Indoline-2,3-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:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD Guideline 474 with deviations. No data on GLP.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Only 4 animals/sex/group; The individual data of the animals are not presented.

GLP compliance:

no

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Indoline-2,3-dione

EC Number:

202-077-8

EC Name:

Indoline-2,3-dione

Cas Number:

91-56-5

Molecular formula:

C8H5NO2

IUPAC Name:

2,3-dihydro-1H-indole-2,3-dione

Details on test material:

- Name of test material (as cited in study report): isatin
- Analytical purity: ≥99.0 %

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: State University of Londrina (Paraná, Brazil).
- Age at study initiation: 7-8 week old.
- Weight at study initiation: 30 g
- Housing: individually in polypropylenecages.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Duration of treatment / exposure:

24 hours.

Frequency of treatment:

Single dose.

Post exposure period:

24h.

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 100, 150 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

4 males and 4 females/group for each treatment.

Control animals:

yes

Positive control(s):

Cyclophosphamide (50 mg/kg b.w., i.p.).

Examinations

Tissues and cell types examined:

Other: all animals were weighed at the beginning and end of each treatment.

Details of tissue and slide preparation:

SACRIFICE
24 h after treatment, the animals were anesthetized and euthanized with a 1:1 mixture of 2% xylazine HCl and 10% ketamine HCl.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
For the peripheral blood cell micronucleus test, blood samples were obtained from the tail veins of mice, before gavage (T0) and 36 (T1), 48 (T2), and 72 (T3) h post-gavage.

DETAILS OF SLIDE PREPARATION:
Bone marrow micronucleus test: Both femurs were immediately removed and the bones were freed from muscles. The epiphyses were cut and the bone marrow was flushed with fetal calf serum. The cell suspension was centrifuged at 900 rpm for 10 min and the supernatant was discarded. A small drop of resuspended cell pellet was spread onto a clean glass slide, which was air-dried and fixed in absolute methanol for 10 min. The smears were stained with Leishman's eosin blue for detection of micronucleated polychromatic erythrocytes (MNPCE). For each animal, three slides were prepared, and 2000 polychromatic erythrocytes (PCE) were counted to determine the frequency of MNPCE.

Micronucleus test of peripheral blood cells: Glass slides were heated to 70 ºC on a hot-plate, and an aqueous solution of acridine orange (10 µg, 1 mg/ml) was placed on each slide and spread evenly over the surface with the end of a second well-cleaned slide. After drying, the slides were kept in the dark at room temperature for at least 24 hours. The cell preparations were examined under a fluorescence microscope with a blue (488 nm) excitation filter and yellow (515 nm) emission (barrier) filter and using an immersion objective.

Evaluation criteria:

Bone marrow micronucleus test: to evaluate bone marrow toxicity, the ratio of PCE/PCE + NCE (normochromatic erythrocytes) was calculated by counting a total of 200 erythrocytes.
Micronucleus test of peripheral blood cells: Reticulocytes (1000 per treated animal) were analyzed and the proportion of micronucleated cells was determined.

Statistics:

Parametric (ANOVA/Tukey) and non-parametric (Kruskal-Wallis/Dunn's post hoc test) tests were used, according to the nature of the data distribution.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: for all doses (50, 100 and 150 mg/kg b.w.) and times of evaluation (36, 48, and 72 h), no significant (P < 0.05) increases in peripheral blood cell micronucleus frequency were seen, compared to the negative control group.

Any other information on results incl. tables

Table 1. Frequency of micronucleated reticulocytes (MNRETs) in a total of 1000 analyzed cells per mouse (n = 8 mice per group) to evaluate the mutagenicity of acute treatments of three different doses of isatin and the positive and negative control groups.

Treatments (mg/kg b.w.)	T0 (0 h)		T1 (36 h)		T2 (48 h)		T3 (72 h)
	MNRETs	Mean ± SD/animal	MNRETs	Mean ± SD/animal	MNRETs	Mean ± SD/animal	MNRETs	Mean ± SD/animal
Water	21	2.6 ± 0.52	15	1.9 ± 0.64^a	17	2.1 ± 0.64^a	19	2.1 ± 0.83^a
CPA
50	15	1.9 ± 0.64	180	22.5 ± 2.07^b	107	13.4 ± 1.68^b	41	5.1 ± 1.64^b
Isatin
50	17	2.1 ± 0.83	13	1.6 ± 0.92^a	14	1.7 ± 0.46^a	14	1.7 ± 0.70^a
100	13	1.6 ± 0.74	15	1.9 ± 0.64^a	16	2.0 ± 0.75^a	14	1.7 ± 0.89^a
150	13	1.6 ± 0.74	27	3.4 ± 1.19^a	13	1.6 ± 0.74^a	11	1.4 ± 0.74^a

Mean ± SD: mean ± standard deviation; water: negative control; CPA: positive control. DC: damaged cells.

Means with the same letter do not differ statistically (P< 0.05).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
After acute treatment with istatin, mutagenic effects were not seen.

Executive summary:

The present study evaluates the genotoxic and mutagenic effects of acute (24 h) exposure to isatin in vivo, using the micronucleus test in accordance with the OECD Guideline 474. To assess the genotoxic and mutagenic effects of isatin, male and female mice were administered a single dose of one of the three concentrations (50, 100, and 150 mg/kg b.w.), by gavage, for 24 h. The negative control group received only water, by gavage, and the positive control group was treated with Cyclophosphamide (50 mg/kg b.w.) intraperitoneally (i.p.) for 24 h. For the peripheral blood cell micronucleus test, blood samples were obtained from the tail veins of mice, before gavage and 36, 48, and 72 h post-gavage. The animals were euthanized and both femurs were immediately removed to take sample of bone marrow. For each animal, three slides were prepared, and 2000 polychromatic erythrocytes (PCE) were counted to determine the frequency of MNPCE. At all doses, after acute treatment with isatin, mutagenic effects were not seen.