2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria:

Key: Ames test, S. typhimurium/ E. coli, with and without metabolic activation: negative (GLP, similar to OECD 471, 1999)

Sup: Ames test, S. typhimurium, with and without metabolic activation: negative (non-GLP, similar to OECD 471, 1980)

 

Cytogenicity in mammalian cells:

Key: Chromosome aberration, chinese hamster lung cell line CHL/U: negative (GLP, similar to OECD 473, 2000)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines on Industrial Chemicals (1997), Guideline under the Japanese Industrial Safety and Health Law (1988, 1997)

GLP compliance:

yes

Remarks:

Japanese GLP on Industrial Chemicals (1984, 1988) GLP under the Japanese Industrial Safety and Health Law (1988)

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains) and trp operon (for E. coli strain).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from the liver of male Sprague-Dawley rats which were intraperitoneally injected with phenobarbital (PB) at 30, 60, 60 and 60 mg/kg every 24 hours, and 5,6-benzoflavone at 80 mg/kg at the third PB injection.

Test concentrations with justification for top dose:

5000, 2500, 1250, 625, 313 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: A solvent selection test indicated that the test substance was insoluble in water for injection (abbrev. DW; Otsuka Pharmaceutical Factory) and DMSO, but uniformly suspended in DMSO at 50 mglmL. No heat, decoloration or foaming was observed when mixing with DMSO. Therefore, DMSO was selected as the solvent.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide, sodium azide, N-ethyl-N' -nitro- N-nitrosoguanidine, 9-aminoacridine hydrochloride, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 2

OTHER
- Negative control: dimethylsulfoxide DMSO

Positive controls
- without S9 mix:
• 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2): 0.01 µg/plate (TA 100)
• sodium azide (NaN3): 0.5 µg/plate (TA 1535)
• N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 µg/plate (E.coli WP2uvrA)
• 9-aminoacridine hydrochloride (9-AA): 80 µg/plate (TA 1537)

- with S9 mix:
2-aminoanthracene (2-AA):
• 0.5 µg/plate (TA 98)
• 1 µg/plate (TA 100)
• 2 µg/plate (TA 1535 and TA 1537)
• 10 µg/plate (E.coli WP2uvrA)

Evaluation criteria:

The test substance was judged to be mutagenic (or positive) when the mean number of revertant colonies at any concentration increased two or more times than that of the corresponding negative control for at least one tester strain either in the presence or absence of S9 mix, and when the number of revertant colonies increased dose-dependently and reproducibly.

Statistics:

The data was not analyzed statistically.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 19.5 µg/plate and over in the absence of S9 mix and at 78.1 µg/plate in the presence of S9 mix, precipitates were observed on the agar plates in the dose-finding and main test.

RANGE-FINDING/SCREENING STUDIES:
- A dose-finding test was conducted at 5000, 1250, 313, 78.1, 19.5, 4.88, and 1.22 µg/plate, and it showed the number of revertant colonies was smaller than twice that of the corresponding negative (solvent) control in all the tester strains regardless of the presence or absence of S9 mix. Microbial toxicity was not observed in any of the tester strains regardless of the presence or absence of S9 mix. According to these results, the main test was conducted.

Standard plate test (313 - 5000 µg/plate)
Strain	Metabolic activation system	Mean revertants in Controls	Maximum revertant factor	dose dependency	Assessment
TA 100	no	110	1,0	no	negative
	yes	105	1,1	no	negative
TA 1535	no	11	1	no	negative
	yes	12	1,1	no	negative
WP2uvrA	no	31	1,1	no	negative
	yes	39	1,0	no	negative
TA 98	no	21	1,0	no	negative
	yes	27	1,1	no	negative
TA 1537	no	10	1	no	negative
	yes	10	1,8	no	negative

In the test, the number of revertant colonies was smaller than twice that of the corresponding negative (solvent) control in all the tester strains regardless of the presence or absence of S9 mix.

Conclusions:

The results suggest that the test substance is not mutagenic under the conditions of this study.

