(1,2-dioxoethylene)bis(iminoethylene) bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro-Ames Assay

Negative with and without metabolic activation.

Genetic toxicity in vitro-Chromosome aberration assay

Negative with and without metabolic activation in Chinese hamster V79 cells.

An Genetic toxicity in vitro in vitro-Mammalian cell assay (Mouse Lymphoma)

Negative with and without metabolic activation in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed in recognised procedure. GLP status not specified in the study report.

Qualifier:

according to guideline

Guideline:

other: Test method conformed to the procedure published by Ames et al. Mut Res 31:347-364, 1975

Deviations:

not specified

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

None specified

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

39, 78, 156, 312, 625 μg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide and ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: nitroflourene; 2-aminoanthracene

Details on test system and experimental conditions:

Induction of Rat Liver Enzymes for Activation
Preparation of animals:
-male rats – 200-300 gm each (Charles River, Wistar strain, Charles River Breeding Lab., Inc.)
-single injection of Aroclor 1254 (diluted in corn oil to a conc. of 200 mg/ml) I.P. at a rate of 500 mg/kg five days before sacrifice.
-Drinking water given ad libitum & Purina Laboratory Chow until 12 hours before sacrifice.
-On fifth day of induction, rats were killed by cervical separation decapitated and bled.
Preparation of liver homogenate fraction (S-9):
-All steps are at 0-4°C using cold, sterile solutions and glassware.
-Liver placed in beakers containing 0.15M KCl (approx. 1 ml/gm of wet liver) for weighing.
-After weighing, livers are transferred to a beaker containing 0.15 M KCl (3 ml/gm wet liver) minced with sterile scissors, and homogenized in a Potter-Elvejham apparatus with a Teflon pestle.
-Homogenate centrifuged for 10 minutes at 9000 x g (8700 rpm).
-Supernatant decanted and saved (S-9 fraction).
-Fresh S-9 fractions are distributed in 2 ml portions.
-Quickly frozen and stored at -80°C.
Preparation of S-9 Mix:
S-9 Mix contains per ml:
S-9 (0.04-0.1 ml) used 50 μl/ml
MGCl2 8μM
KCl 33 μM
Glucose-6-phosphate 5μM
NADP 4Μm
Sodium phosphate Buffer pH 7.4 100μM
S-9 Mix is freshly prepared each day, filter sterilized, and kept on ice before and during use.

Mutagenesis Assays on Plates
Media:
Top Agar: 0.6% Difco Agar; 0.5% NaCl
Autoclaved in 100 ml volumes and kept at room temperature before use.
Agar melted in steam and 10 ml of a sterile solution of 0.5 μM 1-Histidine HCl-0.5 μM biotin is added to the molten top agar and mixed thoroughly by gentle swirling.
Minimal-glucose agar medium in Vogel-Bonner Medium E: 1.5% Difco Agar; 2.0% Glucose

Mutagenesis Assay Procedure with S-9 Mix
Added in order to 2ml molten top agar at 45°C
-0.1 ml of overnight nutrient broth culture of bacterial tester strain
-Sample to be tested - 10μl
-0.5 ml of the S-9 Mix.
-S-9 Mix should not be left at 45°C for more than a few seconds.
The contents are mixed by rotating the tube between the palms.
The contents of the tube are then poured on minimal glucose agar plates.
Uniform distribution of the top agar is accomplished by gently tilting and rotating the uncovered plate, and then setting down to harden.
Mixing, pouring and distributing should take less than 20 seconds.

Mutagenic Assay Without Mammalian Microsomes
Same as above.

Evaluation criteria:

None specified

Statistics:

None specified

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, 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:

not specified

Additional information on results:

Inspection of the data reveals no evidence of mutagenicity in any of the strains at any concentration of the test chemical.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

In Vitro Assay of Uniroyal Compound TVIC-B-5909 Using Salmonella typhimurium (Averages)

Compound	Metabolic Activation	μg of Compound Added per Plate	Histidine-Positive Revertants per Plate
			TA-98	TA-100	TA-1535	TA-1537	TA-1538
Negative Control	- +		16 33	142 191	8 11	6 5	-- 20
TVIC-B-5909	- - - - - + + + + +	39 78 156 312 625 39 78 156 312 625	13 15 17 12 15 30 29 28 32 21	116 121 128 136 149 153 139 158 138 175	4 8 9 6 10 8 10 6 8 8	2 3 4 6 2 4 5 4 5 6	-- -- -- -- -- 15 20 16 14 16
MMNG	- -	10 5	-- --	751 740	659 640	11 14	-- --
9-Aminoacridine	- -	20 10	-- --	-- --	-- --	25 7	-- --
Nitroflourene	- -	20 10	412 351	-- --	-- --	-- --	-- --
2-Aminoanthracene	+ +	15 5	-- 556	-- 373	16 --	13 --	135 --
DMSO	- +		21 29	158 171	9 10	5 7	-- 18
Ethanol	-		--	--	--	7	--

 

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

The compound assayed was found to be non-mutagenic.

Executive summary:

The purpose of the study is to determine whether the compound (TVIC) elicited a mutagenic response in microorganism. An Aroclor 1254-stimulated, rat-liver-homogenate metabolic activation system was included in the assay procedure to provide metabolic steps that the bacteria are either incapable of conducting or that they do not carry our under the assay conditions.

The test method conformed to the procedure published by Ames et al. Mut Res 31:347-364, 1975.

Compound TVIC was examined for mutagenic activity with Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, TA 1538. Each assay was performed in the presence and in the absence of a metabolic activation system. The compound assayed was found to be non-mutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2017 to 17 November 2017

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:

OECD Guidelines for Testing of Chemicals, Section 4, No. 473, “In Vitro Mammalian Chromosome Aberration Test”, 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation No 440/2008 of 30 May 2008 B.10. "Mutagenicity – In Vitro Mammalian Chromosome Aberration Test" as amended by Commission Regulation 2017/735 of 14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Specific details on test material used for the study:

No further details specified in the study report.

Target gene:

structural chromosome aberrations in somatic and/or germ cells.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79: Chinese hamster lung, male
ECACC Cat. No.: 86041102
Lot No.: 10H016
Date of working lot: 31 July 2015
Supplier: ECACC (European Collection of Cells Cultures)
Morphology: Fibroblast

The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10°C (for short-term storage) or in liquid nitrogen (long-term storage). The stock was checked for mycoplasma infection. No infection of mycoplasma was noted.

Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution was used for cell detachment to subculture (cells were rinsed with 1X PBS before detachment). The laboratory cultures were maintained in 150 cm2 plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air. The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1% (v/v) Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B) and 10% (v/v) heat-inactivated fetal bovine serum (DMEM-10, culture medium). When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study). During the treatments, the serum content of the medium was reduced to 5% (v/v) (DMEM-5).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test.
Chromosome Aberration Assay 1: 2000, 666.7, 222.2, 74.1, 24.7 and 8.2 µg/mL (experiment with and without metabolic activation).
Chromosome Aberration Assay 2: 2000, 666.7, 222.2, 74.1, 24.7 and 8.2 µg/mL (experiment with and without metabolic activation).

Vehicle / solvent:

Based on the result of the trial formulations of the test item, which was performed at the Test Facility, Acetone was selected for vehicle (solvent) of the study. The vehicle was compatible with the survival of the cells and the S9 activity.
Name: Acetone
Supplier: VWR
Lot No.: 15J060514 / 17B284019
Expiry date: 31 October 2020 / 28 February 2022
Storage conditions: Room temperature

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

External Metabolic Activation System
An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under the study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary.
Many substances only develop mutagenic potential after they are metabolised. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).
In the experiments with metabolic activation in this study, a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used as an appropriate metabolic activation system.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Citoxlab Hungary Ltd. according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.

TEST PROCEDURE
Toxicity and Concentration Selection
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test.
In this Preliminary Toxicity Test, two assays were performed. In Assay A, cells were treated for 3-hours in the presence and absence of S9-mix with a 20-hour harvesting time. In Assay B, cells were treated for 20 hours in the absence of S9-mix with a 20-hour harvesting time.
The assays were performed with a range of test item concentrations to determine cytotoxicity. Treatment was performed as described for the main test. However, single cultures were used and positive controls were not included. Visual examination of the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed at the end of the treatment period.
At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC (Relative Increase in Cell Counts).

Chromosome Aberration Assays
The Chromosome Aberration Assays were conducted as two independent experiments (Assay 1 and Assay 2) in the presence and in the absence of metabolic activation. In Assay 1, 3-hour treatment was performed with and without metabolic activation (in the presence and absence of S9 mix); cells were harvested 20-hour after the beginning of the treatment.
In Assay 2, a 3-hour treatment was performed with metabolic activation (in the presence of S9 mix) and 20-hour without metabolic activation (in the absence of S9 mix) in duplicate cultures; cells were harvested 20-hour after the beginning of the treatment.

