4-(4-isopropoxyphenylsulfonyl)phenol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-02-24 to 2009-07-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2008-05-30

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

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:

4-(4-isopropoxyphenylsulfonyl)phenol

EC Number:

405-520-5

EC Name:

4-(4-isopropoxyphenylsulfonyl)phenol

Cas Number:

95235-30-6

Molecular formula:

C15H16SO4

IUPAC Name:

4-[4-(propan-2-yloxy)benzenesulfonyl]phenol

Details on test material:

- Name of test material: D-8;
- Molecular formula: C15 H16 O4 S;
- Molecular weight: 292.3;
- Physical state: powder,
- Analytical purity: 99.5 %;
- Purity test date: 2008-09-17;
- Lot/batch No.: IHI-006;
- Expiration date of the lot/batch: 2011-09-12
- Stability under test conditions: 36 months;
- Storage condition of test material: Keep away from direct sunlight. Store in a cool dry place. Keep away from oxidizing agents, and high temperature.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi Coop Ltd., 1103 Budapest, Cserkesz u. 90;
- Age at study initiation: young adult mice, 7 - 8 weeks;
- Weight at study initiation: Preliminary test: Males: 30.3 - 35.1 g; Females: 26.5 - 28.5 g; Main test: Males: 31.6 - 35.4 g;
- Assigned to test groups randomly: All animals were sorted according to body weight by computer and divided to weight ranges.
- Fasting period before study: No
- Housing: Group caging (5 animals/cage, except of highest dose, where 7 animals/cage (incl. 2 reserve mice))
- Cage type: II type polypropylene/polycarbonate
- Diet: Animals received ssniff® SM R/M-Z+H “Autoclavable complete feed for rats and mice” produced by ssniff Spezialdiäten GmbH (D-59494 Soest/ Germany) ad libitum;
- Water: tap water from the municipal supply from 500 mL bottle, ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C;
- Humidity: 30 - 70 % R.H.;
- Air changes: 15 - 20 air changes/h by central air-condition system;
- Photoperiod: 1h hours light from 6:00 a.m. to 6:00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Polyethylene glycol 400 (PEG 400)

Details on exposure:

Justification of the dose selection: Groups of two male and female mice were treated on one occasion by oral gavage at dose levels of 500, 1000, 1500 and 2000 mg/kg bw/day. The treatment volume was 10 mL/kg bw/day. Animals were examined regularly for toxic signs and mortalities. On the basis of results of preliminary toxicity test, doses for the Mouse Micronucleus Test were: 500, 1000 and 2000 mg/kg bw/day. The preliminary study indicates no sex difference in toxicity, then one sex (male) was used in the main study.
The test/vehicle control items were administered by oral gavage on one occasion. In the low and mid dose groups the sampling was made once at 24 hours after treatment. In the high dose and vehicle control groups, mice were treated at 24 and 48 hours before sampling. Five male animals per dose group were used for sampling on each occasion. Cyclophosphamide (positive control) was administered intraperitoneally one occasion at a treatment volume of 10 mL/kg bw/day. Sampling was performed 24 hours after the treatment and five male animals were used for sampling. The mice were examined regularly for visible signs of reactions to treatment, immediately after dosing, and at intervals until sacrifice.

Duration of treatment / exposure:

One occasion

Frequency of treatment:

Once

Post exposure period:

24 hours (24 and 48 hours for negative control and high-dose test item)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

Basis: actual ingested

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Basis: actual ingested

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Basis: actual ingested

No. of animals per sex per dose:

5 males per dose (additional 5 males in negative control and high-dose test item group)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide: 60 mg/kg bw/day

Examinations

Tissues and cell types examined:

The animals from each group were weighed and bone marrow was obtained from two exposed femurs of the mice from every dose- time point immediately after sacrificing. The bone marrow was flushed with foetal bovine serum (5 mL). After vortex mixing, the cell suspension was concentrated by centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides. Slides were then dried at room temperature.

