A mixture mainly based on: 2,3-dihydro-6-(2-hydroxy-2-methyl-1-oxopropyl)-1,1,3-trimethyl-3-[4-(2-hydroxy-2-methyl-1-oxopropyl)phenyl]-1H-indene; 2,3-dihydro-5-(2-hydroxy-2-methyl-1-oxopropyl)-1,1,3-trimethyl-3-[4-(2-hydroxy-2-methyl-1-oxopropyl)phenyl]-1H-indene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

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:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Appearance: clear amber coloured, solids
Storage: -20°C in the dark
Date received: 5 March 2002

Target gene:

Histidine locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat S9

Test concentrations with justification for top dose:

Experiment 1: 750, 1000, 1500, 2500, 3000, 5000, & 10000 µg/plate
Experiment 2: 93.75, 187.5, 375, 750, 1500, 3000, 5000, & 10000 µg/plate

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: Glutaraldehyde & 2·aminoanthracene

Details on test system and experimental conditions:

Test article solutions were prepared by weighing the test articles when frozen and then dissolving test material at room temperature in absolute ethanol, with the aid of vortexing and wanning at 37°C in the Experiment 1 and Experiment 2 (pre-incubation treatments) but with no aids to dissolution in Experiment 2 (plate incorporation treatnlents), immediately prior to assay to give the maximum required treatment solution concentration. These solutions were not filter-sterilised and further dilutions were made using absolute ethanol. Solubility assessments were performed during Experiment l at the time of formulation and al so following addition of the test article formulations to the test system. The test al1icle solutions were protected from light and used within approximately 5¼. hours of the initial formulation of the test article. 0.1 mL additions of test article were used for ali plate-incorporation treatments and 0.05 mL additions were used for pre-incubation treatments.

Controls
Control treatments were performed using the same addition volumes per plate as the test article treatments, 0.1 mL (or 0.05 mL for pre-incubation positive control treatments), Negative controls comprised treatments with the solvent absolute ethanol. Further control treatments employing solvents used in the formulation of positive controI chemicals were also included (as appropriate), The positive controI chemicals were supplied and used as tabulated below:

Chemical Stock* concentration (µg/mL)** Final concentration (µg/plate) Use
Strain(s) S9
2·nilrofluorene (2NF) 50 5.0 TA98 -
Sodium azide (NaN3) 20 2.0 TA100, TA1535 -
9·aminoacridine (AAC) 500 50.0 TA1537 -
Glutaraldehyde (GLU) 250 25.0 TA102 -
Benzo[a]pyrene (B[a]P) 100 10.0 TA98 +
2·aminoanthracene (AAN) 50 5.0 TA100, TA1535, TA1537 +
200 20.0 TA102 +


* With the exception of NaN3 and GLU, which were prepared in water, all slock solutions were prepared in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO). AlL stock solulions were stored in aliquots al 1-10°C in the dark, with the exception of B[a]P which was stored in aliquots at -80°C in the dark.
** For pre-incubation treatments, stock solutions of the positive contrai compounds were twice the concentration stated. This enabled the volume additions to be reduced to 0.05 ml (thus avoiding solvent-induced toxicity) and achievement of the final concentrations per plate as detailed above.

Metabolic activation system
The mammalian Iiver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254 and obtained from Molecular Toxicology Incorporated, USA. Batches of MolTox™ S-9 were stored frozen at -80°C, and thawed just prior to incorporation into the top agar. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Preparation of 10% S-9 mix and buffer solutions
Quantities \Vere prepared according to the following ratios per 100 mL:

lngredient Concentration Quantity (mL)
10% S-9 mix Buffer solution
Sodium phosphate buffer pH 7.4 500mM 20 20
Glucose-6-phosphate (disodium) 180 mg/mL 0.845 -
NADP (disodium) 25 mg/mL 12.6 -
Magnesium chloride 250mM 3.2 -
Potassium chlaride 150mM 22 -
L-histidine HCI (in 250 mM MgCI2) 1 mg/mL 4 4
d-biolin 1 mg/mL 4.88 4.88
S-9 as detailed above 10 -
Water - to volume to volume

Bacteria
Five bacterial strains of Salmonella typhimurium (TA98, TA100, TA1535, TAI537 and TA102) were used in this study. All the tester strains, with the exception of strain TA102, Were originally obtained from the UK NCTC. Strain TA102 was originally obtained from Glaxo Group Research Limited. For all assays, bacteria were cultured for 10 hours at 37±1 °C in nutrient broth (containing ampicillin for strains TA98 and
TAIOO and ampicillin and tetracycline for strain TAI02). Incubation was carried out in a shaking incubator. Bacteria were taken from vials of frozen cultures, which had been checked for strain characteristics of histidine dependence, uvrB character, rfa character and resistance to ampicillin (TA98 and T A 100) or ampicillin plus tetracycline (TA102). Checks were carried out according to Maron and Ames (1975) and De Serres and Shelby (1979). All experimentation commenced within 2 hours of the end of the incubation period.

Mutation Experiment 1
The test material was tested in strain TA100, at the concentrations detailed previously. Triplicate plates without and with S-9 mix were used. Negative (solvent) and positive controls were included in quintuplicate and triplicate respectively, without and with S-9 mix. The platings for the plate-incorporation treatments were achieved by the following sequence of additions to 2.5 mL molten agar at 46±1 °C:
• 0.1 mL bacterial culture
• 0.1 mL test article solution or controI
• 0.5 mL 10% S-9 mix or buffer solution

followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±I°C in the dark for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant coloni es were counted (see Colony counting).

For the pre-incubation treatments, quanhhes of test article or control solution (volumes reduced to 0.05 mL), bacteria and S-9 mix or buffer solution detailed above, were mixed together and incubated for 1 hour at 37± 1°C, with shaking, before the addition of 2.5 mL molten agar at 46±1 °C. Plating ofthese treatrnents then proceeded as for the normal plate-incorporation procedure.