Treatment of the Cells
For the cytogenetic experiments, 1-3 day old cultures (more than 50 % confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 105 cells/dish concentration and incubated for approximately 24 hours at 37C in 10 mL of culture medium (DMEM-10). Duplicate cultures were used for each test item concentration or controls.
After the seeding period, the medium was replaced with 9.90 or 9.95 mL treatment medium (DMEM-5) in case of experiments without metabolic activation or with 9.40 or 9.45 mL treatment medium (DMEM-5) + 0.5 mL S9-mix in case of experiments with metabolic activation.
Cells were treated with different concentration test item solutions, untreated, negative (vehicle) or positive control solution (treatment volume: 100 µL/dish in case of the untreated and positive control; 50 µL/dish in case of the test item and negative control) for the given period of time at 37C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1% (v/v) Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.
Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) from the beginning of treatment.
Solubility of the test item in the final treatment medium was visually examined at the beginning and end of the treatment in each case. Measurement of pH and osmolality was also performed at the end of the treatment period in both main tests.
For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC (Relative Increase in Cell Counts).

Preparation of Chromosomes
2-2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 µg/mL). The cells were swollen with 0.075 M KCl hypotonic solution for 4 minutes, then were washed in fixative (Methanol : Acetic-acid 3 : 1 (v : v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells* was dropped onto clean microscope slides and air-dried. The slides were stained with 5% Giemsa solution, air-dried and coverslips were mounted. At least three slides were prepared for each culture.
*Note: Fixed cells were stored frozen in case if any additional action was required (as documented in the raw data and reported). After the finalization of the report, the remaining frozen cell suspension samples will be discarded.

Examination of Slides
The stained slides were given random unique code numbers at the Test Facility by a person who was not involved in the metaphase analysis. The code labels covered all unique identification markings on the slides to ensure that they were scored without bias.
The metaphase analysis was conducted under the control of the Principal Investigator. When the metaphase analysis was completed for each test, the slide codes were broken and the number of metaphases with aberrations (excluding gaps) and the types of aberrations for each culture were presented in tables.
At least 150* metaphases with 222 chromosomes (centromeres) from each culture (replicate) were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
*Note: The examination of slides from a culture was halted when 25 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.

The aberrations are defined in the following way:

Gap: small unstained lesion smaller than the width of a chromatid and with minimal misalignment of the chromatid(s)
Break: unstained lesion larger than the width of a chromatid, or with clear misalignment
Exchange: breakage and reunion of chromatids within a chromosome, or between chromosomes
Chromatid-type: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids
Chromosome-type: structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.
Fragments could arise from breakage and exchange events. When the origin of a fragment was clear, it was recorded under that category (e.g. a dicentric chromosome with a fragment was recorded as one chromosome exchange event). When the origin of the fragment was not clear, it was recorded as a chromatid break. Metaphases with more than five aberrations (excluding gaps) were recorded as showing multiple damage.
Additionally, the number of polyploid and endoreduplicated cells was scored. Polyploid metaphases are defined as metaphases with approximate multiples of the haploid chromosome number (n), other than the diploid number (i.e. ca. 3n, 4n etc). Endoreduplicated metaphases have chromosomes with 4, 8, etc. chromatids. Marked reductions in the numbers of cells on the slides were recorded if needed.
The vernier co-ordinates of at least five metaphases (with aberrations, where possible) were recorded for each culture.
The metaphase analysis was conducted in compliance with Good Laboratory Practice as required by the United Kingdom GLP Compliance Regulations 1999 (SI 1999 No. 3106, as amended 2004, SI No. 0994) and which are in compliance with the OECD Principles of Good Laboratory Practice (as revised in 1997). These Principles are in conformity with other international GLP regulations.
When the metaphase analysis has been performed, the original raw data (record sheets) for the metaphase analysis and the microscope slides are shipped back to the Test Facility (Citoxlab Hungary Ltd). After the slide reading phase of the study had ended, the Principal Investigator issued a Work Phase Report.

Rationale for test conditions:

In accordance with Test Guidelines.

Evaluation criteria:

The assay is considered valid, if the following criteria are met:
-The negative (vehicle) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
-The positive controls induce increases in the aberration frequency, which are significant.

The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
-Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
-The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
-The increases are statistically significant.
-The increases are not associated with large changes in pH or osmolality of the treated cultures.

The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.

The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed.

Statistics:

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Vehicle and Concentration Selection
Based on the available solubility information (trial formulations of the test item performed at the Test Facility), Acetone at 400 mg/mL concentrations was selected for vehicle (solvent) of the study. The highest examined concentration in the preliminary test was 2000 µg/mL.
Two Concentration Selection Cytotoxicity Assays (Assay A: 3-hour treatment with and without metabolic activation, 20-hour harvesting time; and Assay B: 20-hour treatment without metabolic activation, 20-hour harvesting time) were performed as part of the study to establish an appropriate concentration range for the Chromosome Aberration Assays.
A total of ten test concentrations between 2000 and 3.906 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay. Treatment concentrations for the chromosome aberration assays were selected on the basis of results of the performed Concentration Selection Cytotoxicity Assays according to the OECD guideline instructions (up to the maximum recommended concentration and/or up to the solubility limit).

Chromosome Aberration Assays
In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 2000, 666.7, 222.2, 74.1, 24.7 and 8.2 µg/mL (experiment with and without metabolic activation).
In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 2000-222.2 µg/mL concentration range with metabolic activation and in the 2000-74.1 µg/mL concentration range without metabolic activation. Furthermore, insolubility (minimal amount) was detected at the end of the treatment period in the final treatment medium in the 74.1-8.2 µg/mL concentration range with metabolic activation and in the 24.7 and 8.2 µg/mL concentrations without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 222.2, 74.1 and 24.7 µg/mL (a total of three) were chosen for evaluation in the experiment with metabolic activation and concentrations of 74.1, 24.7 and 8.2 µg/mL (a total of three) were chosen for evaluation in the experiment without metabolic activation.

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 2000, 666.7, 222.2, 74.1, 24.7 and 8.2 µg/mL (experiment with and without metabolic activation).
In Assay 2, similarly to the first experiment, insolubility was detected at the end of the treatment period in the final treatment medium in the 2000-222.2 µg/mL concentration range with metabolic activation and in the 2000 and 666.7 µg/mL concentrations without metabolic activation. Furthermore, insolubility (minimal amount) was detected at the end of the treatment period in the final treatment medium in the 74.1-8.2 µg/mL concentration range with metabolic activation and in the 222.2-24.7 µg/mL concentration range without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in nay samples of this assay. Therefore, concentrations of 222.2, 74.1 and 24.7 µg/mL (a total of three) were evaluated in the experiment with metabolic activation, and concentrations of 666.7, 222.2 and 74.1 µg/mL (a total of three) were evaluated in the experiment without metabolic activation.
None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.
Polyploid metaphases (1-7) were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments, but their incidence was not related to treatment with NAUGARD® XL-1. One endoreduplicated metaphases were detected in the performed experiments in one case in the positive control.

VALIDITY OF THE STUDY
The tested concentrations in the chromosome aberration assays were selected based on the results of the preliminary experiments. Insolubility was detected in all experiments with and without metabolic activation; while cytotoxicity was not detected in any experiment with and without metabolic activation. The evaluated concentration ranges of Assay 1 and Assay 2 were considered to be adequate, as they covered the range from insolubility to little insolubility.
Three test item concentrations were evaluated in each experiment.
The spontaneous aberration frequencies of the negative (vehicle) controls in the performed experiments were in harmony the general historical control range of the testing laboratory.
In the performed experiments, the positive control substances (Cyclophosphamide (CP) in the experiments with metabolic activation and Ethyl methanesulfonate (EMS) in the experiments without metabolic activation) caused the expected statistically significant increase in the number of cells with structural chromosome aberrations demonstrating the sensitivity of the test system in each assay.
The study was considered to be valid.

