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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

402-990-3

EC Name:

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

Cas Number:

163702-01-0

Molecular formula:

C26H32O4

IUPAC Name:

Reaction mass of 2-hydroxy-1-{3-[4-(2-hydroxy-2-methylpropanoyl)phenyl]-1,1,3-trimethyl-2,3-dihydro-1H-inden-5-yl}-2-methylpropan-1-one and 2-hydroxy-1-{1-[4-(2-hydroxy-2-methylpropanoyl)phenyl]-1,3,3-trimethyl-2,3-dihydro-1H-inden-5-yl}-2-methylpropan-1-one

Test material form:

solid

Remarks:

Form specified in RSS.

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

Method

Target gene:

Histidine locus

Species / strain

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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.

Applicant's summary and conclusion

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).