Mutation Experiment 2
The test material was tested for mutation in five strains of Salmonella typhimurium (T A98, TAI00, TA1535, TA1537 and TA102), using standard plate-incorporation and pre-incubation methodologies, at the concentrations detailed previously, using triplicate plates without and with S-9. Negative (solvent) controls were included in each assay, in quintuplicate without and with S-9. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S-9. The activity of the S-9 mix used in each experiment was confirmed by AAN or B[a]P treatrnents (again in triplicate) of the strains in the presence of S-9. Platings were achieved as described above.
In Experiment 2 the plate-incorporation and pre-incubation treatments were performed on separate occasions.

Colony counting
Colonies were counted electronically using a Seescan Colony Counter (Seescan Plc) or manually where confounding factors such as split agar or the presence of microcolonies affected the accuracy of the automated counter. The background lawn was inspected for signs of toxicity.

Evaluation criteria:

Acceptance criteria
The assay was considered valid if the following criteria were met:
1. the mean negative control counts fell within the normal ranges as defined in Appendix 4.
2. the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation.
3. no more than 5% of the plates were lost through contamination or some other unforeseen event.

Evaluation criteria
The test article was considered to be mutagenic if:
1. the assay was valid (see above)
2. Dunnett's test gave a significant response (p 0.01) and the data set(s) showed a significant dose correlation
3. the positive responses described above were reproducible.

Statistics:

Treatment of data
IndividuaI plate counts for each test article from both experiments were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined.

The accepted normal ranges for mean numbers of spontaneous revertants on solvent controI plates for this laboratory are presented in Appendix 4. Data for this laboratory are consistent with ranges of spontaneous revertants per plate considered acceptable elsewhere. The accepted normal ranges for mean numbers of induced revertants on positive controI plates for this laboratory are presented in Appendix 5. The ranges quoted are based on a large volume of historical contro I data accumulated from experiments where the correct strain and assay functioning were considered to have been confirmed.

For evaluation of data there are many statistical methods in use, and several are acceptable. The m-statistic was calculated to check that the data were Poisson-distributed (1989), and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Toxicity, solubility and dose selection
Details of all treatment solution concentrations and fina ltest material doses are provided in the Test article section.

An initial experiment was carried out in strain TA100 only, using final concentrations of test material at 750, 1000, 1500, 2500, 3000, 5000 and 10000 µg/plate, plus solvent and positive controls. For treatments with both test articles, plate-incorporation and pre-incubation methodologies in the presence and the absence of S-9 were used.

Following plate-incorporation treatments with the first batch of test material, evidence of toxicity in the form of a marked decrease in revertant numbers was observed at the top dose treatment in the presence and the absence of S-9. No toxicity was observed following any of the treatments in either the presence or the absence of S-9 following plate-incorporation treatments with the second batch of test material. Following pre-incubation treatments with the first batch of test material, toxicity ranging from a slight thinning of the background bacterial lawn to a complete killing of the test bacteria was observed for ali treatments in the absence of S-9. No clear evidence of toxicity was observed following pre-incubation treatments with this test article in the presence of S-9. Toxicity in the form of slight thinning of the background bacterial lawn and/or a marked decrease in revertant numbers was observed following the top two pre-incubation treatments with the second batch of test material in the presence and absence of S-9.

In Experiment l, both test articles were fully soluble in absolute ethanol at all concentrations formulated, however following addition to the aqueous assay system precipitation was observed at all dose treatments. Following the three day plate incubation, this precipitation was less pronounced. Precipitate was observed following treatments of 1500 µg/plate and above in the absence of S-9 and following treatments of 2500 µg/plate and above in the presence of S-9 using plate-incorporation with both test articles. Precipitate was observed following treatments of 1500 µg/plate and above for the first batch of test material and 1000 µg/plate and above for the second batch of test material in the absence of S-9 using pre-incubation methodology. Precipitate was observed following treatments of 2500 µg/plate and above for the first batch of test material and 1500 µg/plate and above for the second batch of test material in the presence of S-9 using pre-incubation methodology.

Due to the fact that precipitation was observed over most of the treatment doses in Experiment 1, and that toxicity was observed following all first batch of test material pre-incubation treatments in the absence of S-9, an amended dose series was employed for all Experiment 2 treatments. The treatment doses used were 93.75, 187.5,375,750, 1500,3000,5000 and 10000 µg/plate. In this way it was hoped that at least three of the treatment doses would not be affected by precipitation or toxicity following treatment. Treatment of all five strains was perfonned using plate-incorporation and pre-incubation treatments in the presence and absence of S-9.

Following plate-incorporation treatments, a slight thinning of the background bacterial lawn was observed following the top dose treatment with the fist batch of test material in strain TA102 in the presence and the absence of S-9 and following the top dose treatment of the second batch of test material in strain TA102 in the absence of S-9 only. Precipitation of both test articles was observed following treatments at and above 1500 µg/plate in the absence of S-9 and at or above 3000 µg/plate in the presence of S-9. Therefore, four and five doses were unaffected by precipitation respectively.

Following pre-incubation treatments with the first batch of test material in the absence of S-9, toxicity ranging from a slight thinning of the background bacterial lawn to complete killing of the test bacteria was observed at either 187.5 or 375 µg/plate and above with all of the strains except TA1535. While this only provided one or two doses unaffected by toxicity, as the toxicity did not appear to be dose-related between 187.5 µg/plate and the higher doses in most cases, and generally there were not any significant differences between the solvent controI counts and the lowest doses affected by toxicity, it was considered that a thorough assessment of mutagenicity had been perfonned. Toxicity was noted following the top three dose treatments of strains TA1537 and TA102 only with this test article in the presence of S-9. Evidence of toxicity was observed following treatments of all strains with the second batch of test material in the absence of S-9. This toxicity was most extensive following treatments with strain TA102, where doses of 750 µg/plate and above were affected, thus providing three doses which were not affected by toxicity.
No evidence of toxicity was observed following any of the treatments with this test article in the presence of S-9. Precipitation of both test articles was observed following pre-incubation treatments of 750 µg/plate and above in both the presence and the absence of S-9. This therefore provided three doses unaffected by precipitation.

