Benzoic acid, 4-[(1-oxodecyl)oxy]-

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: other: cytogenetic damage

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed GLP and OECD guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Korea Food & Drug Administration, Notification No. 1999-61, Standards for Toxicity Study of Drugs, etc., 1999.12.22.

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

DOBA

IUPAC Name:

DOBA

Constituent 2

Reference substance name:

Benzoic acid, 4-[ (1-oxodecyl) oxy]-

EC Number:

617-941-3

Cas Number:

86960-46-5

Molecular formula:

C17H24O4;

IUPAC Name:

Benzoic acid, 4-[ (1-oxodecyl) oxy]-

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Strain: NMRI
Charles River Laboratories
Research Models and Services Germany GmbH
Sandhofer Weg 7, 97633 Sulzfeld, Germany
Number of animals: 72 (36 males and 36 females)
Acclimation: minimum 5 days
Initial age at start
of experiment: 9 - 10 weeks
Initial Body Weight
at Start of Treatment: males: mean value 36.7 g (SD +/- 1.9 g)
females: mean value 30.2 g (SD +/- 1.8 g)

ENVIRONMENTAL CONDITIONS
Housing: single / group
Cage Type: Makrolon Type II/III, with wire mesh top
(EHRET GmbH, 79302 Emmendingen, Germany)
Bedding: granulated soft wood bedding
(Rettenmaier & Söhne GmbH + Co. KG,
73494 Rosenberg, Germany)
Feed: pelleted standard diet, ad libitum
(Harlan Laboratories B.V.
Postbus 6174, 5960 AD Horst, The Netherlands)
Water: tap water, ad libitum,
(Gemeindewerke, 64380 Rossdorf, Germany)
Environment: temperature 22 + 2 °C
relative humidity 45 - 65 %
artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: June 30, 2011 To: July 18, 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Name: 30% DMSO / 70% PEG 400
Supplier: VWR
64295 Darmstadt, Germany
Catalogue no.: DMSO: 1.02931.1000 / PEG 400: 1.09726.0100
Batch: DMSO: K41607131109 / PEG 400: K34767126
Expiry Date: February 23, 2011
Route and Frequency
of Administration: orally, once
Volume administered: 10 mL/kg b.w.

Details on exposure:

orally

Frequency of treatment:

once

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, and 2000 mg/kg b.w.
Basis:

No. of animals per sex per dose:

2 males, 2 females used in the pre-experiment
72 (36 males and 36 females) used in the main experiment

Control animals:

yes

Positive control(s):

Name: CPA; Cyclophosphamide
Supplier: Fisher Scientific GmbH
61130 Nidderau, Germany
Catalogue no.: 203960010
Batch: A0277203
Expiry Date: December, 2011
Dissolved in: sterile water
Dosing: 40 mg/kg b.w.
Route and Frequency
of Administration: orally, once
Volume administered: 10 mL/kg b.w.
Solution prepared on day of administration.

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.

Evaluation criteria:

Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
All animals per test group were evaluated as described.
The study was considered valid as the following criteria are met:
- at least 5 animals per group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the vehicle control.

Statistics:

nonparametric Mann-Whitney test; p <0.05

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

ruffled fur

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Toxic Symptoms

RESULTS OF RANGE-FINDING STUDY
In a pre-experiment 4 animals (2 males, 2 females) received once orally a single dose of 2000 mg/kg b.w. DOBA formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 2000 mg/kg b.w. expressed toxic reactions as shown in the table:

toxic reactions observed: ruffled fur
hours post-treatment: 1 / 2-4 / 6 / 24 / 48
number of animals (male/female): 0/0 - 0/0 - 1/0 - 1/0 - 0/0

On the basis of these data 2000 mg/kg b.w. was chosen as highest treatment dose.



RESULTS OF DEFINITIVE STUDY

In the main experiment for the high dose groups 12 males and 12 females received orally a single dose of 2000 mg/kg b.w. DOBA formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 mL/kg b.w..
The animals treated with 2000 mg/kg b.w. did not express any toxic reactions. The 6h observation interval was missed out.
For the mid dose group six males and six females received orally a single dose of 1000 mg/kg b.w. DOBA formulated in 30% DMSO / 70% PEG 400, respectively. The volume administered was 10 mL/kg b.w..
The animals treated with 1000 mg/kg b.w. expressed toxic reactions as shown in the table:

toxic reactions observed: ruffled fur
hours post-treatment: 1 / 2-4 / 24
number of animals (male/female): 0/0 - 3/5 - 0/0


For the low dose group six males and six females received orally a single dose of 500 mg/kg b.w. DOBA formulated in 30% DMSO / 70% PEG 400, respectively. The volume administered was 10 mL/kg b.w..
All treated animals for the low dose group (the 6h observation interval was missed out) as well as the animals treated with the vehicle control (30% DMSO / 70% PEG 400) did not express any toxic reactions.

Any other information on results incl. tables

Summary of Micronucleus Test Results

test group	dose mg/kg b.w.	sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000erythocytes
vehicle	0	24	0.117	0 -5	1171
test item	500	24	0.175	2 -10	1260
test item	1000	24	0.133	0 -5	1252
test item	2000	24	0.158	1 -6	1208
positive control	40	24	2.354	30 - 78	1197
test item	2000	48	0.108	1 -5	1317

Biometry

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Negative control versus test group	Significance	p
500 mg DOBA/kg b.w.; 24 h	-	0.0959
1000 mg DOBA /kg b.w.; 24 h	-	0.3420
2000 mg DOBA /kg b.w.; 24 h	-	0.0957
40 mg CPA/kg b.w.; 24 h	+	<0.0001
2000 mg DOBA /kg b.w.; 48 h	n.t.	-

      -     =    not significant
      +    =    significant
      n.t. =    not tested, as the mean micronucleus frequency was not above the vehicle
                    control value

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

The test item DOBA was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in 30% DMSO / 70% PEG 400, which was also used as vehicle control. The volume administered was 10 mL/kg b.w.once orally. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Six males and six females per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000, and 2000 mg/kg b.w..

48 h preparation interval: 2000 mg/kg b.w..

As estimated by a pre-experiment 2000 mg DOBA per kg b.w. (the maximum guideline-recommended dose) was suitable.

The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that DOBA did not have any cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with DOBA were below or near to the value of the vehicle control group and all within the historical vehicle control data range.

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.