Summarized results of the Concentration Selection Cytotoxicity Assay A experiment without metabolic activation

Test group	Dose (μg/mL)	S9-mix	Treatment / sampling time	Cell number (total)	RICC (%)*	Observations beginning / end of treatment
Untreated control	-	-	3/20	6.25E+06	140	normal / normal (pH: 7.4; osm: 345 mmol/kg)
Negative (vehicle) control	-	-	3/20	5.15E+06	100	normal / normal (pH: 7.4; osm: 347 mmol/kg)
NAUGARD® XL-1	2000	-	3/20	5.33E+06	106	discoloured medium^#, precipitate, oily film / discoloured medium^#, precipitate, oily film (pH: 7.4; osm: 352 mmol/kg)
	1000	-	3/20	4.90E+06	91	precipitate^#, oily film / precipitate^#, oily film (pH: 7.4; osm: 349 mmol/kg)
	500	-	3/20	6.00E+06	131	precipitate^#, oily film / precipitate^#, oily film (pH: 7.4; osm: 348 mmol/kg)
	250	-	3/20	5.95E+06	129	precipitate^#, oily film / precipitate^#, oily film (pH: 7.4; osm: 349 mmol/kg)
	125	-	3/20	5.20E+06	102	precipitate^#, oily film / oily film (pH: 7.4; osm: 350 mmol/kg)
	62.5	-	3/20	5.75E+06	122	precipitate^#, oily film / oily film (pH: 7.4; osm: 348 mmol/kg)
	31.25	-	3/20	5.43E+06	110	oily film^#/ oily film^#(pH: 7.4; osm: 348 mmol/kg)
	15.625	-	3/20	5.40E+06	109	oily film^#/ oily film^#(pH: 7.4; osm: 351 mmol/kg)
	7.813	-	3/20	5.68E+06	119	oily film^#/ oily film^#(pH: 7.4; osm: 349 mmol/kg)
	3.906	-	3/20	5.18E+06	101	normal / normal (pH: 7.4; osm: 353 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                  #: minimal amount

RICC: Relative Increase in Cell Counts                                                         osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Summarized results of the Concentration Selection Cytotoxicity Assay A experiment with metabolic activation

Test group	Dose (μg/mL)	S9-mix	Treatment / sampling time	Cell number (total)	RICC (%)*	Observations beginning / end of treatment
Untreated control	-	+	3/20	5.55E+06	90	normal / normal (pH: 7.4; osm: 344 mmol/kg)
Negative (vehicle) control	-	+	3/20	5.90E+06	100	normal / normal (pH: 7.4; osm: 345 mmol/kg)
NAUGARD® XL-1	2000	+	3/20	4.78E+06	68	discoloured medium^#, precipitate, oily film / discoloured medium^#, precipitate, oily film (pH: 7.4; osm: 342 mmol/kg)
	1000	+	3/20	4.65E+06	64	precipitate^#, oily film / precipitate^#, oily film (pH: 7.4; osm: 349 mmol/kg)
	500	+	3/20	5.68e+06	94	precipitate, oily film / precipitate, oily film (pH: 7.4; osm: 347 mmol/kg)
	250	+	3/20	5.08E+06	76	precipitate^#, oily film / precipitate^#, oily film (pH: 7.4; osm: 350 mmol/kg)
	125	+	3/20	5.70E+06	94	precipitate^#, oily film^#/ oily film^#(pH: 7.4; osm: 345 mmol/kg)
	62.5	+	3/20	5.30E+06	83	oily film^#/ oily film^#(pH: 7.4; osm: 343 mmol/kg)
	31.25	+	3/20	5.23E+06	81	oily film^#/ oily film^#(pH: 7.4; osm: 344 mmol/kg)
	15.625	+	3/20	5.60E+06	91	oily film^#/ oily film^#(pH: 7.4; osm: 350 mmol/kg)
	7.813	+	3/20	5.94E+06	73	oily film^#/ oily film^#(pH: 7.4; osm: 345 mmol/kg)
	3.906	+	3/20	5.13E+06	78	normal / normal (pH: 7.4; osm: 346 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                  #: minimal amount

RICC: Relative Increase in Cell Counts                                                         osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Summarized results of the Concentration Selection Cytotoxicity Assay B experiment without metabolic activation

Test group	Dose (μg/mL)	S9-mix	Treatment / sampling time	Cell number (total)	RICC (%)*	Observations beginning / end of treatment
Untreated control	-	-	20/20	5.90E+06	118	normal / normal (pH: 7.4; osm: 348 mmol/kg)
Negative (vehicle) control	-	-	20/20	5.38E+06	100	normal / normal (pH: 7.4; osm: 354 mmol/kg)
NAUGARD® XL-1	2000	-	20/20	5.58E+06	107	discoloured medium^#, precipitate, oily film / discoloured medium^#, precipitate, oily film (pH: 7.4; osm: 354 mmol/kg)
	1000	-	20/20	5.08E+06	90	precipitate, oily film / precipitate, oily film (pH: 7.4; osm: 348 mmol/kg)
	500	-	20/20	5.20E+06	94	precipitate^#, oily film / precipitate^#, oily film (pH: 7.4; osm: 345 mmol/kg)
	250	-	20/20	5.35E+06	99	precipitate^#, oily film / oily film^#(pH: 7.4; osm: 346 mmol/kg)
	125	-	20/20	5.43E+06	102	precipitate^#, oily film / oily film^#(pH: 7.4; osm: 342 mmol/kg)
	62.5	-	20/20	5.38E+06	100	precipitate^#, oily film / oily film^#(pH: 7.4; osm: 346 mmol/kg)
	31.25	-	20/20	5.43E+06	102	oily film^#/ oily film^#(pH: 7.4; osm: 345 mmol/kg)
	15.625	-	20/20	5.38E+06	100	oily film^#/ oily film^#(pH: 7.4; osm: 345 mmol/kg)
	7.813	-	20/20	4.98E+06	86	oily film^#/ normal (pH: 7.4; osm: 346 mmol/kg)
	3.906	-	20/20	5.70E+06	111	normal / normal (pH: 7.4; osm: 350 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                  #: minimal amount

RICC: Relative Increase in Cell Counts                                                        osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Cytotoxicity results of the Chromosome Aberration Assay 1 experiment without metabolic activation

Test group	Dose (μg/mL)	S9-mix	Treatment / sampling time	Cell number (total)	RICC (%)*	Observations beginning / end of treatment
Untreated control	-	-	3/20	6.35E+06	117	normal / normal (pH: 7.4; osm: 353 mmol/kg)
Negative (vehicle) control	-	-	3/20	5.70E+06	100	normal / normal (pH: 7.4; osm: 347 mmol/kg)
NAUGARD® XL-1	2000	-	3/20	5.25E+06	88	discoloured medium^#, precipitate, oily film / discoloured medium^#, precipitate, oily film (pH: 7.4; osm: 347 mmol/kg)
	666.7	-	3/20	5.53E+06	95	precipitate^#, oily film / precipitate^#, oily film (pH: 7.4; osm: 350 mmol/kg)
	222.2	-	3/20	5.58E+06	97	oily film / oily film (pH: 7.4; osm: 354 mmol/kg)
	74.1	-	3/20	5.75E+06	101	oily film / oily film (pH: 7.4; osm: 353 mmol/kg)
	24.7	-	3/20	5.70E+06	100	oily film^#/ oily film^#(pH: 7.4; osm: 351 mmol/kg)
	8.2	-	3/20	5.78E+06	102	oily film^#/ oily film^#(pH: 7.4; osm: 358 mmol/kg)
Positive control (1 μL/mL EMS)	-	-	3/20	3.63E+06	45	normal / normal (pH: 7.4; osm: 349 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                 #: minimal amount

RICC: Relative Increase in Cell Counts                                                        osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Cytotoxicity results of the Chromosome Aberration Assay 1 experiment with metabolic activation

Test group	Dose (μg/mL)	S9-mix	Treatment / sampling time	Cell number (total)	RICC (%)*	Observations beginning / end of treatment
Untreated control	-	+	3/20	5.08E+06	98	normal / normal (pH: 7.4; osm: 347 mmol/kg)
Negative (vehicle) control	-	+	3/20	5.15E+06	100	normal / normal (pH: 7.4; osm: 347 mmol/kg)
NAUGARD® XL-1	2000	+	3/20	5.50E+06	111	discoloured medium^#, precipitate, oily film / discoloured medium^#, precipitate, oily film (pH: 7.4; osm: 345 mmol/kg)
	666.7	+	3/20	5.58E+06	113	precipitate^#, oily film / precipitate^#, oily film (pH: 7.4; osm: 345 mmol/kg)
	222.2	+	3/20	6.15E+06	131	oily film / oily film (pH: 7.4; osm: 354 mmol/kg)
	74.1	+	3/20	5.53E+06	112	oily film^#/ oily film^#(pH: 7.4; osm: 342 mmol/kg)
	24.7	+	3/20	5.60E+06	114	oily film^#/ oily film^#(pH: 7.4; osm: 345 mmol/kg)
	8.2	+	3/20	5.58E+06	113	oily film^#/ oily film^#(pH: 7.4; osm: 348 mmol/kg)
Positive control (6 μg/mL CP)	-	+	3/20	3.63E+06	53	normal / normal (pH: 7.4; osm: 341 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                  #: minimal amount