Mutation
From the data it can be seen that mean solvent control counts fell within the normal historical ranges. The positive control chemicals ali induced large increases in revertant numbers in the appropriate strains, which feli within or above the normal historical ranges. Less than 5% of plates were lost, leaving adequate numbers of plates at all treatments. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

The mutation data were evaluated as foliows:
A small but statisticaliy significant increase in revertant numbers was observed foliowing the first batch of test material pre-incubation treatment of strain TA 100 in the absence of S-9 in Experiment 1 and foliowing TA98 treatments with the second batch of test material in the absence of S-9 in Experiment 2 when the data were analysed at the 1% levei using Dunnett's test. As these increases were not reproduced on a second experimental occasion and as the increases were very smali in magnitude and did not show any evidence of a dose response they were not considered to be indicative of mutagenic activity.

No first and second batch of test material treatments of any of the remaining test strains in any of the experiments resulted in a statisticaliy significant increase in revertant numbers when the data were analysed at the 1% ievel using Dunnett's test. This study was therefore considered to have provided no indication of any test material mutagenic activity.

Conclusions:

It was concluded that the test material did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 10000 µg/plate (a precipitating dose) in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

It was concluded that the test material did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 10000 µg/plate (a precipitating dose) in the absence and in the presence of a rat liver metabolic activation system (S-9).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 January 1988 - 29 March 1988

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:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Identification: Article A - Oligo Propanone Derivative
Physical Description: Brown solid
Date Received: January 6, 1988

Target gene:

thymidine kinase (TK) locus of L5178Y TK+/- mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Derived from the Fischer L5178Y line of D. Clive. Stocks

Metabolic activation:

with and without

Metabolic activation system:

rat liver enzymes (S9 fraction) and an energy-producing system, CORE (nicotinamide adenine dinucleotide phosphate and isocitric acid) prepared at pH 7.5

Test concentrations with justification for top dose:

In the first study the assay was initiated with dose levels ranging from 10.0 µg/ml to 250.0 µg/ml, but due to excessive toxicity only concentrations
up to 125 .0 µg/ml were cloned.

Vehicle / solvent:

Ethanol
Test articles of most physiochemical classes can be evaluated in the mouse lymphoma mutation assay. Solid and liquid test articles were dissolved, if possible, at ten-times the highest desired concentration in culture medium without serum or water and lower 10x concentrations were then prepared by serial dilution with culture medium without serum. If the test article was incompletely soluble in culture medium or water, organic solvents were tried in the order of dimethylsulfoxide (DMSO), ethanol, and acetone.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

DOSING PROCEDURE

Test articles of most physiochemical classes can be evaluated in the mouse lymphoma mutation assay. Solid and liquid test articles were dissolved, if possible, at ten-times the highest desired concentration in culture medium without serum or water and lower 10x concentrations were then prepared by serial dilution with culture medium without serum. If the test article was incompletely soluble in culture medium or water, organic solvents were tried in the order of dimethyl sulfoxide (DMSO), ethanol, and acetone. A solution in an organic solvent was serially diluted with that solvent and then diluted 1:100 into culture medium to test for precipitate formation. If incomplete solubility was obtained with all solvents, the chosen vehicle was the one giving a higher solubility and/ or the best dispersion characteristics.

Preparations of test article in the solvent were prepared fresh each day for biological testing. Treatments were initiated by making 1:10 dilutions of medium soluble stocks or 1:100 dilutions of organic solutions into culture medium containing the cells. The volume of culture medium used in activation studies was reduced to compensate for the volume of S9 mix used.


RANGE FINDING CYTOTOXICITY TESTING

After the selection of ethanol as a suitable vehicle, a wide range of test article concentrations was tested for cytotoxicity, starting with a maximum applied dose of 250.0 µg /m l. Ten concentrations spanning a 3-log concentration range were used.

Cells were quantitatively seeded into a series of tubes at 6E+06 cells per tube. The volume during treatment with the test article and throughout the overnight growth period was 10 ml. After an exposure time of 4 hours, the cells were washed and a viable cell count was obtained the next day. Relative toxicities, expressed as the reduction in growth compared to the growth of untreated cells, were used to select ten doses for mutagenicity testing, covering the range from approximately 0% to 90% reduction in cell growth com-pared to the solvent controls. Treatment conditions chosen for both the nonactivation and activation portions of the mutation assay covered a 25-fold range from 10.0 µg/ml to 250.0 µg / ml. Although ten doses have been selected to initiate a mutation experiment, the objective was to carry five doses through the entire experiment. This procedure compensates for daily variations in cellular toxicity and helps to ensure the choice of at least 4 doses approximately spaced in the relative growth range of approximately 10- 100%.


MUTAGENICITY TESTING

Nonactivation Assay:
The assay procedure was based on that reported by Clive and Spector (1975). The cleansed cells were seeded into a series of tubes at 6E+06 cells per tube. The volume during treatment with the test article and throughout the expression period was 10 ml and 20 ml per tube, respectively. Dosed tubes were closed and placed in a shaker incubator at approximately 37°C for an exposure period of 4 hours. Afterwards, the cells were washed twice, resuspended in growth medium, and returned to the incubator as closed-tube cultures for two days to allow recovery, growth and expression for the induced TK-/- phenotype. Cell densities were determined on Day 1 (about 24 hours after treatment) and adjusted to 3E+05 cells/ml to maintain optimal growth rates. If the cells failed to multiply to a density of 4E+05 cells/ml, the cultures were returned to the shaker incubator without being diluted.
On Day 2, cell counts again were determined, and appropriate cultures were selected for cloning and mutant selection.

