1,3-divinylimidazolidin-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

In vitro: Ames test: The test substance was not mutagenic in the Ames test under the experimental conditions chosen. HPRT test: The test substance was not mutagenic in the HPRT test under the experimental conditions chosen. Chromosome aberration test: Under the test conditions described -, the test substance induced structural chromosome aberrations. Therefore, the test substance was considered as clastogenic in vitro. In vivo: Micronucleus test: The test substance was considered to be negative in the in vivo micronucleus test. UDS: The test substance was considered to be negative in the in vivo UDS assay.

Link to relevant study records

Reference

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:

1995-03-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

March 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 92/69

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, WIGA, Sulzfeld, Germany
- Assigned to test groups randomly: yes, under following basis: computer program
- Weight at study initiation: 26.6 g (mean)
- Housing: Makrolon cages in groups of 5 separately according to sex
- Diet: Standardized pelleted feed (Kliba Haltungsdiaet, Klingentalmuehle AG, Kaiseraugst, Switzerland), ad libitum
- Water: Drinking water from bottles, ad libitum
- Acclimation period: One week

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70%
- Air changes: Fully air-conditioned rooms
- Photoperiod: Day/night rhythm was 12 hours

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: DMSO

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Male and female animals per sacrifice interval were given test substance dissolved in DMSO.
All test substance formulations were prepared immediately before administration.

Duration of treatment / exposure:

24 hours (75, 150, 300 mg/kg bw)
48 hours (300 mg/kg bw)

Frequency of treatment:

single intraperitoneal administration

Post exposure period:

None

Remarks:

Doses / Concentrations:
75, 150, 300 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPP) and vincristine (VCR)

- Route of administration: Intraperitoneal
- Doses / concentrations: 20 mg/kg bw (CPP) and 0.15 mg/kg bw (VCR) in a volume of 10 mL/kg bw

Tissues and cell types examined:

Yes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity deaths were observed down to a dose of 400 mg/kg body weight 300 mg/kg body weight was survived by all animals but led to clinical signs such as piloerection and squatting posture and the general state of the animals was poor.


TREATMENT AND SAMPLING TIMES:
- The two femora were prepared from the animals, and all soft tissues were removed.
After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37'C (about 2 mL/femur).

- The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended.

- 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.


DETAILS OF SLIDE PREPARATION:
The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes.

After being rinsed twice in purified water and clarified in xylene, the preparations were embedded in Corbit-Balsam .


METHOD OF ANALYSIS:

Microscopic

In general, 1000 polychromatic erythrocytes (PCE) from each of the male and female animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE), which occur, are also scored. The following parameters are recorded:

- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes

This ratio indicates an influence of the test substance specifically on the bone marrow:
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D= cell diameter)


Clinical examinations

After the administration of the test substance the animals were examined for any evident clinical signs of toxicity.

Evaluation criteria:

- The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.

- The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals .

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG ).
The number of micronuclei in polychromatic erythrocytes was analyzed.

A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p <=0 .05, ** for p <= 0 .01) were printed with the group means in the tables. This test was performed one-sided.

This analysis was done separately for each sex and combined for both sexes.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

An inhibition of erythropoiesis induced by the treatment of mice with the test substance was detected at a dose of 300 mg/kg body weight as indicated by the ratio of polychromatic to normochromatic erythrocytes groups.

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 400, 300, 150, 75 mg/kg bw

- Clinical signs of toxicity in test animals: Deaths were observed at a dose of 400 mg/kg bw. 300 mg/kg bw was tolerated by all animals but led to clinical signs such as piloerection and squatting posture and the general state of the animals was poor .
- Evidence of cytotoxicity in tissue analyzed: No


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: None
- Ratio of PCE/NCE: See table below
- Clinical signs of toxicity in test animals: Only squatting posture induced by the vehicle DMSO; piloerection was found after treatment of the animals with 150 mg/kg or 300 mg/kg body weight. One animal of the 300 mg/kg group died 1 hour after test substance administration.