RICC: Relative Increase in Cell Counts                                                        osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Cytotoxicity results of the Chromosome Aberration Assay 2 experiment without metabolic activation

Test group	Dose (μg/mL)	S9-mix	Treatment / sampling time	Cell number (total)	RICC (%)*	Observations beginning / end of treatment
Untreated control	-	-	20/20	3.78E+06	97	normal / normal (pH: 7.4; osm: 354 mmol/kg)
Negative (vehicle) control	-	-	20/20	3.85E+06	100	normal / normal (pH: 7.4; osm: 359 mmol/kg)
NAUGARD® XL-1	2000	-	20/20	3.75E+06	97	discoloured medium^#, precipitate, oily film / discoloured medium^#, precipitate, oily film (pH: 7.4; osm: 354 mmol/kg)
	666.7	-	20/20	3.85E+06	100	discoloured medium^#, precipitate^#, oily film / precipitate^#, oily film (pH: 7.4; osm: 356 mmol/kg)
	222.2	-	20/20	4.00E+06	105	precipitate^#, oily film / oily film^#(pH: 7.4; osm: 370 mmol/kg)
	74.1	-	20/20	3.68E+06	94	oily film / oily film^#(pH: 7.4; osm: 350 mmol/kg)
	24.7	-	20/20	3.98E+06	104	oily film^#/ oily film^#(pH: 7.4; osm: 346 mmol/kg)
	8.2	-	20/20	4.00E+06	105	oily film^#/ normal (pH: 7.4; osm: 359 mmol/kg)
Positive control (0.4 μL/mL EMS)	-	-	20/20	2.43E+06	52	normal / normal (pH: 7.4; osm: 356 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                  #: minimal amount

RICC: Relative Increase in Cell Counts                                                        osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Cytotoxicity results of the Chromosome Aberration Assay 2 experiment with metabolic activation

Test group	Dose (μg/mL)	S9-mix	Treatment / sampling time	Cell number (total)	RICC (%)*	Observations beginning / end of treatment
Untreated control	-	+	3/20	2.75E+06	94	normal / normal (pH: 7.4; osm: 344 mmol/kg)
Negative (vehicle) control	-	+	3/20	2.88E+06	100	normal / normal (pH: 7.4; osm: 347 mmol/kg)
NAUGARD® XL-1	2000	+	3/20	2.68E+06	90	discoloured medium^#, precipitate, oily film / discoloured medium^#, precipitate, oily film (pH: 7.4; osm: 343 mmol/kg)
	666.7	+	3/20	3.10E+06	111	discoloured medium^#, precipitate, oily film / precipitate^#(pH: 7.4; osm: 336 mmol/kg)
	222.2	+	3/20	2.78E+06	95	precipitate^#, oily film / oily film (pH: 7.4; osm: 338 mmol/kg)
	74.1	+	3/20	2.91E+06	103	oily film^#/ oily film^#(pH: 7.4; osm: 345 mmol/kg)
	24.7	+	3/20	2.78E+06	95	oily film^#/ oily film^#(pH: 7.4; osm: 335 mmol/kg)
	8.2	+	3/20	3.08E+06	110	oily film^#/ oily film^#(pH: 7.4; osm: 340 mmol/kg)
Positive control (6 μg/mL CP)	-	+	3/20	2.00E+06	56	normal / normal (pH: 7.4; osm: 348 mmol/kg)

*: compared to the negative (vehicle) control (0.5% Acetone)                  #: minimal amount

RICC: Relative Increase in Cell Counts                                                        osm: osmolality

Note: Duplicate counts were performed at each counting.

 

Summary table of Chromosome Aberration Assay 1 without metabolic activation

Concentration (μg/mL) [Number of analyzed cells]	Time of Treatment / Sampling	RICC^# (%)	Insolubility^##	Mean % aberrant cells^###
NAUGARD® XL-1 without metabolic activation (-S9)
Untreated control	3h / 20h	117	-	NE
Negative (vehicle) control [300]	3h / 20h	100	-	2.0
2000 μg/mL	3h / 20h	88	+^b	NE
666.7 μg/mL	3h / 20h	95	+	NE
222.2 μg/mL	3h / 20h	97	+	NE
74.1 μg/mL [300]	3h / 20h	101	+	0.3
24.7 μg/mL [300]	3h / 20h	100	+^a	1.7
8.2 μg/mL [300]	3h / 20h	102	+^a	4.3
Positive control [300]	3h / 20h	45	-	8.3***

Negative (vehicle) control: (0.5% Acetone)

Positive control (-S9): Ethyl methanesulfonate, 1 μL/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

a: Minimal amount

b: discoloured medium

#: compared to the negative (vehicle) control)

##: in the final treatment medium at the end of the treatment

###: excluding gaps

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

 

Summary table of Chromosome Aberration Assay 1 with metabolic activation

Concentration (μg/mL) [Number of analyzed cells]	Time of Treatment / Sampling	RICC^# (%)	Insolubility^##	Mean % aberrant cells^###
NAUGARD® XL-1 with metabolic activation (+S9)
Untreated control	3h / 20h	98	-	NE
Negative (vehicle) control [300]	3h / 20h	100	-	4.0
2000 μg/mL	3h / 20h	111	+^b	NE
666.7 μg/mL	3h / 20h	113	+	NE
222.2 μg/mL [300]	3h / 20h	131	+	2.7
74.1 μg/mL [300]	3h / 20h	112	+^a	2.0
24.7 μg/mL [300]	3h / 20h	114	+^a	3.0
8.2 μg/mL	3h / 20h	113	+^a	NE
Positive control [101]	3h / 20h	53	-	50.5***

Negative (vehicle) control: (0.5% Acetone)

Positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

a: Minimal amount

b: discoloured medium

#: compared to the negative (vehicle) control)

##: in the final treatment medium at the end of the treatment

###: excluding gaps

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

 

Summary table of Chromosome Aberration Assay 2 without metabolic activation

Concentration (μg/mL) [Number of analyzed cells]	Time of Treatment / Sampling	RICC^# (%)	Insolubility^##	Mean % aberrant cells^###
NAUGARD® XL-1 without metabolic activation (-S9)
Untreated control	20h / 20h	97	-	NE
Negative (vehicle) control [300]	20h / 20h	100	-	4.3
2000 μg/mL	20h / 20h	97	+^b	NE
666.7 μg/mL [300]	20h / 20h	100	+	2.3
222.2 μg/mL [300]	20h / 20h	105	+^a	4.7
74.1 μg/mL [300]	20h / 20h	94	+^a	2.3
24.7 μg/mL	20h / 20h	104	+^a	NE
8.2 μg/Ml	20h / 20h	105	-	NE
Positive control [119]	20h / 20h	52	-	42.0***

Negative (vehicle) control: (0.5% Acetone)

Positive control (-S9): Ethyl methanesulfonate, 0.4 μL/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

a: Minimal amount

b: discoloured medium

#: compared to the negative (vehicle) control)

##: in the final treatment medium at the end of the treatment

###: excluding gaps

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

 

Summary table of Chromosome Aberration Assay 2 with metabolic activation

Concentration (μg/mL) [Number of analyzed cells]	Time of Treatment / Sampling	RICC^# (%)	Insolubility^##	Mean % aberrant cells^###
NAUGARD® XL-1 with metabolic activation (+S9)
Untreated control	3h / 20h	94	-	NE
Negative (vehicle) control [300]	3h / 20h	100	-	4.0
2000 μg/mL	3h / 20h	90	+^b	NE
666.7 μg/mL	3h / 20h	111	+	NE
222.2 μg/mL [300]	3h / 20h	95	+	2.3
74.1 μg/mL [300]	3h / 20h	103	+^a	3.3
24.7 μg/mL [300]	3h / 20h	95	+^a	4.7
8.2 μg/mL	3h / 20h	110	+^a	NE
Positive control [55]	3h / 20h	56	-	90.9***

Negative (vehicle) control: (0.5% Acetone)

Positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

RICC: Relative Increase in Cell Counts

a: Minimal amount

b: discoloured medium

#: compared to the negative (vehicle) control)

##: in the final treatment medium at the end of the treatment

###: excluding gaps

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

 

HISTORICAL CONTROL DATA

3h/20h treatment / sampling time without S9-mix

	Aberration rate (phases with aberration in %)
	Negative control		Positive control (EMS)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	2.71	1.20	22.67	18.02
SD	1.65	0.82	12.43	8.16
Range	0-7	0-3	4-63	4-40
n	46	46	40	40

3h/20h treatment / sampling time with S9-mix

	Aberration rate (phases with aberration in %)
	Negative control		Positive control (CP)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	3.24	1.45	73.00	69.50
SD	1.57	0.95	23.51	25.64
Range	0-8	0-4	21-100	21-100
n	43	43	21	21

SD = standard deviation

Range = min.-max. values

n = number of experiments

EMS = Ethyl methanesulfonate

CP = Cyclophosphamide

 

Notes:

1. In the period of 2008-2009, NNDA (N-Nitrosodimethylamine) was used as positive control substance in the experiments with metabolic activation. Mean aberration frequency for NNDA was 22.91 (including gaps) and 18.07 (excluding gaps) in 22 experiments.