The assay treatment conditions consisted of four vehicle control cultures, four positive control cultures, and ten treatment levels (two cultures per dose level). Several of the treated cultures may be eliminated during the expression period. Five or six doses were selected for mutant analysis, inducing a wide range of toxic action so that moderately toxic treatments (approximately 50% relative growth or greater) and highly toxic treatments (approximately 10% to 20% relative growth) were represented, if possible.
Tubes with cell densities less than approximately 3E+05 cells/ml on Day 2 were not considered for analysis.

Each tube culture selected for analysis was sampled to obtain cells for exposure to the selection agent and to determine the cloning efficiency of the population. A total sample size of 3E+06 cells was seeded in soft agar plates with selection medium to select for mutants. This sample was distributed into three 100 mm dishes such that each dish contained approximately 1E+06 cells.
The cloning efficiency was determined by serially diluting the sample and seeding each of three dishes with approximately 200 cells in cloning medium. All dishes were placed in an incubator at approximately 37°C with approximately 5% C02/95% humidified air for colony development. After eleven days in the incubator, colonies were counted on an Artek Model 880 counter fitted with a ten-turn potentiometer. The smallest detectable colony was between 0.2 and 0.3 mm in diameter, depending on its position in the agar matrix.


Activation Assay:
The activation assay was performed independently with its own set of negative and positive controls. The procedure was identical to the nonactivation assay except for the addition of the S9 fraction of rat liver homogenate and necessary cofactors during the four-hour treatment period. The cofactors consisted of nicotinamide adenine dinucleotide phosphate (NADP, sodium salt) and isocitric acid.

Indicator Cells
The mouse lymphoma cell line, L5178Y TK+/- 3.7.2C, used in this assay was derived from the Fischer L5178Y line of D. Clive. Stocks were maintained in liquid nitrogen and laboratory cultures were grown in a shaker incubator at approximately 37°C to maintain population doubling times of approximately 10 hours. Laboratory cultures were periodically checked for karyotype stability and for the absence of mycoplasma contamination. To reduce the negative control frequency (spontaneous frequency) of TK-/- mutants to as low a level as possible, the cell cultures were exposed to conditions which select against the TK-/- phenotype. Cells were maintained in cleansing medium for one day, placed in recovery medium for one day and then returned to culture medium. Cleansed cultures were used to initiate mutation assays from three to eight days after having been removed from cleansing medium.

Media

Cells used during experimental studies were maintained in Fischer's mouse leukaemia medium supplemented with L-glutamine, penicillin G, streptomycin sulfate, pluronic F6B, sodium pyruvate, and horse se rum (10% by volume), hereafter referred to as culture medium or growth medium. Cleansing medium used for reducing the frequency of TK-/- mutants prior to experimental studies consisted of culture medium with 1.2E-05 M thymidine, 3.7E-05 M hypoxanthine, 1.0E-04 M glycine, and 2.2E-07 M methotrexate. Recovery medium was cleansing medium with the methotrexate component removed. Cloning medium consisted of RPMI 1640 supplemented with L-glutamine, sodium pyruvate, antibiotics, and 13.6% horse serum, with the addition of agar in phosphate buffered saline (PBS) to a final concentration of 0.39% to achieve a semi solid state. Selection medium was cloning medium containing 3 µg/ml of TFT.

Evaluation criteria:

See 'any other infromation on materials' section.

Statistics:

An experimental mutant frequency will be considered acceptable for evaluation only if the relative cloning efficiency is 10% or greater and the total number of viable clones exceeds about 60 . These limits avoid problems with the statistical distribution of scorable colonies among dishes.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

250 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

250 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Range finding Cytotoxicity Assay

Article A - Oligo Propanone Derivative was tested in the preliminary cytotoxicity assay both with and without S9 metabolic activation. Ten dose levels were used in each case ranging from 0.5 µg/ml to 250.0 µg/ml. In the preliminary cytotoxicity assay without activation high toxicity was obtained at 125.0 µg/ml (16.1 % relative survival) and lethality at 250.0 µg/ml. The test article appeared to be slightly more toxic when tested with S9 metabolic activation, resulting in high toxicity at 62.5 µg/ml (19.0% relative survival) and excessive toxicity at 125.0 µg/ml (1.7% relative survival). These results were used to select ten dose levels for use in the mutation assays. Treatment levels chosen covered a 25-fold range from 10.0 µg/ml to 250.0 ~g/ml in an effort to cover a wide range of toxic action and to compensate for daily variations in cellular toxicity.

In compliance with regulatory guidelines, the Sponsor authorized two independent studies using two cultures per dose level to be performed with and without metabolic activation. The results of these trials are shown in Tables 1-5.

Mutation Assay Without Metabolic Activation

In the first study the assay was initiated with dose levels ranging from 10.0 µg/ml to 250.0 µg/ml, but due to excessive toxicity only concentrations up to 125 .0 µg/ml were cloned (Table 1). At these concentrations a percentage relative growth of 73.8 to 5.8 was induced. In order for a treatment to be considered mutagenic in this assay, a mutation frequency exceeding 47.1E-06 was required. In this trial the mutation frequencies showed a slightly dose-related increase, with one of the cultures at 125.0 µg/ml just exceeding the minimum criterion of mutagenesis. This increase was not confirmed by the duplicate cultures and was obtained at excessive toxicity < 10% relative growth) and should not be viewed as clearly indicative of mutagenic activity.