Test substance analysis

The stability of a comperable batch (26300/9) in the vehicle over a period of 4 hours was verified analytically.

 

Test substance preparation analysis

Depending on the dose, about 83 - 104% of the theoretical values could be determined analytically.

 

Feed, water bedding analysis

Were all in the expected range.

 

 

Table 1: Summary table (males+ females): Polychromatic and normochromatic erythrocytes

	Interval 24 hours				Interval 48 hours
	Total No. of PCE´s	NCE´s/PCE´s	MN(0/00) PCE´s	MN (0/00) NCE´s	Total No. of PCE´s	NCE´s/PCE´s	MN(0/00) PCE´s	MN (0/00) NCE´s
Vehicle DMSO	10000	3918	1.2	0.5	10000	4262	1.7	0.5
75 mg/kg bw	10000	4319	1.0	0.7	-	-	-	-
150 mg/kg bw	10000	4712	1.9	0.4	-	-	-	-
300 mg/kg bw	9000	5584	1.7	0.5	10000	7520	2.5	1.2
CCP 20 mg/kg bw	5000	2333	11.0	0.0	-	-	-	-
VCR 0.15 mg/kg bw	5000	3472	59.4	0.9	-	-	-	-

 

Conclusions:

Interpretation of results (migrated information): negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Key studies

 

Ames test

BASF SE, study no.: 40M0031/934045, 1994

 

A bacterial reverse mutation test similar to OECD 471 was conducted under GLP conditions. The test substance was tested for its mutagenic potential in Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98 at the dose range of 20 µg - 5000 µg/plate.

Standard plate test and preincubation test both with and without metabolic activation (Aroclor induced rat liver S-9 mix) were performed.

No precipitation of the test substance was found. A weakly bacteriotoxic effect was observed only with the strains TA 1535 at 5000 µg/plate and

and with TA 100 at and above 500 µg/plate.

An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

Thus, the test substance was not mutagenic in the Ames test under the experimental conditions chosen.

 

HPRT test

BASF SE, study no.: 50M0031/934041, 1993

 

The test substance was tested according to ECD 476 and under GLP conditions for its ability to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster Ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without exogenous metabolic activation, using identical procedures. Test concentrations in the range of 0.01- 5 mg/mL were tested. The cells were treated with the test substance for 4 hours in serum-free medium after a 20-24 hours attachment period.

Reduced cloning efficiencies were found 18-20 hours post exposure to the test substance at 5 mg/mL in experiment 1 and at concentrations ≥ 2.15 mg/mL in experiment 2.

No biologically significant increases in mutant frequency were observed in either of the experiments with and without metabolic activation. It is concluded that the test substance was not mutagenic under the test conditions employed in this in vitro test system.

 

 

Chromosome aberration test

BASF SE, study no.: 32 M 0031/939003, 1994

 

The test substance was investigated for induction of structural and numerical chromosome aberrations in cultures of the V79 cell line in vitro (lung fibroblasts of a Chinese hamster) according to OECD guideline 473 under GLP conditions.

 

Concentrations between 625 to 5000 µg/mL were investigated. No clear cytotoxic effect was seen at any concentration. Negative (solvent) and positive controls (cyclophosphamide and mitomycin C) were tested in parallel. The cultures were treated for 4 h with S9 mix. Without S9 mix the treatment lasted 18 and 28 h. The total culture duration after adding the substance was 18 and 28 h. A metabolising system (S9 mix) from the livers of Sprague-Dawley rats pretreated with Aroclor 1254 was added to half of the cultures. The investigation comprised 2 independent experiments. 2 parallel cultures (slide cultures in Quadriperm dishes) were set up per experiment and per experimental point. After 18 h 1250, 2500 and 5000 µg/mL both with and without S9 mix were examined for chromosome aberrations. After 28 h the same concentrations were examined in experiment 2 and only 5000 µg/mL in experiment 1.100 diploid metaphases (22 ± 1 chromosomes) were analysed per culture.