2. In studies performed before the updated OECD guideline (2014) 2000 metaphases were scored for chromosomal aberration per samples. Minimum and maximum values reflect the total number of aberrant cells in 2000 metaphases. Furthermore, in those studies counting for a positive control sample was halted when 15 aberrant cells were counted.

 

20h/28h treatment / sampling time without S9-mix

	Aberration rate (phases with aberration in %)
	Negative control		Positive control (EMS)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	2.58	1.13	34.97	30.96
SD	1.81	0.86	10.94	9.80
Range	0-8	0-4	5-76	5-68.2
n	43	43	43	43

3h/20h treatment / sampling time with S9-mix

	Aberration rate (phases with aberration in %)
	Negative control		Positive control (CP)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	3.07	1.47	52.63	48.80
SD	1.64	1.07	23.98	24.49
Range	0-8	0-5	7-93.8	6-93.8
n	43	43	21	21

SD = standard deviation

Range = min.-max. values

n = number of experiments

EMS = Ethyl methanesulfonate

CP = Cyclophosphamide

 

Notes:

1. In the period of 2008-2009, NNDA (N-Nitrosodimethylamine) was used as positive control substance in the experiments with metabolic activation. Mean aberration frequency for NNDA was 22.91 (including gaps) and 18.07 (excluding gaps) in 22 experiments.

2. In studies performed before the updated OECD guideline (2014) 2000 metaphases were scored for chromosomal aberration per samples. Minimum and maximum values reflect the total number of aberrant cells in 2000 metaphases. Furthermore, in those studies counting for a positive control sample was halted when 15 aberrant cells were counted.

3. These historical controls were used for this study due to the lack of the 20h/20h treatment/sampling time without S9-mix historical control. 

Conclusions:

The test item NAUGARD® XL-1 was tested for potential clastogenic activity using the Chromosome Aberration Assay. The study included two Concentration Selection Cytotoxicity Assays and two Chromosome Aberration Assays.
The performed experiments were considered to be valid and to reflect the real potential of the test item to cause structural chromosomal aberrations in the cultured V79 Chinese hamster cells used in this study.
Treatment with the test item did not result in a statistically and biologically significant, reproducible, dose-dependent increase in the frequency of the cells with structural chromosome aberrations without gaps either in the presence or absence of a metabolic activation system which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

In conclusion, NAUGARD® XL-1 did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, NAUGARD® XL-1 was considered as not clastogenic in this test system.

Executive summary:

NAUGARD® XL-1 was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in Acetone and it was examined up to cytotoxic concentrations according to the OECD guideline recommendations. In independent Chromosome Aberration Assays using duplicate cultures, at least 300 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each evaluated test item treated, negative (vehicle) and positive control sample.

In Chromosome Aberration Assay3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 2000, 666.7, 222.2, 74.1, 24.7 and 8.2 µg/mL (experiment with and without metabolic activation).

In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 2000-222.2 µg/mL concentration range with metabolic activation and in the 2000-74.1 µg/mL concentration range without metabolic activation. Furthermore, insolubility (minimal amount) was detected at the end of the treatment period in the final treatment medium in the 74.1-8.2 µg/mL concentration range with metabolic activation and in the 24.7 and 8.2 µg/mL concentrations without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 222.2, 74.1 and 24.7 µg/mL (a total of three) were chosen for evaluation in the experiment with metabolic activation and concentrations of 74.1, 24.7 and 8.2 µg/mL (a total of three) were chosen for evaluation in the experiment without metabolic activation.

 

In Chromosome Aberration Assay3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 2000, 666.7, 222.2, 74.1, 24.7 and 8.2 µg/mL (experiment with and without metabolic activation).

In Assay 2, similarly to the first experiment, insolubility was detected at the end of the treatment period in the final treatment medium in the 2000-222.2 µg/mL concentration range with metabolic activation and in the 2000 and 666.7 µg/mL concentrations without metabolic activation. Furthermore, insolubility (minimal amount) was detected at the end of the treatment period in the final treatment medium in the 74.1-8.2 µg/mL concentration range with metabolic activation and in the 222.2-24.7 µg/mL concentration range without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in nay samples of this assay. Therefore, concentrations of 222.2, 74.1 and 24.7 µg/mL (a total of three) were evaluated in the experiment with metabolic activation, and concentrations of 666.7, 222.2 and 74.1 µg/mL (a total of three) were evaluated in the experiment without metabolic activation.

None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.

Polyploid metaphases (1-7) were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments, but their incidence was not related to treatment with NAUGARD® XL-1. One endoreduplicated metaphases were detected in the performed experiments in one case in the positive control.

The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; at least three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

In conclusion, NAUGARD® XL-1 did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, NAUGARD® XL-1 was considered as not clastogenic in this test system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 November 2017 to 14 December 2017

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:

OECD Guidelines for the Testing of Chemicals, Section 4, No. 490, "In Vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene", 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008 of 30 May 2008, B.17. "Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test" (Official Journal L 142, 31/05/2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)

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):

Cell line: L5178Y TK+/- 3.7.2 C mouse lymphoma
Product No.: CRL-9518
Lot No.: 1661603 / 60797977
Supplier: American Type Culture Collection (Manassas, Virginia, USA)
Date of receipt: 27 February 2014
Date of working lot: 17 May 2017 (MP14)
The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection. Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma. For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assays were selected on the available information from other in vitro genetic toxicology studies.
Assay 1, 3-hour treatment with metabolic activation: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 µg/mL,
Assay 1, 3-hour treatment without metabolic activation: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 µg/mL,
Assay 2, 3-hour treatment with metabolic activation: 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 µg/mL,
Assay 2, 24-hour treatment without metabolic activation: 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 µg/mL.

Vehicle / solvent:

Based on the available information, Acetone was selected for vehicle of the study as the test item was insoluble in other, most frequently used vehicles (Distilled water or Dimethyl sulfoxide).
Acetone was used for vehicle (solvent) of the test item. In each case, sterile vehicle was prepared and used for formulation. Data of the chemical used in the study are shown below:
Name: Acetone
Supplier: VWR
Lot No.: 17B284019
Appearance: Clear colourless liquid
Expiry date: 28 February 2022
Storage conditions: Room temperature

Dimethyl sulfoxide was used for vehicle (solvent) of the positive control chemicals. Data of the chemical used in the study are shown below:
Name: Dimethyl sulfoxide
Abbreviation: DMSO
Supplier: Sigma-Aldrich Co.
Lot No.: 016F304002
Appearance: Clear colourless liquid
Expiry date: 31 May 2021
Storage conditions: Room temperature, protected from humidity

Untreated negative controls:

yes

Remarks:

Untreated control

Negative solvent / vehicle controls:

yes

Remarks:

Acetone; DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

TEST PROCEDURE
Principles of Dose Selection
Treatment concentrations for the mutation assays were selected on the available information from other in vitro genetic toxicology studies.
As no cytotoxicity had been observed in those studies, no preliminary experiment was performed. The highest selected concentration was the recommended maximum concentration (2 mg/mL), and several concentrations (separated by factor of two) were additionally selected to properly cover the concentration range from insolubility to little or no insolubility. A total of 10 concentrations were selected for Assay 1. Concentrations for Assay 2 were slightly changed based on the actual results of
Assay 1.