The independent repeat test was initiated with concentrations of the test article from 10.0 µg /m l to 150.0 µg/ml, but due to excessive toxicity only concentrations up to 80.0 µg/ml were cloned (Table 2). The concentrations assayed for mutant analysis induced a percentage relative growth of 70.7 to 10.6. The minimum criterion for mutagenesis in this assay (63.3E-06 ) was just exceeded by one of the cultures of 40.0 µg /ml and both cultures of 60.0 µg/ml and 80.0 µg/ml. This trial was therefore evaluated as weak positive. Because the weak mutagenic response in this trial was not confirmed in the first trial, an additional trial was initiated. The additional mutagenicity test was initiated with concentrations from 10.0 µg/ml to 150.0 µg /m l, but due to excessive toxicity only concentrations up to 100.0 µg/ml were cloned (Table 3). At these concentrations a percentage relative growth of 93.7 to 10.5 was induced, while none of the cultures exceeded the minimum criterion of mutagenesis (60.5E-06 ). The small increases in mutant frequency in the second trial were not confirmed in first trial and in the additional trial and therefore the test article was considered nonmutagenic without metabolic activation.

Mutation Assay With Metabolic Activation

In the presence of a metabolic activation system the first assay was initiated with dose levels from 10.0 µg/ml to 250.0 µg /ml, but due to excessive toxicity only concentrations up to 60.0 µg/ml were cloned (Table 4). At these concentrations a percentage relative growth of 103.7 to 7.0 was induced. In order for a treatment to be considered mutagenic in this assay, a mutation frequency exceeding 94.7E-06 was required. In this trial the mutation frequencies showed a slight dose-related increase, with one of the cultures at 60.0 µg/ml just exceeding the minimum criterion of mutagenesis. This increase was not confirmed by the duplicate culture and was obtained at excessive toxicity < 10% relative growth) and should not be viewed as clearly indicative of mutagenic activity. The requirement to obtain treatments in the range of 10 to 20% relative growth was waived because a dose increment of 1.5 caused excessive toxicity. The independent repeat study (Table 5) was initiated with concentrations of the test article from 5.0 µg/ml to 125.0 µg/ml, but due to excessive toxicity only concentrations up to 60.0 µg/ml were cloned. The concentrations that were assayed for mutant analysis induced a percentage relative growth of 103.4 to 9.0, while none of the cultures exceeded the minimum criterion of mutagenesis (121.7E-06). This trial was therefore evaluated as negative.

In the assays used in this evaluation, the average cloning efficiencies of the negative controls varied from 93.0% to 101.1% without activation and from 89.7% to 90.3% with activation, which demonstrated excellent cloning conditions for the assays. The mutant frequency of all negative and positive controls were acceptable and within the historical range for this laboratory.

DATA PRESENTATION

The screened doses, cell counts, and mutant and viable colony counts are analyzed and presented in tables.

The suspension growth of each culture is calculated as (Day 1 Cell Count/3) x (Day 2 Cell Count/3) when the cultures are split back to 3E+05 cells/ml after the daily count. If the cell count is less than 4E+05 cells/ml, the culture is not split back and the cell count is substituted for 3 in the denominator of the next daily count. The suspension growth is calculated for each solvent control and then averaged. Relative suspension growth values are derived by dividing the suspension growth values by the average solvent control value and multiplying by 100%.

The average cloning efficiency for the solvent controls in an assay is the average number of viable colonies for the controls, divided by 600 and multiplied by 100%. In the tables, the cloning efficiency of each culture is expressed relative to the average solvent control cloning efficiency. Whenever the number of cells seeded for viable colony counts differs from 600, the calculation of the relative cloning efficiency is adjusted by the factor (600/cells seeded).

A percent relative growth value is calculated as (relative suspension growth) x (relative cloning efficiency/l00). Corrected values for the relative cloning efficiency are used in the cases where the number of cells seeded for viable colonies differs from 600.

The mutant frequency is calculated as the ratio of mutant colonies to viable colonies times 2E-04. This calculation is unaffected by changes in the number of cells seeded for viable count because the number of cells seeded for mutant selection is changed by the same factor. Thus, as an example, if 500 cells are seeded for viable count, 2.5E-06 cells are seeded for mutant selection; the 10-4 factor remains constant.

Conclusions:

The test article, Article A - Oligo Propanone Derivative, was assayed in L5178Y mouse lymphoma cells from 10.0 µg/ml to 125.0 µg/ml without metabolic activation and from 5.0 µg/ml to 60.0 µg/ml with S9 metabolic activation, inducing a wide range of toxicities. Under nonactivation conditions small dose-related increases in mutant frequency were induced in one trial, just exceeding the minimum criterion of rnutagenesis. However, these small increases could not be confirmed in two other trials. In the presence of a rat liver activation system no significant increases in the mutant frequency were induced. The test article, Article A - Oligo Propanone Derivative, was therefore evaluated as nonmutagenic in the Mouse Lymphoma Forward Mutation Assay both without and with S9 metabolic activation, according to our evaluation criteria.

Executive summary:

The test article, Article A - Oligo Propanone Derivative, was evaluated as non-mutagenic in the Mouse Lymphoma Forward Mutation Assay both without and with S9 metabolic activation, according to our evaluation criteria.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: Article A - Oligo Propanone Derivative,
Physical Description: brown solid (brown crystals after grinding)
Date Received: September 8, 1987

Target gene:

Histidine locus

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

1.0, 10.0, 100.0, 500.0, 1000.0, 2500.0, 5000.0, & 10,000.0 µg

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Remarks:

Solvent control used as negative

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: Quinacrine mustard & 2-Anthramine

Details on test system and experimental conditions:

Indicator Microorganisms
The Salmonella typhimurium strains used are alI histidine autotrophs by virtue of mutations in the histidine operon. When these histidine-dependent cells are grown in a minimal media petri plate containing a trace of histidine, only those cells that revert to histidine independence (his+) are able to form colonies. The trace amount of histidine allows all the plated bacteria to undergo a few divisions; this growth is essential for mutagenesis to occur. The his+ revertants are easily scored as colonies against the slight background growth. The spontaneous mutation frequency of each strain is relatively constant, but when a mutagen is added to the agar the mutation frequency is increased 2- to 100-fold. Cells which grow to form colonies on the minimal media petri plates are therefore assumed to have reverted, either spontaneously or by the action of a test substance to his+ genotype.