Details of tissue and slide preparation:

Subsequently the slides were stained as follow:
1. Fixed for a minimum of 5 minutes in methanol and allowed to air-dry.
2. Stained with Giemsa solution (99 mg/mL distilled water) for 25 minutes.
3. Rinsing in distilled water.
4. Drying at room temperature (at least 12 hours).
5. Coating with EZ-mounting.

Evaluation criteria:

The frequencies of micronucleated polychromatic erythrocytes in animals in the test and positive control groups were compared to the values found in the corresponding negative control group.
The test item would have been considered to have shown genotoxic activity in this study if the following criteria had been met:
- increases in the frequency of micronucleated polychromatic erythrocytes were observed in treated animals compared to the corresponding negative controls.
- the increases were dose-related
- the increases were statistically significant.

Statistics:

Statistically analysis was performed using Kruskall Wallis Non Parametric ANOVA test.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Clinical signs and Mortality

Preliminary Toxicity Test (Pretest)

A preliminary toxicity test was performed to identify the appropriate maximum dose level for the main test. Groups of two male and female mice were treated on one occasion by oral gavage at dose levels of 500, 1000, 1500 and 2000 mg/kg bw/day. The treatment volume was 10 mL/kg bw/day. Animals were examined regularly for toxic signs and mortalities. The animals treated with dose levels of D-8 expressed mortality as shown in the table:

D-8 (mg/kg bw./day)	Number of Animals	Mortality
500	2 males and 2 females	0 males and 0 females
1000	2 males and 2 females	0 males and 0 females
1500	2 males and 2 females	0 males and 0 females
2000	2 males and 2 females	0 males and 0 females

No adverse reactions to treatment were observed in the mice dosed 500 and 1000 mg/kg body weight/day. One male animal dosed 1500 mg/kg body weight showed moderate decrease in activity, narrow palpebra 3, 4 hours after the treatment and a slight decrease in activity between 4 and 48-hour after the treatment. The other male and two female were symptom free in this dose group. One male animal dosed 2000 mg/kg body weight showed a moderate and a slight decrease in activity.The symptom was observed between one and four hours after the treatment. The other male and two female were symptom free in this dose group.

Micronucleus Test :

Based on the results of the preliminary toxicity test, the doses of the MNT were following: 500, 1000 and 2000 mg/kg bw/day.

The mice were examined for visible signs of reactions to treatment, immediately after dosing and at intervals until sacrifice.

No animals died during the study.

No adverse reactions to treatment were observed in the mice of the vehicle and positive control groups. The mice dosed at the 500, 1000 mg/kg body weight/day dose level were symptom-free during the study.

One male animal dosed 2000 mg/kg body weight showed slight piloerection, slight narrow palpebra and slight decrease in activity on the day of treatment. The other male in this dose group showed slight narrow palpebra and slight, moderate decrease in activity on the day of treatment. In this animal slight and moderate piloerection were observed during the study. No adverse reactions to treatment were observed in the other mice dosed at the 2000 mg/kg body weight/day.

No notable effect of treatment on body weights was observed.

Applicant's summary and conclusion

Conclusions:

The frequencies of micronucleated polychromatic erythrocytes (MPCEs) for the negative and positive control mice were within acceptable ranges and compatible with the historical control data. No biologically significant increases in the frequency of MPCEs were seen in the groups of mice treated with D-8 compared to the vehicle control group. D-8 did not show any genotoxic activity in this Mouse Micronucleus Test.

Executive summary:

Potential mutagenic activity of D-8 was examined in bone marrow of male CRL: NMRI BR mice according to EU Method B.12. The doses of the test item for the Micronucleus Test were determined according to a preliminary oral toxicity test performed within this study, the doses selected were 500, 1000 and 2000 mg/kg body weight/day of D-8. The single oral administration of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight of D-8 did not induce any biologically relevant increase in the frequency of MPCEs in male mice at either 24 or 48 hours after the treatment compared to the vehicle control. No biologically significant increases in the frequency of MPCEs were seen in the groups of mice treated with D-8 compared to the vehicle control group. D-8 did not show any genotoxic activity in this mouse micronucleus test.