Main Mutation Assays
In Assay 1, cells were treated for 3 hours in the presence and absence of S9-mix. In Assay 2, cells were treated for 3 hours in the presence of S9-mix and for 24 hours in the absence of S9-mix.
A suitable volume* of RPMI-5 medium, vehicle (solvent), test item formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or 1.0 mL of 150 mM KCl (in case of 3-hour treatment without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatments, 107 cells were placed in each of a series of 75 cm2 sterile flasks. For the 24-hour treatment, 6E+6 cells were placed in each of a series of 25 cm2 sterile flasks. The treatment medium contained a reduced serum level of 5% (v/v) RPMI-5.
*Note: Treatment volume of 0.1 mL for a final volume of 20 mL (5 µL/mL) was used for Acetone vehicle (solvent) control and test item formulations; while treatment volume of 0.2 mL for a final volume of 20 mL (10 µL/mL) was used for RPMI-5 medium (untreated control), DMSO vehicle (solvent) control and positive control solutions.
Duplicate cultures were used for each treatment. Cultures were visually examined at the beginning and end of treatments. During the treatment period, cultures were incubated at 37°C ± 1°C (approximately 5% CO2 in air). Gentle shaking was used during the
treatments. Measurement of pH and osmolality was also performed after the treatment period.
Then cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue culture medium and suspended in at least 20 mL RPMI-10. The number of viable cells in the individual samples was counted manually using a haemocytometer. Where sufficient cells survived, cell density was adjusted to a concentration of 2E+5 cells/mL. Cells were transferred to flasks for growth through the expression period (maximum 30 mL of suspension) or diluted to be plated for survival.

Plating for Survival
Using a multi-channel pipette, 0.2 mL of the final concentration of each culture were placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 °C ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks (12 days). Wells containing viable clones were identified by eye using background illumination and counted.

Expression Period
To allow expression of TK- mutations, cultures were maintained in flasks for 2 days. During the expression period, subculturing was performed daily. On each day, cell density was adjusted to a concentration of 2x105 cells/mL (whenever possible) and transferred to flasks for further growth.
On completion of the expression period, at least six test item treated samples, untreated, negative (vehicle) and positive controls were plated for determination of viability and 5-trifluorothymidine (TFT) resistance.

Plating for Viability
At the end of the expression period, the cell density in the selected cultures was determined and adjusted to 1E+4 cells/mL with RPMI-20 for plating for a viability test.
Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 °C ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks (12 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for -trifluorothymidine (TFT) resistance
At the end of the expression period, the cell concentration was adjusted to 1E+4 cells/mL. TFT (300 µg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 µg/mL. Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four, 96-well microplates (384 wells) at 2E+3 cells per well.
Microplates were incubated at 37 °C ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks (12 days) and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed to obtain information on the possible mechanism of action of the test item, if any.

Rationale for test conditions:

In accordance with Test Guidelines.

Evaluation criteria:

The test item was considered to be clearly positive (mutagenic) in this assay if all the following criteria were met:
1. At least one concentration exhibited a statistically significant increase (p<0.05) compared with the concurrent negative (vehicle) control and the increase was biologically relevant (i.e. the mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle/solvent) control value).
2. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).
3. The increase was concentration-related (p < 0.05) as indicated by the linear trend analysis.
The test item was considered clearly negative (non-mutagenic) in this assay if in all experimental conditions examined there was no concentration related response or, if there is an increase in MF, but it did not exceed the GEF. Then, test item was considered unable to induce mutations in this test system.

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software.

The negative (vehicle/solvent) control log mutant frequency (LMF) was compared to the LMF of each treatment concentration, based on Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTATION ASSAYS
In the mutation assays, cells were exposed to the test item for 3 hours with or without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The cells were plated for determination of survival data and in parallel subcultured without test item for approximately 2 days to allow expression of the genetic changes. At the end of the expression period, cells were allowed to grow and form colonies for approximately 2 weeks (12 days) in culturing plates with and without selective agent (TFT) for determination of mutations and viability.

Assay 1
In Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 µg/mL in both experiments.
In Assay 1, no large changes in pH or osmolality were detected. Insolubility / minimal amount of insolubility was observed in the final treatment medium at the end of the treatment in the 2000-62.5 µg/mL concentration range in both experiments.
In the presence of S9-mix (3-hour treatment), no marked cytotoxicity of the test item was observed. Thus, an evaluation was made using data of seven highest concentrations (concentration range of 2000-31.25 µg/mL. No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. In overall, this experiment was considered as being negative.
In the absence of S9-mix (3-hour treatment), no marked cytotoxicity of the test item was observed. An evaluation was made using data of the eight highest concentrations (concentration range of 2000-15.625 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment was considered as being negative.

Assay 2
In Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 24-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Based on the results of Assay 1, treatment concentrations were 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 µg/mL in both experiments.
In Assay 2, no large changes in pH or osmolality were detected. Insolubility / minimal amount of insolubility was observed in the final treatment medium at the end of the treatment in the 2000-74.07 µg/mL concentration range in both experiments.
In the presence of S9-mix (3-hour treatment), similarly to the first assay, no marked cytotoxicity of the test item was observed. An evaluation was made using data of all the six concentrations (concentration range of 2000-8.23 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment confirmed the negative result of the first test with metabolic activation.
In the absence of S9-mix (24-hour treatment), no marked cytotoxicity of the test item was observed. An evaluation was made using data of all the six concentrations (concentration range of 2000-8.23 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis . This experiment was considered as being negative.

Minor increases in the mutation frequency were observed sporadically in Assays 1 and 2; however, they were without any statistical significance and the difference between the observed values and the relevant solvent control value did not exceed the global evaluation factor, so they were considered as biologically not relevant increases, just showing the biological variability of the test system.

VALIDITY OF THE MUTATION ASSAYS
Untreated, negative (vehicle/solvent) and positive controls were run concurrently in the study. The spontaneous mutation frequency of the negative (vehicle/solvent) and untreated controls were in the recommended range in all cases.
The positive controls (Cyclophosphamide in the presence of metabolic activation and
4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays. All of the positive control samples in the performed experiments fulfilled at least one of the relevant OECD criteria.
The plating efficiencies for the negative (vehicle) control of the test item and untreated control samples at the end of the expression period (PEviability) were within the acceptable range (65-120%) in all assays.
The number of test concentrations evaluated was at least six in each case, which met the acceptance criteria about the minimum number of evaluated concentrations.
The tested concentration range in the study was considered to be adequate as the highest evaluated concentration was the recommended maximum concentration (2000 µg/mL) and the selected range covered concentrations with insolubility to no insolubility. Lower test concentrations were spaced by a factor of two (Assay 1) or three (Assay 2).
Suspension growth value of the untreated and negative (vehicle) control were in line with the recommended range in all cases (the minimal difference in Assay 2 without metabolic activation was considered as acceptable based on the observed mutation frequency values).
The overall study was considered to be valid.

Survival Results of Assay 1 (I.)

(3-hour treatment in the presence of S9-mix)

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells/total number of wells	Plating Efficiency (PE)	Relative Survival^§ (%RS)	Relative Total Growth (%RTG)
+	3	A1	2000 μg/mL	82/384	0.965	80	69
			1000 μg/mL	96/384	0.866	96	107
			500 μg/mL	62/384	1.140	95	66
			250 μg/mL	97/384	0.860	69	66
			125 μg/mL	77/384	1.004	82	72
			62.5 μg/mL	82/384	0.965	90	76
			31.25 μg/mL	62/384	1.140	97	100
			15.625 μg/mL	56/384	1.203	94	n.d.
			7.813 μg/mL	74/384	1.029	83	n.d.
			3.906 μg/mL	104/384	0.816	82	n.d.
			Vehicle control	51/384	1.262	100	100
			Vehicle control for CP	53/384	1.238	108	84
			Untreated control	89/384	0.914	82	95
			Positive control (CP)	178/384	0.481	36	35

A1 = Assay 1

+ = in the presence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide (4 μg/mL)

ND = No data (Cells did not survive the treatment or expression period)

n.d. = no data (Sample was not plated for viability and mutagenicity)

^§= Relative survival values (%) corrected with the post treatment cell concentrations.

 

Survival Results of Assay 1 (II.)

(3-hour treatment in the absence of S9-mix)

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells/total number of wells	Plating Efficiency (PE)	Relative Survival^§ (%RS)	Relative Total Growth (%RTG)
-	3	A1	2000 μg/mL	87/384	0.928	84	54
			1000 μg/mL	76/384	1.012	92	76
			500 μg/mL	81/384	0.973	95	70
			250 μg/mL	73/384	1.038	103	78
			125 μg/mL	85/384	0.942	95	105
			62.5 μg/mL	103/384	0.822	85	80
			31.25 μg/mL	95/384	0.873	100	90
			15.625 μg/mL	89/384	0.914	93	83
			7.813 μg/mL	77/384	1.004	109	n.d.
			3.906 μg/mL	85/384	0.942	86	n.d.
			Vehicle control	94/384	0.880	100	100
			Vehicle control for NQO	76/384	1.012	109	97
			Untreated control	80/384	0.980	115	87
			Positive control (NQO)	104/384	0.816	88	72

A1 = Assay 1

- = in the absence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide (0.15 μg/mL)

n.d. = no data (Sample was not plated for viability and mutagenicity)

^§= Relative survival values (%) corrected with the post treatment cell concentrations.