Executive summary:

In a GLP conform study conducted similar to OECD guideline 471, the potential of the test substance (purity: 99.3 weight-%) to induce gene mutations was investigated in Salmonella typhimurium strains TA 100, TA 1535, TA1537 and TA 98, and in Escherichia coli WP2uvrA. The test was performed with phenobarbital and benzoflavone induced rat liver S9-mix and without microsomal activation at a dose range up to 5000 µg/plate. At 19.5 µg/plate, and over that dose in the absence of S9 mix, and at 78.1 µg/plate in the presence of S9 mix, precipitates were observed on the agar plates. Microbial toxicity was not observed in any of the tester strains regardless of S9 mix being present or absent. 

The number of revertant colonies was smaller than twice that of the corresponding negative (solvent) control in all the tester strains regardless of the presence or absence of S9 mix. These results suggest that the test substance is not mutagenic under the conditions of the study.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

acceptable restrictions: missing strain TA 102 to detect crosslinking agents, second independent trial is missing

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no bacterial strains (E.coli WP2 or S.thyphimurium TA102) to detect cross-linking mutagens.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

0, 4, 20, 100, 500 and 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF (Dimethylformamid)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see detailed information below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 4

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect: (slight decrease in the number of his+ revertants) was observed with some strains at 2500 µg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect: (slight decrease in the number of his+ revertants) was observed with some strains at 2500 µg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect: (slight decrease in the number of his+ revertants) was observed with some strains at 2500 µg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect: (slight decrease in the number of his+ revertants) was observed with some strains at 2500 µg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect: (slight decrease in the number of his+ revertants) was observed with some strains at 2500 µg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: incomplete solubility of the test substance at the highest dose of 2500 µg/plate.

Standard plate test (4 - 2500 µg/plate)

Strain	Metabolic activation system	Mean revertants in Controls	Maximum revertant factor	dose dependency	Assessment
TA 98	no	28	1.0	no	negative
	yes	33	1.0	no	negative
TA 100	no	140	1.0	no	negative
	yes	150	1.0	no	negative
TA 1535	no	13	1.1	no	negative
	yes	15	1.1	no	negative
TA 1537	no	6	1.0	no	negative
	yes	5	1.6	no	negative
TA 1538	no	12	1.0	no	negative
	yes	22	1.1	no	negative

Conclusions:

The test substance is not mutagenic in the Ames test under the experimental conditions chosen here.

Executive summary:

In the Ames test comparable to OECD guideline 471, the test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i. e. Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 1538, TA 98. The study was performed prior to the introduction of GLP and the OECD testing guideline, but is comparable both in design and reporting. In deviation to the current version of the OECD testing guideline 471, the design did not include a tester strain for crosslinking. The investigations were performed with Aroclor 1254-induced rat liver S-9 mix and without microsomal activation at a dose range up to 2500 µg/plate. At the highest dose of 2500 µg/plate the limit of complete solubility was reached.

A weak bacteriotoxic effect (slight decrease in the number of his⁺revertants) was observed with some strains at 2500 µg/plate.

An increase in the number of his⁺revertants could not be observed either without S-9 mix or after addition of a metabolizing system. Therefore, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

other: Guideline: Japanese Guidelines on Industrial Chemicals (1997)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

Type and identity of media: Eagle's minimum essential medium

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared from the liver of male Sprague-Dawley rats treated with phenobarbital (PB; 30, 60, 60, 60 mg/kg every 24 hours, respectively, i.p.) and 5,6-benzoflavone (80 mg/kg i.p. at the third PB injection)

Test concentrations with justification for top dose:

1250, 2500 and 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: physiological saline (50 mg/mL was suspended uniformly in physiological saline)
- Justification for choice of solvent/vehicle: In the preliminary test for the selection of the solvent, the test substance was insoluble in DMSO and acetone at 250 mg/mL. lt was not suspended uniformly in 1 % sodium carboxymethylcellulose at 50 mg/mL. lt was suspended uniformly in physiological saline (saline) at 50 mg/mL. According to these results, saline was selected as the solvent for the test substance.

