell lysis

e) Not recorded

 

Mitotic index of human lymphocyte cultures treated with Lowinox® 22IB46

in cytogenetic assay 1A

Lowinox® 22IB46 concentration (μg/ml)	Number of metaphases^a)
	Absolute	Number of cells scored	Percentage of control
With metabolic activation (+S9-mix)
3 h exposure time, 24 h fixation time
Control^b) 5 30 40 50 60 70 80 CP; 10μg/ml	61 – 57 53 – 49 41 – 44 42 – 40 38 – 42 37 – 34 22 – 24 18 – 15 29 – 35	1000 – 1002 1000 – 1002 1005 – 1005 1008 – 1003 1000 – 1002 1008 – 1000 1006 – 1000 1005 – 1000 1002 – 1002	100 86 72 69 68 60 39 28 54

a) Duplicate cultures

b) Dimethyl sulfoxide

 

Chromosome aberrations in human lymphocyte cultures treated with Lowinox® 22IB46 in the absence of S9-mix in the

first cytogenetic assay (3 h exposure time, 24 h fixation time)

Conc	DMSO (1.0% v/v)			5μg/ml			25μg/ml			50μg/ml			MMC-C 0.5μg/ml
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic Index (%)	100			89			65			37			65
No. of Cells scored	150	150	300	150	150	300	150	150	300	150	150	300	150	150	300
No. of Cells with aberrations (+gaps) a)	1	0	1	1	0	1	1	1	2	1	0	1	31	30	***) 61
No. of Cell with aberrations (- gaps)	1	0	1	1	0	1	0	1	1	1	0	1	30	29	***) 59
g'
g'’							1						1	3
b'	1							1					14	11
b'’													13	15
m'													1	3
m'’				1
exch.													8	5
dic
d'
misc.										ma	poly			poly
Total aberr (+ gaps)	1	0		1	0		1	1		10	0		37	37
Total aberr (- gaps)	1	0		1	0		0	1		10	0		36	34

a) Abbreviations used for various types of aberrations are listed in APPENDIX 2

misc = (miscellaneous) aberrations not belonging to the ones mentioned above.

The numerical variation polyploidy (poly) was not counted as an aberration.

*) Significantly different from control group (Fisher’s exact test), *P<0.05, **P<0.01 or ***P<0.001.

 

Chromosome aberrations in human lymphocyte cultures treated with Lowinox® 22IB46 in the absence of S9-mix in

cytogenetic assay 1A (3 h exposure time, 24 h fixation time)

Conc	DMSO (1.0% v/v)			5μg/ml			40μg/ml			70μg/ml			CP 10μg/ml
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic Index (%)	100			86			69			39			54
No. of Cells scored	150	150	300	150	150	300	150	150	300	150	150	300	150	150	300
No. of Cells with aberrations (+gaps) a)	1	0	1	1	0	1	1	0	1	1	0	1	31	24	***) 55
No. of Cell with aberrations (- gaps)	1	0	1	1	0	1	0	0	0	1	0	1	30	21	***) 51
g'							1
g'’													2	3
b'	1												7	12
b'’										1			19	16
m'				1									2
m'’	3												1
exch.													3	3
dic
d'													1
misc.					endo									poly
Total aberr (+ gaps)	4	0		1	0		1	0		1	0		35	34
Total aberr (- gaps)	4	0		1	0		0	0		1	0		33	31

a) Abbreviations used for various types of aberrations are listed in APPENDIX 2

misc = (miscellaneous) aberrations not belonging to the ones mentioned above.

The numerical variations endo-reduplication (endo) and polyploidy (poly) was not counted as an aberration.

*) Significantly different from control group (Fisher’s exact test), *P<0.05, **P<0.01 or ***P<0.001.

 

Mitotic index of human lymphocyte cultures treated with Lowinox® 22IB46

in the second cytogenetic assay

Lowinox® 22IB46 concentration (μg/ml)	Number of metaphases^a)
	Absolute	Number of cells scored	Percentage of control
Without metabolic activation (-S9-mix)
24 h exposure time, 24 h fixation time
Control^b) 5 15 25 35 45 55 MMC-C; 0.2μg/ml MMC-C; 0.3μg/ml	82 – 80 77 – 73 71 – 70 70 – 65 62 – 66 46 – 39 12 – 11 51 – 48 20 – 24	1000 – 1000 1008 – 1000 1006 – 1003 1000 – 1000 1000 – 1007 1002 – 1000 1000 – 1000 1001 – 1002 1003 – 1000	100 93 87 83 79 52 14 61 27
48 h exposure time, 48 h fixation time
Control^b) 5 15 25 35 45 55 MMC-C; 0.1μg/ml MMC-C; 0.15μg/ml	84 – 78 78 – 75 76 – 70 55 – 56 53 – 51 17 – 9 0 – 0 55 – 47 42 – 39	1000 – 1000 1000 – 1000 1000 – 1000 1000 – 1009 1004 – 1003 1000 – 1001 1000 – 1000 1002 – 1010 1000 – 1000	100 94 90 69 64 16 0 63 50

a) Duplicate cultures

b) Dimethyl sulfoxide

 

