Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

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:

2009-10-14 - 2009-10-17

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:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Based on the fact that acute oral LD50 of male and female mice were all greater than 5000 mg/kg, experimental groups were determined to be 5000 mg/kg, 2500 mg/kg, 1250 mg/kg, a negative control group (com oil, 20 m!/kg), and a positive control group (cyclophosphamide, 40 mg/kg).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

EC Number:

236-109-7

EC Name:

Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

Cas Number:

13170-05-3

Molecular formula:

C22H27AlO5

IUPAC Name:

aluminum hydroxide bis(4-tert-butylbenzoate)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At normal room temperature and humidity in the lightproof sample storeroom

Test animals

Species:

mouse

Strain:

other: KM mice

Details on species / strain selection:

Mouse is prior recommended for the mammalian erythrocyte micronucleus test according to " OECD Guidelines for the Testing of Chemicals (2004) " 474.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shanghai SILAIKE Experimental Animal Co. Ltd. Animal Production Certification: SCXK (Hu) 2007-0005. Animal Usage Certification: SYXK (Hu) 2007-0008.
- Weight at study initiation: body weight between 25~30g,
- Fasting period before study: yes, 3-4h
- Housing: Animals were housed in the plastic cages (L46> - Diet (e.g. ad libitum): laboratory diet was provided by Suzhou Shuangshi Laboratory Animal Feed Science and Technology Ltd. (Certification: Su (E) Sishengzi (2002) 006)
- Water (e.g. ad libitum): Laboratory drinking water was freely for accessing during the test period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): The room is illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Sample preparation was following the dosage design to weigh the test sample of all groups. Then 5000mg, 2500mg and 1250mg of the test sample were weighed and corn oil was added to 20ml and mixed extensively to form the test sample.

Details on exposure:

The test sample of various doses was administered to the mice by double oral gavages (24h's interval). All mice were weighed and the gavage volume was 0.4ml/20g bw.Mice were divided randomly into 5 groups by body weight, 30 for each group, half of each sex. Experimental groups were divided as 5000 mg/kg, 2500 mg/kg, 1250 mg/kg (high dosage, middle dosage, and low dosage) and a negative control (corn oil, 20 ml/kg) and a positive control group (cyclophosphamide 40 mg/kg).

Duration of treatment / exposure:

24h or 48h after single application

Frequency of treatment:

single application

Post exposure period:

24h, 36h, 48h after the last gavage.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

high dosage

Doses / concentrations 2

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Remarks:

middle dosage

Doses / concentrations 3

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Remarks:

low dosage

No. of animals per sex per dose:

15 / sex / dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: cyclophosphamide 40mg/kg

Examinations

Tissues and cell types examined:

femur bone marrow erythrocytes

Details of tissue and slide preparation:

10 mice per group, half of each sex, were executed by cervical dislocation at 24h, 36h, 48h after the last gavage. Femur marrow was fetched out and mixed with bovine serum. Slides were smeared, fixed and stained by Giemsa's solution.
Microscopic Observation: 2000 polychromatic erythrocytes (PCE) of each mouse were counted and the number of PCE containing micronucleus was recorded, and then rate of cell containing micronucleus was calculated as parts per thousand. As 200 polychromatic erythrocytes (PCE) were counted, normal chromatic erythrocytes (NCE) were also counted to obtain the value of PCE/RBC.

Evaluation criteria:

ACCEPTABILITY OF ASSAY
A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (WiIcoxon Rank Sum Test, two-sided test at P (0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.

In this study 10 mice per group, half of each sex, were executed by cervical dislocation at 24h, 36h, 48h after last gavage. Femur marrow was fetched out. Observed under the microscope, 2000 polychromatic erythrocytes (PCE) of each mouse were counted and number of PCE containing micronucleus was recorded, and then rate of cell containing micronucleus was calculated as parts per thousands. As 200 polychromatic erythrocytes (PCE) were counted, normal chromatic erythrocytes (NCE) were also counted to obtain the value of PCE/RBC.

Statistics:

Compared with the negative group, rate of cell containing micronucleus of the dosage groups was analyzed by Chi Square test.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Mammalian Erythrocyte Micronucleus Test of the test substance, Bis(4-(tert-butyl)benzoate-0) hydroxyaluminium (CAS No. 13170-05-3) was negative. The test substance is not considered to be mutagenic.

Executive summary:

Bis(4-(tert-butyl)benzoate-0) hydroxyaluminium was tested in the Micronucleus Test in mice according to OECD 474 under GLP, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Mice were divided randomly into 5 groups, 30 animals for each group, half of each sex. Experimental groups were determined to be 5000 mg/kg, 2500 mg/kg, 1250 mg/kg, a negative control group (com oil, 20 ml/kg), and a positive control group (cyclophosphamide, 40 mg/kg). The test sample of various doses, the positive control and the negative control were administered to the mice by double oral gavages (24h's interval). The gavage volume was 0.4 ml/20g bw. 10 mice per group, half of each sex, were executed by cervical dislocation at 24h, 36h, 48h after last gavage. The rate of cell containing micronucleus of the dosage groups was analyzed compared with the negative group.

Mammalian Erythrocyte Micronucleus Test of the test substance, Bis(4-(tert-butyl)benzoate-0) hydroxyaluminium (CAS No. 13170-05-3) was negative. The test substance is not considered to be mutagenic.