The Salmonella tyhimurium strains used in this assay were obtained from Dr. Bruce Ames, University of California at Berkeley. The following strains are used:

The aforementioned strains have, in addition to the mutation in the histidine operon, a mutation (rfa-) that leads to defective lipopolysaccharide coat, a deletion that covers genes involved in the synthesis of vitamin biotin (bio-) and in the repair of ultraviolet (uv) -induced DNA damage (uvrB-). The rfa- mutation makes the strains more permeable to many large molecules. The uvrB- mutation decreases repair of some types of chemically or physically damaged DNA and thereby enhances the strain's sensitivity to some mutagenic agents.

The resistant transfer factor plasmid (R factor) pKM10l in TA-98 and TA-100 is believed to cause an increase in error-prone DNA repair that leads to many more mutations for a given dose of most mutagens. In addition, plasmid pKM101 confers resistance to the antibiotic ampicillin, which is a convenient marker to detect the presence of plasmid in the cells .

The indicator strains were kept at 4°C on complete medium plates supplemented with ampicillin (25 µg/ml) for TA-98 and TA-l00, to ensure stable maintenance of plasmid pKM101. New stock culture plates are made as often as necessary from frozen master cultures or from single colony reisolates that were checked for their genotypic characteristics (his, rfa, uvrB, bio) and for the presence of the plasmid.

- Media:
For daily use, an inoculum from stock culture plates is grown overnight at 37°C in Oxoid Media #2 (nutrient broth) and used in the mutagenicity test. The minimal media plates for the selection of histidine revertants consisted of the Vogel Bonner Medium E (Vogel and Bonner, 1956) with 2% glucose and 1.5% bactoagar. The overlay agar contained the following per 100 ml volume; 0.6 gms of purified agar, 10 ml of 0.5mM L-Histidine-0.5mM Biotin and 0.5 9 NaCl according to the method of Ames, et al (1975).

- Activation system:

1. S9 Homogenate:
A 9,000 x 9 supernatant prepared from 5prague-Oawley adult male rat liver induced by Aroclor 1254 (described by Ames et al., 1975) was purchased commercially and used in this assay.

2. S9 Mix:
Components Concentration per Milliliter S9 Mix
NADP (sodium salt) 4 µmoles
D-glucose-6-phosphate 5 µmoles
MgC12 8 µmoles
KCl 33 µmoles
Sodium phosphate buffer pH 7.4 100 µmoles
Organ homogenate from rat liver (59 fraction) 100 µliters

- Control Compounds:
1. A negative control, consisting of the solvent used for the test material, was assayed concurrently with the test material. The solvent control was employed for each indicator strain and was used in the absence and presence of 59 mix.
The solvent was tested at a single concentration equal to the maximum volume used to administer the highest dose of the test artic1e or at the concentration indicated in the Resu1ts Table(s) of this report. The solvent used to prepare the stock solution of the test article is given below. A11 dilutions of the test article were made using this solvent.

2. Positive Control Articles:
Strain specific positive controls and positive controls to ensure the efficacy of the S9 mixture were assayed concurrently with the test material. The following positive controls were employed in the assays:

Assay Chemical Concentrations per plate
Solvent (µg) Strains Salmonella
Nonactivation Sodium azide (SA) Water 10.0 TA-1535, TA-100
2-Nitrofluorene (NF) Dimethyl sulfoxide 10.0 TA-1538, TA-98
Quinacrine mustard (QM) Dimethyl sulfoxide 5.0 TA-1537
Activation 2-Anthramine (2AA) Dimethyl sulfoxide 2.5 For all strains

METHODS:
A. Dosage Selection:
Doses were selected for the actual assay based on a preliminary dose rangefinding test with the strain TA-100. Fourteen doses of the test material, using two-fold dilutions from 10,000.00 /µg per plate were used in this dose selection assay.

B. Dose Rangefinding Test:
To a sterile test tube containing 2.0 ml of overlay agar (placed in a 43°-45°C water bath) the following were added:

• 0.05 - 0 . 10 ml of a solution of the test material to give the appropriate dose.
• 0.1 to 0.2 ml of indicator organism.
• 0.5 ml of 0.2M phosphate buffer, pH 7.4.
This mixture was swirled gently and then poured on to minimal agar plates (see II.C; Media). After the overlay agar had set, the plates were incubated at about 37°C for approximately two days. The number of colonies growing on the plates were counted and recorded.

A reduction in the number of revertants, appearance of microcolonies, or clearing of the background lawn on the test material treated plates as compared to the solvent control plates were considered as indications of toxicity by the test material.

C. Mutagenicity Testing
The procedure used is based on the paper published by Ames et al, 1975) and was performed as follows:

1. Nonactivation Assay
To a sterile test tube placed in a 43°C-45°C water bath the following were added in order:
(a) 2.00 ml of overlay agar (see II . C; Media).

(b) 0.05 - 0.10 ml of a solution of the test chemical to yield the appropriate dose.

(b) 0 . 2 ml of indicator organism.
(d) 0.50 ml of 0.2M phosphate buffer, pH 7.4.
This mixture was swirled gently and then poured onto minimal agar plates (see II . C; Media) . After the top agar had set, the plates were incubated at about 37°C for approximately two days. The number of his+ revertant colonies growing on the plates were counted and recorded.