Mitotic index of human lymphocyte cultures treated with Lowinox® 22IB46

in cytogenetic assay 2A

Lowinox® 22IB46 concentration (μg/ml)	Number of metaphases^a)
	Absolute	Number of cells scored	Percentage of control
Without metabolic activation (-S9-mix)
48 h exposure time, 48 h fixation time
Control^b) 5 15 25 30 35 40 45 50 MMC-C; 0.1μg/ml MMC-C; 0.15μg/ml	30 – 33 26 – 29 28 – 25 25 – 25 32 – 22 24 – 28 21 – 20 12 – 12 4 – 1 23 – 9 14 – 12	1006 – 1018 1017 – 1018 1012 – 1017 1010 – 1011 1024 – 1015 1000 – 1006 1011 – 1015 1005 – 1002 1003 – 1007 1009 – 1028 1011 – 1016	100 87 84 79 86 83 65 38 8 67 41

a) Duplicate cultures

b) Dimethyl sulfoxide

 

Chromosome aberrations in human lymphocyte cultures treated with Lowinox® 22IB46 in the absence of S9-mix in the

second cytogenetic assay (24 h exposure time, 24 h fixation time)

Conc	DMSO (1.0% v/v)			5μg/ml			35μg/ml			45μg/ml			MMC-C 0.2μg/ml
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic Index (%)	100			93			79			52			61
No. of Cells scored	150	150	300	150	150	300	150	150	300	150	150	300	150	150	300
No. of Cells with aberrations (+gaps) a)	0	2	2	0	2	2	0	0	0	0	3	3	33	43	***) 76
No. of Cell with aberrations (- gaps)	0	2	2	0	1	1	0	0	0	0	1	1	30	42	***) 72
g'					1						3		4	1
g'’														1
b'		1			1						1		24	42
b'’													7	8
m'		1												1
m'’
exch.													2	8
dic														1
d'
misc.
Total aberr (+ gaps)	0	2		0	2		0	0		0	4		38	62
Total aberr (- gaps)	0	2		0	1		0	0		0	1		34	60

a) Abbreviations used for various types of aberrations are listed in APPENDIX 2

misc = (miscellaneous) aberrations not belonging to the ones mentioned above.

*) Significantly different from control group (Fisher’s exact test), *P<0.05, **P<0.01 or ***P<0.001.

 

Chromosome aberrations in human lymphocyte cultures treated with Lowinox® 22IB46 in the absence of S9-mix in

cytogenetic assay 2A (48 h exposure time, 48 h fixation time)

Conc	DMSO (1.0% v/v)			5μg/ml			40μg/ml			45μg/ml			MMC-C 0.1μg/ml
Culture	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B	A	B	A+B
Mitotic Index (%)	100			87			65			38			67
No. of Cells scored	150	150	300	150	150	300	150	150	300	220	115	335	75	75	150
No. of Cells with aberrations (+gaps) a)	0	0	0	2	0	2	0	2	2	3	5	**) 8	46	45	***) 91
No. of Cell with aberrations (- gaps)	0	0	0	2	0	2	0	2	2	3	5	**) 8	45	44	***) 89
g'														1
g'’													1
b'				2				1		4	4		34	22
b'’								1					7	5
m'
m'’													2
exch.											1		13	23
dic														2
d'
misc.				endo				2poly		3poly	2poly endo		3p	4intra 2p
Total aberr (+ gaps)	0	0		2	0		0	2		4	5		60	59
Total aberr (- gaps)	0	0		2	0		0	2		4	5		59	58

a) Abbreviations used for various types of aberrations are listed in APPENDIX 2

misc = (miscellaneous) aberrations not belonging to the ones mentioned above.

The numerical variations endo-reduplication (endo) and polyploidy (poly) was not counted as an aberration.

*) Significantly different from control group (Fisher’s exact test), *P<0.05, **P<0.01 or ***P<0.001.