 

In experiment 2 in the 18-h cultures without S9 mix a dose-dependent increase in the aberration rate was observed and in those with S9 mix there was a slight increase in the aberration rate. However, in the 28-h cultures both with and without metabolic activation there was a significant increase in the aberration rate at the highest concentration (5000 µg/mL). In experiment 2 this was shown to be dose dependent. At increasing concentrations and longer preparation intervals, apart from gaps, breaks and fragments, additional aberrations occurred, such as exchanges, multiple aberrations and pulverised metaphases.

In the positive controls a marked rise in the incidence of various aberrant metaphases was also observed with known mutagens indicating the sensitivity of the test system. Apart from gaps and breaks mainly chromatid exchanges, dicentric chromosomes and multiple aberrations were observed.

Numerical aberrations in the form of polyploids were also included in the evaluation of the test substance. This parameter was not influenced. Under the test conditions described, the test substance induced structural chromosome aberrations. There was no indication for induction of numerical aberrations.

 

 

 

In vivo

Key study, Micronucleus test

BASF SE, study no.: 26M0043/954009, 1995

 

The test substance was tested for clastogenicity and for spindle poison effects in NMRI mice using the micronucleus test method according to OECD 474 and under GLP conditions

For this purpose, the test substance, dissolved in DMSO was administered once intraperitoneally to male and female animals at dose levels of 75 mg/kg, 150 mg/kg and 300 mg/kg body weight in a volume of 4 mL/kg body weight in each case.

As a negative control, male and female mice were administered merely the vehicle DMSO by the same route. This treatment resulted in frequencies of micronucleated polychromatic erythrocytes within the historical control range.

Both positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic eryhtrocytes containing small or large micronuclei indicating the sensitivity of the test system.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 300 mg/kg body weight and in the vehicle controls. In the test groups of 150 mg/kg and 75 mg/kg body weight and in the positive control groups the 24-hour sacrifice interval was investigated only.

After staining of the preparations 1000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 1000 polychromatic erythrocytes were also registered.

 

The single intraperitoneal administration of the test substance did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always in the same range as that of the negative control in all dose groups and at all sacrifice intervals.

An inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected at a dose of 300 mg/kg body weight indicating that the test substance reacted the target organ.

Thus, under the experimental conditions chosen, the test substance was shown to have no chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis.

 

 

Supporting study, UDS test

BASF SE, study no.: 80M0043/954030, 1996

 

The test substance was tested for its ability to induce DNA repair synthesis (unscheduled DNA synthesis; UDS) in vivo in rat hepatocytes according to OECD 486 and under GLP conditions.

UDS is a repair process which is detected by the incorperation of tritrium-labeled thymidine (3H-thymidine) into the DNA that was not in the normal phase of the DNA synthesis (S-phase) and is measured by autoradiography . Cells undergoing repair are identified by an increase in the number of silver grains overlying the nuclei.

For this purpose, the test substance, dissolved in DMSO, was administered once orally by gavage to male Wistar rats at dose levels of 1000 mg/kg and 2000 mg/kg body weight in a volume of 4 mL/kg body weight in each case.

The animals were felled and hepatocytes were harvested 3 and 16 hours after administration of the test substance.

As a negative control, male rats were administered merely the vehicle DMSO by the same route, which gave frequencies of mean nuclear net grain counts were within the historical control range.

 

The positive control chemical 2-AAF administered orally in a dose of 50 mg/kg body weight demonstrated the expected increase in unscheduled DNA synthesis indicating the sensitivity of the test system.

 

The single oral treatment with the test substance did not lead to a significant increase in the mean number of nuclear grain counts at any dose level or exposure time. Therefore, the test substance is considered to be negative in the present in vivo UDS assay.

Justification for selection of genetic toxicity endpoint
GLP and guideline compliant study

Justification for classification or non-classification

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation (EC) No 605/2014.