A mixture of: esters of C14-C15 branched alcohols with 3,5-di-t-butyl-4-hydroxyphenyl propionic acid; C15 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate; C13 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate

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:

November 11, 1991 to December 18, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Organization for Economic Cooperation and Development Guidelines, Section 4, Subpart 474, Paris 1981 and subsequent revisions.

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Test material form:

liquid

Specific details on test material used for the study:

No further details specified to the study report.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague dawley rats used in accordance with the specified guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system: Sprague Dawley Charles River Crl:CD (SD) BR rat.
Number of animals: Seventy rates (35M + 35F) supplied by the Charkes River Company of Calco (Como), Italy, were used (shipping slip no. 08564, dated November 8, 1991). The animals, 5-weeks old, weighed from 113 to 150g at the time of use.

Accommodation: The rats were housed in a room having the following ambient conditions: temperature 22 °C ±2, relative humidity 55% ±10, about 20 air changes per hour filtered on HEPA 99.97%, artificial lighting with a circadian cycle of 12 hours of light (7 a.m. – 7 p.m.). An automatic standby power supply was brought into operation should the main supply fail.
The rats were kept for the entire duration of the study in wire cages (cm 40.5x38.5x18h) with a stainless steel feeder.
The waste that drops through the wire bottom on a removable paper was periodically disposed of.

Identification system for animals and groups: Five animals were allocated to each cage and were individually identified by colour mark. Each cage bore a tag with indelible identification of experiment number, dosage group, cage number and progressive number of the animals.

Diet and water supply: The animals were fed a diet coded 4RF21GLP, produced by Charles River Italia’s feed licensee Mucedole S.r.l., Settimo Milanese.
The declared contents, on the label, on dry matter basis (moisture 12%). Were
Crude protein 18.5%
Crude fat 3 %
Crude fiber 6%
Ash 7%
The diet was supplemented by the Producer with vitamins and trace elements. 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, other contaminants (e.g. solvents, pesticides) and chemical and physical characteristics.
The acceptance limits of quality of the drinking water are those defined in EEC Directive 80/778.
Contaminants that might interfere with the objectives of the study were not expected to be present either in the diet or the water.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Details on exposure:

Of the test article [ANOX BF] about 21 grams (d=0.93) were suspended omogeneously in 0.5% methylcellulose in order to obtain a concentration of 250 mg/ml.

Duration of treatment / exposure:

Single exposure

Frequency of treatment:

Single dose

Post exposure period:

66 hours

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw (total dose)

Remarks:

20 ml/kg

No. of animals per sex per dose:

Test group: 30 animals (15M/15F)
Negative control: 30 animals (15M/15F)
Poitive control: 10 animals (5M/5F)

Control animals:

yes, concurrent vehicle

other: concurrent positive control (Mitomycin C)

Positive control(s):

Mitomycin C: 8 mg/kkg (10 ml/kg) administered by intraperitoneal route.

Examinations

Tissues and cell types examined:

Bone marrow from femurs taken from test animals.

Details of tissue and slide preparation:

Animals were sacrificed at three intervals after treatment: 18th, 42nd, 66th hours.
At each time, 10 animals (5M + 5F) treated with [ANOX BF] and 10 of the negative controls were sacrificed by cervical dislocation under i.p. anaesthesia with penobarbital.
The animals treated with Mitomycin C were sacrificed after about 42 hours.
The femurs were immediately removed and the bone marrow taken, suspended, and carefully washed in 3 ml of fetal bovine serum.
The suspension was centrifuged at 1,000xg for 10’. The supernatant was carefully taken off and the cell 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, batch no. 08943240, expiry date April 30, 1992) solution for 3 min;
2. washing in 1.779 g/l NaNH4HPO4.4H2O (buffer) (Merck, batch no. 938A194382)
3. immersion in May Gruenwald solution diluted 1:1 with buffer for 2 min., then rinsed twice with buffer.
4. immersion in Giesma (Merck, batch no. 98498177, expiry date October 31, 1992) solution diluted 1:6 with buffer for 7.5 min;
5. rinsing with buffer and carefully dried with filter paper.

The stained slides were coded and read at the microscope (1250 X). For each animal, 2000 polychromatic erythrocytes were counted and scored for micronucleated cells.
Furthermore, the ratio of polychromatic to normochromatic erythrocytes (P/N) was calculated on one slide/animal by counting a total of 1000 polychromatic erythrocytes.

Evaluation criteria:

Not detailed in the study report.

Statistics:

Comparison of the frequency among groups was perfomed with a non-parametric method (Mann Whitney).
Significances are expressed in the following manner:
* p < 0.05
** p < 0.01
*** p < 0.001

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No cytotoxic effects on bone marrow cells were evidenced.

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

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

Applicant's summary and conclusion

Conclusions:

The results of the study indicate that the test article [ANOX BF] administered to Charles River rats by oral route at the dose of 5000 mg/kg, did not induce any statistically significant increase in the frequency of micronucleated cells in the bone marrow, 18, 42 and 66 hours from the administration.
On the basis of the results described, [ANOX BF] is not mutagenic in the micronucleus test.

Executive summary:

Appraisal of the possible genotoxic activity exercised by the test article [ANOX BF] by the micronucleus in the rat.

 

The animals were divided into the following experimental groups:

 

Negative control (vehicle treated animals): 30 (15M + 15F)

Treated with [ANOX BF]: 30 (15M + 15F)

Positive control (Mitomycin C): 10 (5M + 5F)

 

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

 

Animals were sacrificed at three intervals after treatment: 18th, 42nd, 66th hours.

At each time, 10 animals (5M + 5F) treated with [ANOX BF] and 10 of the negative controls were sacrificed by cervical dislocation under i.p. anaesthesia with penobarbital.

The animals treated with Mitomycin C were sacrificed after about 42 hours.

 

The femurs were immediately removed and the bone marrow taken, suspended, and carefully washed in 3 ml of fetal bovine serum. Cell sediment was smeared on microscope slides.

Two slides/animal were prepared.

 

No cytotoxic effects on bone marrow cells were evidenced.

 

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

 

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

 

The results of the study indicate that the test article [ANOX BF] administered to Charles River rats by oral route at the dose of 5000 mg/kg, did not induce any statistically significant increase in the frequency of micronucleated cells in the bone marrow, 18, 42 and 66 hours from the administration.

On the basis of the results described, [ANOX BF] is not mutagenic in the micronucleus test.