Conclusions:

Lowinox® 22IB46 is not clastogenic in human lymphocytes under the experimental conditions described in the report.

Executive summary:

Evaluation of the potential of Lowinox® 22IB46 to induce chromosome aberrations in cultured peripheral human lymphocytes.

 

This report describes the effect of Lowinox® 22IB46 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of Lowinox® 22IB46 was tested in two independent experiments.

 

The study procedures described in this report are in compliance with the following guidelines:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 473: In Vitro Mammalian Chromosome Aberration Test (adopted September 26, 2014).

 

Batch WB44L0016 of Lowinox® 22IB46 was a white to cream coloured powder with a purity of 99.7%. Lowinox® 22IB46 was soluble in dimethyl sulfoxide at concentrations of 20 mg/ml and below but formed a suspension at concentrations of 25 mg/ml and higher.

 

In the first cytogenetic assay, Lowinox® 22IB46 was tested up to 50 and 70 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. Appropriate toxicity was reached at these dose levels.

 

In the second cytogenetic assay, Lowinox® 22IB46 was tested up to 45 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

 

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

Lowinox® 22IB46 did not induce a statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

 

No biologically relevant effects of Lowinox® 22IB46 on the number of polyploid cells and cells with endo-reduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Lowinox® 22IB46 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

 

Finally, it is concluded that this test is valid and that Lowinox® 22IB46 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May 2016 to 20 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 490: "Genetic Toxicology: In Vitro Mammalian Cell Gene Mutation Test Using the Thymidine Kinase Gene", (adopted 28 July 2015).

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Type of assay:

other: Mammalian Cell Gene Mutation Test Using the Thymidine Kinase Gene

Specific details on test material used for the study:

No further details specified in the study report.

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Test System: L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale: Recommended test system in international guidelines (e.g. OECD).
Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1E+6 cells/ml.

Cell culture
Horse serum
Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.

Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).

Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).

Exposure medium
For 3 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat inactivated horse serum (R5-medium).
For 24 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat inactivated horse serum (R10-medium).

Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT) (Sigma).

Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).

Environmental conditions
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 54 – 100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.0 – 37.9°C).
Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8E+6 cells (E+06 cells/ml for 3 hours treatment) or 6E+6 cells (1.25E+5 cells/ml for 24 hours treatment) with a number of test item concentrations increasing by approximately half log steps.
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 15 to 200 μg/ml in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.

1st mutagenicity test
Based on the results of the dose range finding test, the following dose range was selected for the first mutagenicity test in the absence and presence of S9-mix: 0.1, 0.5, 1, 5, 7.5, 10, 15, 17.5, 20, 22.5 and 25 μg/ml exposure medium.
Evaluation of toxicity
In the absence of S9-mix, the dose levels of 17.5 to 25 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the presence of S9-mix, the dose levels of 20 to 25 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies were:
Without S9-mix: 0.1, 0.5, 1, 5, 7.5, 10 and 15 μg/ml exposure medium.
With S9-mix: 0.1, 0.5, 1, 5, 7.5, 10, 15 and 17.5 μg/ml exposure medium.

2nd mutagenicity test
Based on the results of the dose range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 μg/ml exposure medium.
Evaluation of toxicity
The dose levels selected to measure mutation frequencies were: 1, 5, 10, 12.5, 15, 17.5, 20 and 22.5 μg/ml exposure medium.

Vehicle / solvent:

No correction was made for the purity/composition of the test item.
The test item was dissolved in dimethyl sulfoxide (Merck). Lowinox® 22IB46 concentrations were used within 1.5 hour after preparation.
The final concentration of the solvent in the exposure medium was 1% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Study design
Cleansing
Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Merck) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.

Dose range finding test
In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8E+6 cells (E+6 cells/ml for 3 hours treatment) or 6E+6 cells (1.25E+5 cells/ml for 24 hours treatment) with a number of test item concentrations increasing by approximately half log steps. The cell cultures for the 3 hours treatment were placed in sterile 30 ml centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hours treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test item was tested in the absence and presence of S9-mix.
Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 200 μg/ml exposure medium.
For the 3 hours treatment, cell cultures were exposed for to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and finally resuspension in 50 ml growth medium (R10).
For the 24 hours treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and finally resuspension in 20 ml growth medium (R10). The cells in the final suspension were counted with the coulter particle counter or with a microscope in an "Improved Neubauer" haemocytometer.
The surviving cells of the 3 hours treatment were subcultured twice to determine cytotoxicity.
After 24 hours of subculturing, the cells were counted and subcultured again for another 24 hours, after that the cells were counted. The surviving cells of the 24 hours treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25E+5 cells/ ml were counted no subculture was performed.
The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.