 

Survival Results of Assay 2 (I.)

(3-hour treatment in the presence of S9-mix)

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells/total number of wells	Plating Efficiency (PE)	Relative Survival^§ (%RS)	Relative Total Growth (%RTG)
+	3	A2	2000 μg/mL	66/384	1.101	83	71
			666.6 μg/mL	80/384	0.980	80	101
			222.2 μg/mL	63/384	1.130	80	75
			74.07 μg/mL	48/384	1.300	94	71
			24.69 μg/mL	43/384	1.300	94	71
			8.23 μg/mL	62/384	1.140	91	84
			Vehicle control	50/384	1.274	100	100
			Vehicle control for CP	64/384	1.120	90	75
			Untreated control	67/384	1.091	94	101
			Positive control (CP)	136/384	0.649	43	25

A2 = Assay 2

+ = in the presence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide (4 μg/mL)

^§= Relative survival values (%) corrected with the post treatment cell concentrations.

 

Survival Results of Assay 2 (II.)

(24-hour treatment in the absence of S9-mix)

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells/total number of wells	Plating Efficiency (PE)	Relative Survival^§ (%RS)	Relative Total Growth (%RTG)
-	24	A2	2000 μg/mL	75/384	1.021	101	51
			666.6 μg/mL	82/384	0.965	90	75
			222.2 μg/mL	98/384	0.854	67	51
			74.07 μg/mL	93/384	0.886	78	69
			24.69 μg/mL	74/384	1.029	88	98
			8.23 μg/mL	86/384	0.935	86	95
			Vehicle control	83/384	0.957	100	100
			Vehicle control for NQO	79/384	0.988	88	83
			Untreated control	79/384	0.988	107	109
			Positive control (NQO)	219/384	0.351	16	9

A2 = Assay 2

- = in the absence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide (0.1 μg/mL)

^§= Relative survival values (%) corrected with the post treatment cell concentrations.

 

Viability Results of Assay 1 (I.)

(3-hour treatment in the presence of S9-mix)

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells/total number of wells	Plating Efficiency (PE)
+	3	A1	2000 μg/mL	85/384	0.942
			1000 μg/mL	60/384	1.160
			500 μg/mL	119/384	0.732
			250 μg/mL	97/384	0.8600
			125 μg/mL	116/384	0.748
			62.5 μg/mL	121/384	0.722
			31.25 μg/mL	84/384	0.950
			15.625 μg/mL	n.d.	n.d.
			7.813 μg/mL	n.d.	n.d.
			3.906 μg/mL	n.d.	n.d.
			Vehicle control	83/384	0.957
			Vehicle control for CP	100/384	0.841
			Untreated control	79/384	0.988
			Positive control (CP)	156/384	0.563

A1 = Assay 1

+ = in the presence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide (4 μg/mL)

n.d. = no data (Sample was not plated for viability and mutagenicity)

 

Viability Results of Assay 1 (II.)

(3-hour treatment in the absence of S9-mix)

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells/total number of wells	Plating Efficiency (PE)
-	3	A1	2000 μg/mL	90/384	0.907
			1000 μg/mL	97/384	0.860
			500 μg/mL	104/384	0.816
			250 μg/mL	108/384	0.793
			125 μg/mL	66/384	1.101
			62.5 μg/mL	86/384	0.935
			31.25 μg/mL	90/384	0.907
			15.625 μg/mL	100/384	0.841
			7.813 μg/mL	n.d.	n.d.
			3.906 μg/mL	n.d.	n.d.
			Vehicle control	90/384	0.907
			Vehicle control for NQO	101/384	0.835
			Untreated control	107/384	0.799
			Positive control (NQO)	85/384	0.942

A1 = Assay 1

- = in the absence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide (0.15 μg/mL)

n.d. = no data (Sample was not plated for viability and mutagenicity)

 

Viability Results of Assay 2 (I.)

(3-hour treatment in the presence of S9-mix)

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells/total number of wells	Plating Efficiency (PE)
+	3	A2	2000 μg/mL	84/384	0.950
			666.6 μg/mL	67/384	1.091
			222.2 μg/mL	75/384	1.021
			74.07 μg/mL	76/384	1.012
			24.69 μg/mL	76/384	1.012
			8.23 μg/mL	68/384	1.082
			Vehicle control	63/384	1.130
			Vehicle control for CP	106/384	0.805
			Untreated control	62/384	1.140
			Positive control (CP)	175/384	0.491

A2 = Assay 2

+ = in the presence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide (4 μg/mL)

 

Viability Results of Assay 2 (II.)

(24-hour treatment in the absence of S9-mix)

S9-mix	Treatment period (hours)	Study phase	Test item or control concentration	Number of empty wells/total number of wells	Plating Efficiency (PE)
-	24	A2	2000 μg/mL	40/384	1.414
			666.6 μg/mL	75/384	1.021
			222.2 μg/mL	66/384	1.101
			74.07 μg/mL	68/384	1.082
			24.69 μg/mL	66/384	1.101
			8.23 μg/mL	74/384	1.029
			Vehicle control	69/384	1.073
			Vehicle control for NQO	59/384	1.171
			Untreated control	71/384	1.055
			Positive control (NQO)	115/384	0.754

A2 = Assay 2

- = in the absence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide (0.1 μg/mL)

 

Mutagenicity Results of Assay 1 (I.)

(3-hour treatment in the presence of S9-mix)

S9-mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/total number of wells	Dn^2/var(Dn) ◊	Mutation frequency
+	3	2000 μg/mL	671/768	59/768	38/768	0.189	71.6
		1000 μg/mL	645/768	77/768	46/768	0.408	75.2
		500 μg/mL	667/768	62/768	39/768	2.307	96.3
		250 μg/mL	687/768	40/768	41/768	0.005	64.8
		125 μg/mL	689/768	51/768	28/768	0.527	78.4
		62.5 μg/mL	695/768	67/768	37/768	0.084	69.2
		31.25 μg/mL	683/768	49/768	36/768	0.011	61.7
		15.625 μg/mL	n.d.	n.d.	n.d.	n.d.	n.d.
		7.813 μg/mL	n.d.	n.d.	n.d.	n.d.	n.d.
		3.906 μg/mL	n.d.	n.d.	n.d.	n.d.	n.d.
		Vehicle control	680/768	53/768	35/768	--	63.6
		Vehicle control for CP	651/768	72/768	45/768	--	77.0
		Untreated control	683/768	53/768	32/768	--	69.7
		Positive control (CP: 4 μg/mL)	208/768	141/768	319/768	◊◊ 1.87E-11	811.5*

In linear trend analysisβ²/var (β) = 0.28, not significant

* = Statistically significant

◊ = Evaluated by Dunnett’s test for multiple comparisons. Significant if D_n²/var(D_n) >5.48(at p<0.05).

◊◊ = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).

Dn = Differece of log mutant frequency of dose “n” and that of the vehicle control

var(D_n) = variance of D_n                    β = slope of the curve                        var(β) = variance of the slope

+ = in the presence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide

n.d. = no data (Sample was not plated for viability and mutagenicity)

Note: Mutation frequency refers to 10⁶viable cells

 

Mutagenicity Results of Assay 1 (II.)

(3-hour treatment in the absence of S9-mix)

S9-mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/total number of wells	Dn^2/var(Dn) ◊	Mutation frequency
-	3	2000 μg/mL	657/768	73/768	38/768	0.411	86.1
		1000 μg/mL	638/768	62/768	68/768	2.212	107.8
		500 μg/mL	648/768	69/768	51/768	1.789	104.1
		250 μg/mL	683/768	38/768	47/768	0.009	74.0
		125 μg/mL	663/768	58/768	47/768	0.067	66.8
		62.5 μg/mL	680/768	60/768	28/768	0.120	65.1
		31.25 μg/mL	662/768	65/768	41/768	0.210	81.9
		15.625 μg/mL	661/768	71/768	36/768	0.553	89.2
		7.813 μg/mL	n.d.	n.d.	n.d.	n.d.	n.d.
		3.906 μg/mL	n.d.	n.d.	n.d.	n.d.	n.d.
		Vehicle control	674/768	73/768	21/768	--	72.0
		Vehicle control for NQO	676/768	53/768	39/768	--	79.9
		Untreated control	678/768	64/768	26/768	--	74.7
		Positive control (NQO: 0.15 μg/mL)	346/768	208/768	214/768	◊◊ 1.63E-10	423.0*

In linear trend analysisβ²/var (β) = 1.44, not significant

* = Statistically significant

◊ = Evaluated by Dunnett’s test for multiple comparisons. Significant if D_n²/var(D_n) >5.66(at p<0.05).