Negative solvent / vehicle controls:

yes

Remarks:

physiological saline

Positive controls:

yes

Positive control substance:

other: without S9-mix: Mitomycin C (MMC) 0.1 µg/mL (puls treatment) and 0.03 µg/mL (continuous treatment); with S9-mix: Benzo(a)pyrene (BP) 20 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 and 24h
- Fixation time (start of exposure up to fixation or harvest of cells): 6 h exposure: 18 h; 24 h exposure: 0h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 2 h bevore the end of the cell treatment (Final concentration 0.1µg/ml).
STAIN (for cytogenetic assays): 20 min in 3 % Giemsa's solution.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphase cells per plate (i.e. 200 cells for each concentration)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Other:
Structural aberrations:
• chromatid breaks (ctb)
• chromatid exchanges (cte)
• chromosome breaks (csb)
• chromosome exchanges (cse : dicentric, ring, etc.)
• fragmentation (frg)
• Polyploid cells including endoreduplicated cells were scored.
A "gap" is an achromatic region in a single chromatid that is narrower than the width of the chromatid. The gap was distinguished from the other aberrations and was not included to structural aberration.

Evaluation criteria:

A cell having at least one structural chromosomal aberration was classified as an aberrant cell.
Aberrant cells were counted excluding cells having only gaps.
The test substance was judged to have a potential to induce chromosomal aberration as follows.
When both incidence of aberrant cells and that of numerical aberrations are less than 5 %, the test substance is negative.
When either of the incidences is 5 % or more and less than 10 %, the test substance is inconclusive.
When either of the incidences is 10 % or more, the test substance is positive.
When the judgement was negative, the 24-hour continuous treatment was conducted.

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Measurement of 50% inhibition concentration of cell growth

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
According to a preliminary test, concentrations selected for the celI growth inhibition test were 313, 625, 1250, 2500, and 5000 µg/mL for the pulse treatment without S9 mix (-S9 mix) and with S9 mix (+S9 mix). The test substance did not inhibit cell growth more than 50 % in each treatment.

 

Results of the chromosomal aberration study (pulse treatment)
Exposure-Recovery time (h)	Metabolic activation	Treatment (µg/ml)	Total structural aberrant cells (%)	Cell growth index (%)	Number of numerical aberrant cells (%) polyploids	Total aberrant cells (%)
6 - 18	no	Negative control (Saline)	1.0	100	0.0	0.0
6 - 18	yes		0.5	100	0.0	0.0
6 - 18	no	1250 P	0.0	106	0.0	0.0
6 - 18	yes		0.5	101	0.0	0.0
6 - 18	no	2500 P	0.5	108	0.5	0.5
6 - 18	yes		1.0	90	0.0	0.0
6 - 18	no	5000 P	0.0	96	0.5	0.5
6 - 18	yes		1.5	101	0.5	0.5
6 - 18	no	MMC 0.1	56.5	112	0.5	0.5
6 - 18	yes	BP 20	75.0	82	0.0	0.0

 

MMC: Mitomycin C, BP :Benzo(a)pyrene, Saline: Japanese Pharmacopoeia saline

P: Some of the test substance precipitated at the bottom of the plate and the other suspended in the medium at the end of the treatment.

Results of the chromosomal aberration study (continuous treatment)
Exposure-Recovery time (h)	Treatment (µg/ml)	Total structural aberrant cells (%)	Cell growth index (%)	Total aberrant cells (%)
24 - 0	Negative control (Saline)	2.0	100	0.0
24 - 0	1250 P	1.5	106	0.0
24 - 0	2500 P	1.5	112	0.0
24 - 0	5000 P	1.0	74	0.0
24 - 0	MMC 0.1	26.5	73	0.0

 

MMC: Mitomycin C, BP: Benzo(a)pyrene, Saline: Japanese Pharmacopoeia saline

P: Some of the test substance precipitated at the bottom of the plate and the other suspended in the medium at the end of the treatment.

Conclusions:

It was concluded that the test item did not have a clastogenic potential in this CHL/IU cell line system.

Executive summary:

In a GLP conform study conducted according to OECD guideline 473, the test substance (purity: 99.3 weight-%) was assessed in vitro for possible clastogenic activity both in the presence and in the absence of a metabolizing system (S9 mix) using the established cell line (CHL/IU) from the lungs of a female Chinese hamster (Mitsubishi 2000).