Mutagenicity test
The test item was tested in the presence of S9-mix with a 3-hour treatment period and in the absence of S9-mix with 3- and 24-hours treatment periods. Eight doses of Lowinox® 22IB46 were tested in the mutation assay. Except in the first experiment (absence of S9-mix) in which seven dose levels were tested.
The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.

Treatment of the cells
Per culture 8E+6 cells (E+6 cells/ml for 3 hours treatment) or 6E+6 cells (1.25E+5 cells/ml for 24 hours treatment) were used. The cell cultures for the 3 hours treatment were placed in sterile 30 ml centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm.
The cell cultures for the 24 hours treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate.
For the 3 hours treatment, cell cultures were exposed for to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and finally resuspension in 50 ml growth medium (R10).
For the 24 hours treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and finally resuspension in 20 ml growth medium (R10). The cells in the final suspension were counted with the coulter particle counter or with a microscope in an "Improved Neubauer" haemocytometer.

Expression period
For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4E+6 cells (if possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with Lowinox® 22IB46, the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

Determination of the mutation frequency
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96- well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6E+5 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6E+5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection).
The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the absence of S9-mix, the dose levels of 17.5 to 25 μg/ml were too toxic for further testing. In the presence of S9-mix, the dose levels of 20 to 25 μg/ml were too toxic for further testing.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Solubility
Lowinox® 22IB46 precipitated in the exposure medium at concentrations of 154 μg/ml and above. The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose range finding test was 200 μg/ml.

Dose range finding test
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 15 to 200 μg/ml in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.
In the absence of S9-mix, the relative suspension growth was 51% at the test item concentration of 15 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 27 μg/ml and above.
In the presence of S9-mix, the relative suspension growth was 54% at the test item concentration of 15 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 27 μg/ml and above.
The relative suspension growth was 32% at the test item concentration of 15 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 27 μg/ml and above.

Mutation experiment
Based on the results of the dose range finding test, the test item was tested in two mutation assays. The first experiment was performed in the absence and presence of S9-mix with a 3 hour treatment period. The second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period.

First mutagenicity test
Based on the results of the dose range finding test, the following dose range was selected for the first mutagenicity test in the absence and presence of S9-mix: 0.1, 0.5, 1, 5, 7.5, 10, 15, 17.5, 20, 22.5 and 25 μg/ml exposure medium.
Evaluation of toxicity
In the absence of S9-mix, the dose levels of 17.5 to 25 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the presence of S9-mix, the dose levels of 20 to 25 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.1, 0.5, 1, 5, 7.5, 10 and 15 μg/ml exposure medium.
With S9-mix: 0.1, 0.5, 1, 5, 7.5, 10, 15 and 17.5 μg/ml exposure medium.
In the absence of S9-mix, the relative total growth of the highest test item concentration was 32% compared to the total growth of the solvent controls.
In the presence of S9-mix, the relative total growth of the highest test item concentration was 24% compared to the total growth of the solvent controls.
Evaluation of the mutagenicity
No significant increase in the mutation frequency at the TK locus was observed after treatment with Lowinox® 22IB46 either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the Lowinox® 22IB46 treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutagenicity test
To obtain more information about the possible mutagenicity of Lowinox® 22IB46, a second mutation experiment was performed in the absence of S9-mix with a 24-hour treatment period.
Based on the results of the dose range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 μg/ml exposure medium.
Evaluation of toxicity
The dose level of 25 μg/ml was not used for mutation frequency measurement, since this dose level was too toxic for further testing. The dose levels of 0.01 to 1 μg/ml showed no cytotoxicity.
Therefore, the dose levels of 0.01 to 0.5 μg/ml were not regarded relevant for mutation frequency measurement.
The dose levels selected to measure mutation frequencies at the TK-locus were: 1, 5, 10, 12.5, 15, 17.5, 20 and 22.5 μg/ml exposure medium.
The relative total growth of the highest test item was 11% compared to the total growth of the solvent controls.
Evaluation of mutagenicity
Lowinox® 22IB46 did not induce an increase in the mutation frequency at the TK locus.