◊◊ = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).

Dn = Differece of log mutant frequency of dose “n” and that of the vehicle control

var(D_n) = variance of D_n                    β = slope of the curve                        var(β) = variance of the slope

- = in the absence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide

n.d. = no data (Sample was not plated for viability and mutagenicity)

Note: Mutation frequency refers to 10⁶viable cells

 

Mutagenicity Results of Assay 2 (I.)

(3-hour treatment in the presence of S9-mix)

S9-mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/total number of wells	Dn^2/var(Dn) ◊	Mutation frequency
+	3	2000 μg/mL	674/768	49/768	45/768	0.016	68.7
		666.6 μg/mL	681/768	44/768	43/768	0.465	55.1
		222.2 μg/mL	679/768	50/768	39/768	0.124	60.3
		74.07 μg/mL	664/768	70/768	34/768	0.089	71.9
		24.69 μg/mL	655/768	71/768	42/768	0.421	78.6
		8.23 μg/mL	652/768	76/768	40/768	0.255	75.7
		Vehicle control	661/768	69/768	38/768	--	66.4
		Vehicle control for CP	673/768	46/768	79/768	--	82.1
		Untreated control	656/768	72/768	40/768	--	69.2
		Positive control (CP: 4 μg/mL)	241/768	163/768	364/768	◊◊ 1.25E-09	1179.9*

In linear trend analysisβ²/var (β) = 0.13, not significant

* = Statistically significant

◊ = Evaluated by Dunnett’s test for multiple comparisons. Significant if D_n²/var(D_n) >5.24(at p<0.05).

◊◊ = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).

Dn = Differece of log mutant frequency of dose “n” and that of the vehicle control

var(D_n) = variance of D_n                    β = slope of the curve                        var(β) = variance of the slope

+ = in the presence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control CP = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide

Note: Mutation frequency refers to 10⁶viable cells

 

Mutagenicity Results of Assay 2 (II.)

(24-hour treatment in the absence of S9-mix)

S9-mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/total number of wells	Dn^2/var(Dn) ◊	Mutation frequency
-	24	2000 μg/mL	641/768	77/768	50/768	0.005	63.9
		666.6 μg/mL	655/768	52/768	51/768	0.516	78.0
		222.2 μg/mL	656/768	55/768	57/768	0.145	71.6
		74.07 μg/mL	654/768	61/768	53/768	0.277	74.3
		24.69 μg/mL	659/768	68/768	41/768	0.070	69.5
		8.23 μg/mL	671/768	55/768	42/768	0.001	65.6
		Vehicle control	668/768	61/768	39/768	--	65.0
		Vehicle control for NQO	647/768	65/768	56/768	--	73.2
		Untreated control	667/768	64/768	37/768	--	66.8
		Positive control (NQO: 0.1 μg/mL)	174/768	335/768	259/768	◊◊ 6.89E-12	985.1*

In linear trend analysisβ²/var (β) = 0.10, not significant

* = Statistically significant

◊ = Evaluated by Dunnett’s test for multiple comparisons. Significant if D_n²/var(D_n) >5.24(at p<0.05).

◊◊ = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).

Dn = Differece of log mutant frequency of dose “n” and that of the vehicle control

var(D_n) = variance of D_n                    β = slope of the curve                        var(β) = variance of the slope

- = in the absence of S9-mix

Negative (vehicle) control = Acetone

Negative (vehicle) control NQO = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide

Note: Mutation frequency refers to 10⁶viable cells

 

Historical Control Data

(updated on 11 January 2017)

Mutation Frequency of the Negative Controls (2006 – 2016)
	Culture medium			Distilled water
Treatments	3h,S9+	3h,S9-	24h,S9-	3h,S9+	3h,S9-	24h,S9-
Average	94.3	103.6	106.4	90.4	96.6	96.3
SD	26.9	35.3	27.4	22.7	19.0	24.6
Min.	39.3	52.6	41.7	33.4	55.1	43.2
Max.	198.5	235.6	179.1	121.8	125.0	141.1
n	84	43	44	26	13	13
	Dimethyl sulfoxide (DMSO)
Treatments	3h,S9+	3h,S9-	24h,S9-
Average	97.3	97.3	98.9
SD	33.7	38.5	26.8
Min.	44.2	33.7	47.1
Max.	269.9	261.6	159.4
n	101	57	50
Mutation Frequency of the Positive Controls (2006 – 2016)
	Cyclophosphamide			4-Nitroquinoline-N-oxide
Treatment	3h,S9+				3h,S9-	24h,S9-
Average	1178.7				722.2	831.9
SD	524.7				330.0	337.2
Min.	196.1				223.5	245.0
Max.	2642.5				1687.3	1577.6
n	106				58	52

h = hour

SD = Standard Deviation

S9+ = experiment with metabolic activation

S9- = experiment without metabolic activation

n = number of cases

Conclusions:

The Mouse Lymphoma Assay with NAUGARD® XL-1 on L5178Y TK +/- 3.7.2 C cells was considered to be valid and to reflect the real potential of the test item to cause mutations in the cultured mouse cells used in this study.
In this Mouse Lymphoma Assay, treatment with the test item did not result in a statistically significant and biologically relevant increase in the mutation frequency both in the presence and absence of a rat metabolic activation system (S9 fraction) at concentrations up and including the recommended maximum concentration. Therefore, no mutagenic activity of the test item was concluded in the performed experiments.

In conclusion, no mutagenic effect of NAUGARD® XL-1 was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of NAUGARD® XL-1 test item to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

 

Acetone was used as vehicle of the test item in this study. Based on the results of other in vitro studies, the following test item concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with metabolic activation: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 µg/mL,

Assay 1, 3-hour treatment without metabolic activation: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 µg/mL,

Assay 2, 3-hour treatment with metabolic activation: 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 µg/mL,

Assay 2, 24-hour treatment without metabolic activation: 2000, 666.7, 222.2, 74.07, 24.69 and 8.23 µg/mL.

 

In Assays 1-2, there were no large changes in pH or osmolality after treatment. Insolubility / minimal amount of insolubility was observed in the final treatment medium at the end of the treatment in the 2000-62.5 µg/mL concentration range in Assay 1 with and without metabolic activation, and in the 2000-74.07 µg/mL concentration range in Assay 2 with and without metabolic activation.

 

In Assay 1, following a 3-hour treatment with metabolic activation, no marked cytotoxicity of the test item was observed. Thus, an evaluation was made using data of seven highest concentrations (concentration range of 2000-31.25 µg/mL. No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. No dose-response relationship was observed. In overall, this experiment was considered as being negative.

 

In Assay 1, following a 3-hour treatment without metabolic activation, no marked cytotoxicity of the test item was observed. An evaluation was made using data of the eight highest concentrations (concentration range of 2000-15.625 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment was considered as being negative.

 

In Assay 2, following a 3-hour treatment with metabolic activation, similarly to the first assay, no marked cytotoxicity of the test item was observed. An evaluation was made using data of all the six concentrations (concentration range of 2000-8.23 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment confirmed the negative result of the first test with metabolic activation.

 

In Assay 2, following a 24-hour treatment without metabolic activation, no marked cytotoxicity of the test item was observed. An evaluation was made using data of all the six concentrations (concentration range of 2000-8.23 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment was considered as being negative.

 

The experiments were performed using appropriate untreated, negative (vehicle/solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle/solvent) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

 

In conclusion, no mutagenic effect of NAUGARD® XL-1 was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In Vitro Ames Assay:

Compound TVIC was examined for mutagenic activity with Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, TA 1538. Each assay was performed in the presence and in the absence of a metabolic activation system. The compound assayed was found to be non-mutagenic.

In Vitro Chromosome Aberration Assay:

NAUGARD® XL-1 was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in Acetone and it was examined up to cytotoxic concentrations according to the OECD guideline recommendations. In independent Chromosome Aberration Assays using duplicate cultures, at least 300 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each evaluated test item treated, negative (vehicle) and positive control sample.

NAUGARD® XL-1 did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, NAUGARD® XL-1 was considered as not clastogenic in this test system.

In Vitro Mammalian Cell Assay

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of NAUGARD® XL-1 test item to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

In conclusion, no mutagenic effect of NAUGARD® XL-1 was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Justification for classification or non-classification

The substance is "not mutagenic" under The CLP Regulation. European Regulation (EC) No 1272/2008.