4H-3,1-benzoxazine-2,4(1H)-dione

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:

1992-06-09 to 1992-09-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983-05-26

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: EPA, CFR 40, Subpart F-Genetic Toxicity, "In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay.", 1986-07-01

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4H-3,1-benzoxazine-2,4(1H)-dione

EC Number:

204-255-0

EC Name:

4H-3,1-benzoxazine-2,4(1H)-dione

Cas Number:

118-48-9

Molecular formula:

C8H5NO3

IUPAC Name:

4H-3,1-benzoxazine-2,4(1H)-dione

Details on test material:

- Name of test material (as cited in study report): N-Carboxyanthranilsäureanhydrid
- Physical state: solid, beige
- Analytical purity: 99.7%
- Impurities (identity and concentrations): no data
- Purity test date: 1992-05-04
- Lot/batch No.: 41-0176
- Storage condition of test material: room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: River Wiga GmbH Sandhofer Weg 7, D-8741 Sulzfeld 1
- Age at study initiation: at least 10 weeks
- Weight at study initiation: ca. 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes, ca. 18 h prior to dose
- Housing: single, Makrolon Type I cage, with wire mesh top
- Diet: pelleted standard diet, ad libitum (ALTROMIN 1324, D-4937 Lage/Lippe )
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 30-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: polyethylene glycol (PEG 400)
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test article was formulated in PEG 400. All animals received a single standard volume of 10 ml/kg body weight orally.

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

up to 48 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
200, 670, 2000 mg/kg bw
Basis:
actual ingested
24 h preparation interval

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested
48 h preparation interval

No. of animals per sex per dose:

6 males and 6 females per groups treated;
5 males and 5 females per group evaluated

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: orally, once.
- Doses / concentrations: 30 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow cells from the femura

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test articles. The volume to be administered should be compatible with physiological space available.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Six males and six females were assigned to each test group. Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum. The cell suspension was centrifuged at 1500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group.

Evaluation criteria:

A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points.
A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.

Statistics:

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

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

2000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg bw, N-Carboxyanthranilsäureanhydrid formulated in PEG 400. The volume administered was 10 ml/kg bw. The treated animals expressed toxic reactions characterized by reduction of spontaneous activity (up to 72 h post dose), eyelid closure (up to 24 h post dose) and apathy (up to 24 h post dose).
As requested by the sponsor in accordance to the guideline-draft "EEC Directive 79/831, Annex V, B 12" 2000 mg/kg bw of the test article was used as maximum dose in the micronucleus assay.

Any other information on results incl. tables

test group	dose [mg/kg]	sampling time [h]	PCEs with micronuclei [%]	range	PCE / NCE
vehicle	0	24	0.07	0 - 2	1000 / 681
test article	200	24	0.14	0 - 5	1000 / 790
test article	670	24	0.05	0 - 1	1000 / 775
test article	2000	24	0.13	0 - 3	1000 / 713
cyclophosphamide	30	24	2.16	6 - 40	1000 / 810
vehicle	0	48	0.06	0 - 3	1000 / 755
test article	2000	48	0.06	0 - 2	1000 / 786

Applicant's summary and conclusion

Executive summary:

Report summary

This study was performed to investigate the potential of N-Carboxyanthranilsäureanhydrid (i.e. isatoic anhydride) to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was formulated in polyethylene glycol 400. This vehicle was used as negative control. The volume administered orally was 10 ml/kg bw. 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose levels of the test article were investigated:

- 24 h preparation interval: 200, 670 and 2000 mg/kg bw

- 48 h preparation interval: 2000 mg/kg bw

The highest dose recommended by guideline (2000 mg/kg) was estimated by a pre-experiment to be suitable. The animals expressed toxic reactions. In the micronucleus assay 2 out of 24 treated animals died.

After treatment with the test article the number of NCEs was not increased as compared to the corresponding negative controls thus indicating that N-Carboxyanthranilsäureanhydrid had no cytotoxic effectiveness.

In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article and with any dose level used.

30 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronucleus frequency.

CONCLUSION

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, N-Carboxyanthranilsäureanhydrid is considered to be non-mutagenic in this micronucleus assay.