Nonylbenzoate, branched and linear

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04.Mar. 2005 - 06. Jun. 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid isononylester

Cas Number:

670241-72-2

IUPAC Name:

Benzoic acid isononylester

Details on test material:

Name : BENZOIC ACID ISONONYLESTER
Label name : Isononyl benzoat
Batch Number : 04/3945-3947
Expiry date : Not indicated by the Sponsor
Received from : OXENO GmbH
Date received : 21 February 2005
Amount received : 2000 grams
Description : Colourless liquid
Container : Two colourless glass bottles
Storage at RTC : Room temperature
RTC reference number : 9428

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy S.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 7-10 weeks
- Weight at study initiation: between 185.7 and 255.7 grams
- Assigned to test groups randomly: no data
- Fasting period before study: animals were fasted overnight before dosing
- Housing: The animals were housed 5 animals/cage, by sexes, in clear polycarbonate cages measuring 59 x 20 x 39 cm with a stainless steel mesh lid and floor (Type 4: Techniplast). Each cage held absorbent bedding which was inspected daily and changed as necessary.
- Diet (e.g. ad libitum): 4RF21, Mucedola S.r.l., Settimo Milanese, MI, Italy ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%,
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: standard vehicle for these kind of studies
- Concentration of test material in vehicle: 50, 100, 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required): batch no. 103K0107 obtained from Sigma
- Purity:

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Solutions were prepared in corn oil on a weight/volume basis with correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of materials as received. No assay of test item stability, nor its concentration and homogeneity in vehicle were undertaken as not requested by the Sponsor. All dose-levels in this report are expressed to three significant figures.

Duration of treatment / exposure:

24h and 48 h

Frequency of treatment:

Animals were treated once

Post exposure period:

none

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 500, 1000, 2000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

- Positive control: mitomycin C
- Justification for choice of positive control(s): standard positive control for these kind of studies
- Route of administration: intraperitoneal injection
- Doses / concentrations: 2.0 mg/kg

Examinations

Tissues and cell types examined:

polychromatic cells from bone marrow of femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The selection of dose-levels was based on the results obtained in a preliminary toxicity assay. A group of two male and two female rats was dosed by oral gavage with the test item at 2000 and 1000 mg/kg body weight. The animals were observed for 48 hours and sacrificed. Scoring was performed on slides prepared from the femurs of animals.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The vehicle and the test item solutions were administered by oral gavage at the volume-dosage of 10 ml/kg bodyweight. The positive control treatment with Mitomycin-C was administered via intraperitoneal injection at the volume-dosage of 10 ml/kg bodyweight. Animals from vehicle treatment group and the test item treatment groups were fasted overnight
before dosing. Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time.

DETAILS OF SLIDE PREPARATION: Animals were sacrificed at appropriate sampling times as indicated in the experimental schemes. The femurs of animals were removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared to make smears on slides. Three slides from each animal were air-dried, fixed in methanol for 10 minutes and then stained with haematoxylin and eosin, and mounted with Eukitt.

METHOD OF ANALYSIS: The slides were randomly coded by a person not involved in the subsequent microscope scoring. The slides were examined under low power (x 16 objective) and one slide from each animal was selected according to staining and quality of smears. Two thousand polychromatic cells per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time the numbers of normal and micronucleated normochromatic erythrocytes were also recorded.

Evaluation criteria:

Provided that the slides are of an adequate quality and a sufficient number of cells can be scored, it may only be necessary to score one of series. Scoring is effected using a microscope and high-power objective. Immature polychromatic erythrocytes (PCE's) stain a blue-grey colour (since they retain basic ribosomal material for approximately 24 h after enucleation), and can be distinguished from the orange-red normochromatic erythrocytes (NCE's). Erythrocytes lack nuclei, making micronuclei obvious when present; tbe criteria of Schmid (1976) will be used to score micronuclei. At least two thousand polychromatic erythrocytes per animal are scored for the presence of
micronuclei (unlcss thcrc is a marked depression in PCE numbers). At the same time the number of normochromatic erythrocytes is recorded, as we as the number of micronucleated NCE's. The proportion of immature erythrocytes among total erythrocytes gives an indication of the
toxicity of the treatment; a reduction in the proportion indicates inhibition of cell division. The incidence of micronucleated NCE's gives an indication of the pre-treatment status of the animals. Finally, tbe incidence of micronucleated PCE's provides an index of induced genetic damage.