Dose range finding test: Cytotoxicity of Lowinox® 22IB46 (3 hours treatment)

Dose (μg/ml)	Cell count after 24 hours of subculture (cells/ml x 10^5)	Cell count after 48 hours of subculture (cells/ml x 10^5)	SG^(1) (x10^5cells/ml)	RSG^(2) (%)
Without metabolic activation
SC 15 27 48 87 154^(3) 200^(3)	7.5 4.0 0.1^(4) 0.1^(4) 0.1^(4) 0.1^(4) 0.1^(4)	5.7 5.5 CD CD CD CD CD	21 11 0 0 0 0 0	100 51
With metabolic activation
SC 15 27 48 87 154^(3) 200^(3)	6.1 3.7 0.7^(4) 0.1^(4) 0.1^(4) 0.1^(4) 0.1^(4)	5.4 4.8 CD CD CD CD CD	16 9 0 0 0 0 0	100 54

Note: all calculations were made without rounding off

SC = solvent control = dimethyl sulfoxide

CD = cell depth

⁽¹⁾= suspension growth

⁽²⁾= relative suspension growth

⁽³⁾= test item precipitated in the exposure medium

⁽⁴⁾= since less than 1.25 x 10⁵c/ml were present, no subculture was performed

SG = Suspension growth = [Day 1 cell count/1.6 x 10^{5 1)}] x [Day 2 cell count /1.25 x 10^{5 1)}]

¹⁾Or appropriate cell concentration

RSG = [SG_(test)/SG_(control)] x 100

 

Dose range finding test: Cytotoxicity of Lowinox® 22IB46 (24 hours treatment)

Dose (μg/ml)	Cell count after 24 hours of subculture (cells/ml x 10^5)	Cell count after 48 hours of subculture (cells/ml x 10^5)	SG^(1) (x10^5cells/ml)	RSG^(2) (%)
Without metabolic activation
SC 15 27 48 87 154^(3) 200^(3)	10.1 4.6 1.0^(4) 0.1^(4) 0.1^(4) 0.1^(4) 0.1^(4)	5.5 3.8 0.7 0.1 0.1 0.1 0.1	44 14 1 0 0 0 0	100 32 <1 <1 <1 <1 <1

Note: all calculations were made without rounding off

SC = solvent control = dimethyl sulfoxide

 ⁽¹⁾= suspension growth

⁽²⁾= relative suspension growth

⁽³⁾= test item precipitated in the exposure medium

⁽⁴⁾= since less than 1.25 x 10⁵c/ml were present, no subculture was performed

SG = Suspension growth = [Day 0 cell count/1.25 x 10⁵] x [Day 1 cell count/1.25 x 10^{5 1)}] x [Day 2 cell count/1.25 x 10^{5 1)}]

¹⁾Or appropriate cell concentration

RSG = [SG_(test)/SG_(control)] x 100

 

Experiment 1: Cytotoxic and mutagenic response of Lowinox® 22IB46 in the mouse lymphoma L5178Y test system

Dose (μg/ml)	RSG (%)	CE day2 (%)	RCE (%)	RTG (%)	Mutation frequency per 10^6survivors
					Total	( small	large )
Without metabolic activation
3 hours treatment
SC1 SC2 0.1 0.5 1 5 7.5 10 15 MMS	100   121 117 119 108 96 87 33 86	125 91 110 93 97 94 104 89 107 60	100   102 86 89 87 96 82 99 56	100   123 100 106 94 92 71 32 48	68 116 85 126 126 134 107 136 94 899	( 12 ( 22 ( 12 ( 17 ( 22 ( 33 ( 17 ( 14 ( 21 ( 447	54 ) 90 ) 72 ) 105 ) 99 ) 94 ) 87 ) 118 ) 69 ) 333 )
With metabolic activation
3 hours treatment
SC1 SC2 0.1 0.5 1 5 7.5 10 15 17.5 CP	100   101 95 91 86 87 97 73 29 42	85 99 115 78 76 80 75 58 54 76 29	100   124 85 82 87 81 62 58 82 32	100   125 81 75 75 71 61 43 24 14	119 103 98 96 71 91 78 112 72 70 1710	( 37 ( 28 ( 17 ( 22 ( 14 ( 9 ( 18 ( 26 ( 20 ( 22 ( 897	77 ) 71 ) 78 ) 72 ) 56 ) 80 ) 58 ) 84 ) 52 ) 46 ) 606 )

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growht; SC = Solvent Control = dimethyl sulfoxide; MMS = Methymethansulfonate; CP = Cyclophosphamine

 

Experiment 2: Cytotoxic and mutagenic response of Lowinox® 22IB46 in the mouse lymphoma L5178Y test system