2. Activation Assay
The activation assay was run concurrently with the nonactivation assay . The only difference was the addition of 0.5 mI of 59 mix (see II.D.2; Activation 5ystem) in pIace of 0.5 mI of phosphate buffer that was added in nonactivation assays. AlI other details are similar to the procedure for nonactivation assays.

A detailed flow diagram for the plate incorporation assay is provided in Figure 1.

D. Explanatian of Evaluation Procedures far Ames Plate Test Data

Statistical methods are not currently used, and evaluation is based on the criteria included in this report .

Plate test data consist cf direct revertant colony counts obtained fram a set cf selective agar plates seeded \~;th populations of mutant cells suspended in a semisolid overlay. Because the test material and the cells are incubated in the overlay for approximately two days and a few cell divisions occur during the incubation period, the test is semiquantitative in nature. Although these features reduce the quantitation of result, they provide certain advantages not contained in a quantitative suspension test:

• The small number of cell divisions permits potential mutagens to act on replicating DNA, which is often more sensitive than nonreplicating DNA .

• The combined incubation cf the test material and the cells in the overlay permits constant exposure of the indicator cells for approximately two days.

1. Dose-Response Phenomena
The demonstration of dose-related increases in revertant counts is an important criterion in establishing mutagenicity. Since we employ several doses in the actual assay, a dose response would normally be seen with a mutagenic test material. Additional tests may be performed over a narrower dose range if the mutagenic test material fails to exhibit a dose-response in the initial assay. However, occasionally it is difficult to generate a dose-response and the test material wi ll be evaluated based on the available data.

2. Pattern
Because TA-l535 and TA-100 are both derived from the same parental strain (G-46) and because TA - l538 and TA-98 are both derived from the same parental strain (D3052), to some extent there is a built-in redundancy in the microbial assay. In general, the two strains of a set respond to the same mutagen and such a pattern is sought. Generally, if a strain responds to a mutagen in nonactivation tests, it will do so in activation tests. occasionally, exceptions to this pattern may be seen.

3. Reproducibility
If a test material produces a response in a single test which cannot be reproduced in additional runs, the initial positive test data lose significance.

4. Control Tests
Positive and negative control assays are conducted with each experiment and consist of direct-acting mutagens for nonactivation assays and mutagens requiring metabolic bio-transformation in activation assays. Negative controls consist of the test material solvent in the overlay agar, together with the other essential components. The negative control plate for each strain gives a reference point to which the test data are compared. The positive control assay is conducted to demonstrate that the test systems are functional with known mutagens.

E. Evaluation Criteria for Ames Assay
Because the procedures used to evaluate the mutagenicity of the test material were semiquantitative, the criteria used to determine positive effects were inherently sUbjective and were based primarily on a historical data base. Most data sets were evaluated using the following criteria:

l. Strains TA-l535, TA-l537 and TA-l538
Data sets will be evaluated as positive if a dose response is observed over a minimum of three test concentrations and the increase in revertants is equal to or greater than three times the solvent control value at the peak of the dose response. The solvent control should be within the normal range for evaluating the results (see also 111.0.1: Dose response phenomena).

2. Strains TA-98 and TA-l00
Data sets will be evaluated as positive if a dose response is observed over a minimum of three test concentrations and the increase in revertants achieves a doubling of the solvent control value at the peak of the dose response. The solvent control value should be within the normal range for evaluating the results (see also 111.0.1; Dose response phenomena).

The following ranges of revertants for solvent controls are generally considered acceptable:
TA-1535: 8-30
TA-1537: 4-30
TA-1538: 10- 40
TA-98: 20-75
TA-l00: 80-250

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Additional information on results:

Preliminary Dose Rangefinding Studies
Doses for the mutagenesis assays were selected from a preliminary study conducted on the test material at 14 doses from 1.22 to 10,000.0 µg per plate using the strain TA-100. At dosing in the overlay agar, the test material precipitated at 156.25 µg per tube and higher doses . In this preliminary study, the test material did not exhibit toxicity to the indicator strain at any of the doses tested as evidenced by the number of revertants on the minimal plates.
Therefore, the mutation assays were conducted at 8 doses from 1.0 to 10,000.0 µg per plate. The mutation assays were conducted using three plates per dose level.

C. Mutation Assays
Tables 2-9 provide the summary of the test results for the test material and the data are presented as individual plate counts with mean revertants per plate and standard deviation for each treatment and control group. The results of the first assays conducted on the test material at dose levels ranging from 1.0 to 10,000.0 µg per plate in the absence and presence of metabolic activation were positive with the strain TA-l00. Also with the strains TA-1538 and TA-98 without activation and with TA-98 with activation, increases in the numbers of revertants were observed but these increases were not sufficient for a positive evaluation. The original test with the strain TA-1535 was repeated because of contamination (data not shown). The repeat test with this strain was negative.

The independent repeat test confirmed the positive results with the strain TA-l00 both with and without metabolic activation. Again increases with the strains TA-1538 and TA-98 were observed which were not sufficient for a positive evaluation with these strains.

In consultation with the Sponsor, an additional trial was performed under maximum feasible darkness with the strain TA-l00, nonactivation part only. This test again confirmed the positive results of the previous trials.

The positive control treatments in both the nonactivation and S-9 activation assays induced large increases in the revertant numbers with all the indicator strains, which demonstrates the effectiveness of the S-9 activation system and ability of the test system to detect known mutagens.

Conclusions:

The test material, Article A - Oligo Propanone Derivative, exhibited genetic activity with the strain TA-l00 in both the non-activation and activation part of the assays conducted in this evaluation and was considered mutagenic to Salmonella typhimurium indicator organisms under these test conditions according to our evaluation criteria.