Statistics:

Only counts from polychromatic cells are subjected to statistical analysis. Using the original observations (and not the micronueleus frequencies per 1000 cells) a modified chi-squared calculation is employed to compare treated and control groups. The degree of heterogeneity within each group is first calculated, and where it is significant it is taken into account in the comparison between groups. If there is no significant within-group heterogeneity, the chi-squared test is used to compare treated groups with the controls. If there is significant within-groups heterogeneity, then that group is compared with the controls using a variance ratio (F) value calculated from the between-group and within-group Chi-squared values.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Soft faeces were observed for all treated animals at 1000 and 2000 mg/kg bodyweight. No depression of bone marrow erythropoietic cell division was observed. Body weight loss was observed for one male animal only dosed at 2000 mg/kg. Based on the above results, dose-levels of 500, 1000 and 2000 mg/kg bodyweight were selected for the Main assay.

Any other information on results incl. tables

 

Treatment	Dose level (mg/kg)	Incidence of micronucleated PCE´s			PCE (PCE +NCE)  % over the mean negative control value
	M/F	Mean	SE	Range
24 hr sampling time
Vehicle	10 mL/kg	0.8	0.2	2.5	100
Test item	500	0.6	0.1	1.5	100
Test item	1000	0.6	0.1	1.0	95
Test item	2000	0.9	0.2	2.5	99
Mitomycin-C	2.0	6.2 ***	0.7	9.5	88
48 hr sampling time
Vehicle	10 mL/kg	0.6	0.2	1.5	100
Test item	2000	0.8	0.2	2.5	92

PCE = polychromatic erythrocytes, NCE = normochromatic erythrocytes

*** Significantly different at p < 0.001

Mucus in the cage litter was the only sign observed in some animals from the high dose group. No relevant increase in the incidence of micronucleated PCE’s over the control values was observed in any test item treatment group at any sampling time. Pronounced increases in the frequency of micronucleated PCE's were observed in the positive control group, indicating the correct functioning of the test system. The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, a slight inhibitory effect on erythropoietic cell division was observed at the high and intermediate dose-levels of treatment for female animals from the 24 hour sampling time. A slight depression of bone marrow erythropoietic cell division was also observed at the 48 hour sampling time for both male and female animals from the high dose-group.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
On the basis of the results obtained, it is concluded that BENZOIC ACID ISONONYLESTER, administered orally, does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

Executive summary:

The ability of BENZOIC ACID ISONONYLESTER to cause chromosomal damage in vivo was investigated in a micronucleus test in rats. Based on results obtained in a preliminary toxicity test, dose-levels selected were 500, 1000 and 2000 mg/kg bodyweight for male and female animals. The test item was administered orally as indicated in the Study Protocol.

Sprague-Dawley SD rats were dosed once only with the vehicle corn oil, Benzoic Acid Isononylester at the selected dose-levels or with the positive control substance Mitomycin-C. Each group consisted of five male and five female animals with the exception of the control and high-dose group, which included an additional five animals of each sex per group. Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time. Bone-marrow smear slides were made and stained with haematoxylin and eosin stains. The slides were coded prior to scoring and two thousand polychromatic cells per animal were examined for the presence of micronuclei. The results obtained at each sampling time were subjected to statistical analysis using a modified chi-squared test.

Following treatment with Benzoic Acid Isononylester, no statistically significant increase in the incidence of micronucleated PCE's over the control value, was observed in any treatment group. Statistically significant increases in the incidence of micronucleated PCE's over the control values were observed following treatment with the positive control Mitomycin-C, indicating the correct functioning of the test system.

Following treatment with the test item, no remarkable adverse reaction was observed after treatment. A slight depression of bone marrow erythropoietic cell division, was observed at the high and intermediate dose-levels of treatment for female animals from the 24 hour sampling time. A slight depression of bone marrow erythropoietic cell division was also observed at the 48 hour sampling time for both male and female animals from the high dose-group.

It is concluded that, under the reported experimental conditions, BENZOIC ACID ISONONYLESTER administered orally at the selected dose-levels to male and female rats, does not induce micronuclei in the polychromatic erythrocytes.