Dose (μg/ml)	RSG (%)	CE day2 (%)	RCE (%)	RTG (%)	Mutation frequency per 10^6survivors
					Total	( small	large )
Without metabolic activation
24 hours treatment
SC1 SC2 1 5 10 12.5 15 17.5 20 22.5 MMS	100   86 72 57 44 33 23 17 11 89	78 66 69 69 90 115 77 88 70 69 81	100   96 96 125 159 107 123 98 96 113	100   83 69 71 69 36 28 16 11 101	141 134 130 124 78 60 155 74 73 124 535	( 56 ( 50 ( 55 ( 55 ( 32 ( 32 ( 55 ( 34 ( 29 ( 78 ( 298	78 ) 78 ) 69 ) 64 ) 43 ) 26 ) 91 ) 38 ) 43 ) 42 ) 182 )

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growht; SC = Solvent Control = dimethyl sulfoxide; MMS = Methymethansulfonate; CP = Cyclophosphamine

 

Historical control data of the spontaneous mutation frequencies of the solvent controls for the mouse lymphoma assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hours treatment
Mean	83	75	84
SD	22	24	27
n	161	146	210
Upper control limit (95% control limits)	128	126	141
Lower control limit (95% control limits)	37	25	28

SD = Standard deviation

n = Number of observations

 

Historical control data of the mutation frequencies of the positive controls for the mouse lymphoma assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hours treatment
Mean	894	724	1694
SD	247	200	793
n	81	74	108
Upper control limit (95% control limits)	1431	1216	4045
Lower control limit (95% control limits)	356	231	-657

SD = Standard deviation

n = Number of observations

Conclusions:

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Although the mutation frequencies of the solvent control cultures in the second experiment were just above the historical control data range, the observed mutation frequency of these solvent control cultures were within the acceptability criteria of this assay.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The growth rate over the two-day expression period for cultures treated with DMSO was between 20 and 27 (3 hours treatment) and 75 and 82 (24 hours treatment).
In the absence of S9-mix, Lowinox® 22IB46 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
In the presence of S9-mix, Lowinox® 22IB46 did not induce a significant increase in the mutation frequency.
In conclusion, Lowinox® 22IB46 is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

Evaluation of the mutagenic activity of Lowinox® 22IB46 in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells.

 

This report describes the effects of Lowinox® 22IB46 on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hours treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

 

The study procedures described in this report were based on the most recent OECD guideline.

 

Batch WB44L0016 of Lowinox® 22IB46 was a white to cream coloured powder with a purity of 99.7%. The test item was dissolved in dimethyl sulfoxide.

 

In the first experiment, Lowinox® 22IB46 was tested up to concentrations of 15 and 17.5 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was 32 and 24% in the absence and presence of S9-mix, respectively.

 

In the second experiment, Lowinox® 22IB46 was tested up to concentrations of 22.5 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 11%.

 

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay.

 

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, Lowinox® 22IB46 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

 

In the presence of S9-mix, Lowinox® 22IB46 did not induce a significant increase in the mutation frequency.

 

It is concluded that Lowinox® 22IB46 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro - Chromosome Aberration

The report describes the effect of Lowinox® 22IB46 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of Lowinox® 22IB46 was tested in two independent experiments.

Lowinox® 22IB46 was soluble in dimethyl sulfoxide at concentrations of 20 mg/ml and below but formed a suspension at concentrations of 25 mg/ml and higher.

 

In the first cytogenetic assay, Lowinox® 22IB46 was tested up to 50 and 70 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, Lowinox® 22IB46 was tested up to 45 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

 

Lowinox® 22IB46 did not induce a statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

No biologically relevant effects of Lowinox® 22IB46 on the number of polyploid cells and cells with endo-reduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Lowinox® 22IB46 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that Lowinox® 22IB46 is not clastogenic in human lymphocytes under the experimental conditions described in the report.

Genetic Toxicity In Vitro - Mammalian Cell Gene Mutation Test

The report describes the effects of Lowinox® 22IB46 on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hours treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

 

In the first experiment, Lowinox® 22IB46 was tested up to concentrations of 15 and 17.5 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was 32 and 24% in the absence and presence of S9-mix, respectively.

In the second experiment, Lowinox® 22IB46 was tested up to concentrations of 22.5 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 11%.

 

In the absence of S9-mix, Lowinox® 22IB46 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, Lowinox® 22IB46 did not induce a significant increase in the mutation frequency.

It is concluded that Lowinox® 22IB46 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Justification for classification or non-classification

The substance is not classified under CLP as it does not fill the requirements for classification.