Executive summary:

At the request of Flli. Lamberti S.p.a., Hazleton Biotechnologies examined Article A - Oligo Propanone Derivative, for mutagenic activity in the Ames Salmonella/Microsome assays using Salmonella typhimurium strains TA-1535, TA-1537, TA-1538, TA-98 and TA-100. The assays were conducted using three plates per dose level in the presence and absence of a metabolic activation system. The entire assay was performed twice as requested by the Sponsor.

The test material, Article A - Oligo Propanone Derivative, exhibited genetic activity in these assays and was considered mutagenic under these test conditions according to our evaluation criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 November 1988 - 16 December 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

The test article ARTICLE A, batch HE 230, was supplied by the Sponsor in the form of a orange-coloured vitreous solid.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD (SD) BR rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River Company of Calco (Como), Italy

- Weight at study initiation:
102 to 130 g

- Assigned to test groups randomly:
The animaIs were allocated to the experimental groups, caged by five and appropriately identified. No formal randomization was adopted.

- Housing:
Rats were housed in a room expressly equipped for this test only, having the following ambient conditions: temperature 22 ± 2 deg. C, relative humidity 60% ± 20, 13 air changes per hour filtered on HEPA 98%, artificial lighting with a circadian cycle of 12 hours of light. Rats were kept for the entire duration of the study in makrolon cages (cm 42x26x15), each fitted with a stainless steel cover-feed rack. Dust-free poplar/fir chips (Type 700/2000 - Robino S.n.c. - Moncalieri -To), heat processed for resin removal, were used for bedding. The Producer supplies a certificate of analysis for each batch of wood chips. Contaminants that might interfere with the objective of the study were not expected to be present in bedding.

- Diet/water (e.g. ad libitum):
The animals were fed a diet coded 4RF21GLP, produced by Charles River Italia's feed licensee Italiana flangimi, Settimo flilanese. The declared contents, on the Iabel, on dry matter basis (moisture 12%), were
crude protein 21 %
crude fat 3.5 %
crude fibre 6.5 %
ash 9 %
Non-nitrogen extracts 60 %

The diet was supplemented by the Producer with vitamins and microelements. According to the analytical certificates provided by the supplier, the contents of the batch of diet used in this study were within ± 5% of the declared values and contaminants were within the limits proposed by EPA-TSCA 44FR:44053-44093, July 26, 1979).
The animal feed, in compliance with RBM SOPs Is analysed twice a year for bacterial contamination.
The diet was available to the animals "ad libitum".
Filtered water was distributed by means of an automatic watering valve system. The drinking water offered to the animals came from the municipal water main.
The water is periodically analysed for microbiological count, heavy metals and other physical and chemical properties. Contaminants that might interfere with the objectives of the study were not expected to be present either in the diet or in the water.

Route of administration:

oral: gavage

Vehicle:

On the basis of the Sponsor indication, due to the physical characteristics of the test article the material was freezed at -20 °C , then 8 grams were taken and ground by pestle and mortar and dissolved in olive-oil by gently warming to obtain the concentration of 200 mg/ml.
The formulate was prepared just before its use and kept protected from light (container wrapped with metal foil) .

Mitomycin C was dissolved in distilled water lo obtain the concentration of 0 .8 mg/ml.

Details on exposure:

The negative controI and the test article were administered by oraI route (by gavage) , while Mitomycin C was administered by intraperitoneal route.

Duration of treatment / exposure:

1 treatment

Frequency of treatment:

1 treatment

Post exposure period:

17 , 42 or 65 hours

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Negative controI (vehicle-lreated animals): 30 (15M + 15F)
Treated with ARTICLE A: 30 (15M + 15F)
Positive con traI (Mitomycin C): 10 (5M + 5F)

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C; 8 mg/kg

Tissues and cell types examined:

polychromatic erythrocytes

Details of tissue and slide preparation:

The femurs were immediately removed and the bone marrow taken, suspended, and accurately washed in 3 mI of fetal bovine serum.
The suspension was centrifuged at 1,000xg for 10'. The supernatant was carefully taken off and the celI sediment smeared on microscope slides.
Two slides/animal were prepared.

At least 20 hours later, the smears were stained as follows:

1. immersion in May Gruenwald (Merck) solution for 3 min.
2. immersion in May Gruenwald solution diluted 1: 1 with water for 2 min., then rinsed twice with water
3. immersion in Giemsa (Merck) solution diluted 1:6 with water for 10 min.
4. rinsing with water and accurate drying with filter paper
5. washing back surface and slide with methanol

The stained slides were coded and read at the microscope (1250 X).

Evaluation criteria:

For each animaI, 2000 polychromatic erythrocytes were counted and scored for micronucleated cells.

Statistics:

Statistical evaluation: treated groups versus control group.

Data evaluation:
Comparison of the frequency among groups will be performed with a non-parametric method (Mann Whitney) (3).
Slgnificances were expressed in the following manner:

* p < 0.05
** p < 0.01
*** p < 0.001

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

On the basis of the resuIts and from the statistical analysis there is no significant difference between the micronucleus frequency in the treated group in comparison with the controi group, at any sampling time.

The group of animals treated with Mitomycin C showed a statistically different frequency of micronucleated cells in comparison with the control group.

Full data tables attached to background material section.

Conclusions:

The results of this study indicate that the test article ARTICLE A, administered to Charles River rats by oral route at the dose of 2000 mg/kg, did not induce any statistically significant increase in the frequency of micronucleated cells in the bone marrow after 17, 42 and 65 hours from the administration.

Executive summary:

The results of this study indicate that the test article administered to Charles River rats by oral route at the dose of 2000 mg/kg, did not induce any statistically significant increase in the frequency of micronucleated cells in the bone marrow after 17, 42 and 65 hours from the administration.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The substance does not meet the criteria for classification in accordance with the classification, labelling and packaging regulation (EC 1272/2008).