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 vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

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

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:

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

Test animals

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.

Administration / exposure

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

Doses / concentrations

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

Examinations

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

Results and discussion

Test results

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.

Any other information on results incl. tables

Full data tables attached to background material section.

Applicant's summary and conclusion

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.