In a preliminary test, concentrations of 313, 625, 1250, 2500, and 5000 µg/mL (pulse treatment) or 156, 313, 625, 1250, 2500, and 5000 µg/mL (continuous treatment), in the presence or absence of S9 mix, were used to evaluate the cell growth inhibition. No inhibit of cell growth higher than 50 % was observed, both in the pulse and in the continuous treatment protocols.

Therefore, the concentration levels selected for the main chromosomal aberration test were 1250, 2500, and 5000 µg/mL with and without S9 mix. The incidence of cells with structural and numerical aberrant chromosomes was less than 5% in each treatment. The test substance was judged as negative in the pulse treatment and a second test with continuous treatment.

It was concluded that the test item did not have a clastogenic potential in this CHL/IU cell line system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are reliable in vitro studies available to assess the potential of the test substance for gene mutations in bacteria and chromosome aberration in mammalian cells. 

 

Gene mutation in bacteria

 

Key study: Ames test 

In a GLP conform study conducted similar to OECD guideline 471, the potential of the test substance (purity: 99.3 weight-%) to induce gene mutations was investigated in Salmonella typhimurium strains TA 100, TA 1535, TA1537 and TA 98, and in Escherichia coli WP2uvrA (Mitsubishi, 1999). The test was performed with phenobarbital and benzoflavone induced rat liver S9-mix and without microsomal activation at a dose range up to 5000 µg/plate. At 19.5 µg/plate, and over that dose in the absence of S9 mix, and at 78.1 µg/plate in the presence of S9 mix, precipitates were observed on the agar plates. Microbial toxicity was not observed in any of the tester strains regardless of S9 mix being present or absent. 

The number of revertant colonies was smaller than twice that of the corresponding negative (solvent) control in all the tester strains regardless of the presence or absence of S9 mix. These results suggest that the test substance is not mutagenic under the conditions of the study.

 

Supporting study: Ames test

In the Ames test comparable to OECD guideline 471, the test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i. e. Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 1538, TA 98 (BASF SE, 1980). The study was performed prior to the introduction of GLP and the OECD testing guideline, but is comparable both in design and reporting. In deviation to the current version of the OECD testing guideline 471, the design did not include a tester strain for crosslinking. The investigations were performed with Aroclor 1254-induced rat liver S-9 mix and without microsomal activation at a dose range up to 2500 µg/plate. At the highest dose of 2500 µg/plate the limit of complete solubility was reached.

A weak bacteriotoxic effect (slight decrease in the number of his⁺revertants) was observed with some strains at 2500 µg/plate.

An increase in the number of his⁺revertants could not be observed either without S-9 mix or after addition of a metabolizing system. Therefore, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here.

 

 

Cytogenicity in mammalian cells

 

Key study: Chromosome abberation test

In a GLP conform study conducted according to OECD guideline 473, the test substance (purity: 99.3 weight-%) was assessed in vitro for possible clastogenic activity both in the presence and in the absence of a metabolizing system (S9 mix) using the established cell line (CHL/IU) from the lungs of a female Chinese hamster (Mitsubishi 2000).

In a preliminary test, concentrations of 313, 625, 1250, 2500, and 5000 µg/mL (pulse treatment) or 156, 313, 625, 1250, 2500, and 5000 µg/mL (continuous treatment), in the presence or absence of S9 mix, were used to evaluate the cell growth inhibition. No inhibit of cell growth higher than 50 % was observed, both in the pulse and in the continuous treatment protocols.

Therefore, the concentration levels selected for the main chromosomal aberration test were 1250, 2500, and 5000 µg/mL with and without S9 mix. The incidence of cells with structural and numerical aberrant chromosomes was less than 5% in each treatment. The test substance was judged as negative in the pulse treatment and a second test with continuous treatment.

It was concluded that the test item did not have a clastogenic potential in this CHL/IU cell line system.

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 1272/2008. No indication of genotoxicity was observed in the Ames test (similar to OECD 471, GLP) and in the Chromosome Abberation test (similar to OECD 473, GLP). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the seventeenth time in Regulation (EC) No. 2021/849.