2-[(3-aziridin-1-ylpropionyl)methyl]-2-ethylpropane-1,3-diyl bis(aziridine-1-propionate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance has a potential for point mutation-inducing activity in a reverse bacterial mutation assay according to an OECD 471 (Ames) assay.

The test substance is considered to cause structural chromosomal aberrations in cultured mammalian cells according to an in vitro OECD 473 chromosomal aberration assay.

The test substance has a potential to induce forward mutations in mammalian cell lines (HGPRT gen locus) according to OECD 476 HGPRT assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Edition 11

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Celanese Corporation
- Date received: 11 July 1986

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: responsdibility of the sponsor, given
- Solubility and stability of the test substance in the solvent/vehicle: miscible in water, solubility tested in McCoy's 5a culture medium at 500 mg/mL (pH 10)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: 1:100 in to culture medium, 5.0 mg/mL (pH 7.5)

FORM AS APPLIED IN THE TEST solution within McCoy's 5a culture medium

OTHER SPECIFICS:
- measurement of pH in the culture medium to which the test chemical is added:
- stock solution (500 mg/mL): pH 10
- final dilution (5.0 mg/mL): ph 7.5

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-WBL

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Dr. S. Wolff, University of California, Sa. Francisco
- Normal cell cycle time (negative control): 12 - 14h

For cell lines:
- Absence of Mycoplasma contamination: examination of slides, stained with Hoechst 33258 under UV microscopy as well as decrease uptake of BrdUrd in cell cycle delay test
- Number of passages if applicable: 5-8
- Cell cycle length: 12 - 14h
- Modal number of chromosomes: 21 per cell
- Periodically checked for karyotype stability: no

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: McCoy's 5a medium, supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin and streptomycin

Cytokinesis block (if used):

0.1 µg/mL Colcemid was added to cultures (in standard cell culture medium) after washing

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : male Sprague Dawley rat liver
- method of preparation of S9 mix: derived from rats were treated with Arocolor 1254 inducing mixed function of oxidase enzymes

Test concentrations with justification for top dose:

Based on a rangefinding assay a 20h harvest time was chosen for the aberration assay with concentrations ranging from 37.5 µg/mL through 500 µg/mL.


Rangefinding assay:
- without metabolic activation:
- with a test item concentration of 16.7, 50, 167, 500 µg/mL and 1.67, 5.0 mg/mL, precipitate was present
- toxicity in the monolayer was observed at 500 µg/mL through 5.0 mg/mL
- no mitotic cells available at 1.67 and 5.0 mg/mL
- first mitotic division observed at 167 µg/mL and 500 µg/mL; cell cycle delay observed through 16.7 µg/mL
- with metabolic activation.
- precipitate present, toxicity to monolayer, mitotic cells and first mitotic division comparable to testing without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: McCoy's 5a culture medium

- Justification for choice of solvent/vehicle: solubility of test item was given in culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1 (and dose range finding)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1.2 * 10E6
- Test substance added in suspension

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: for testing with metabolic activation: 2h hour exposure period, followed by washing and 17.8h incubation
- Exposure duration/duration of treatment: 17.2h for non-metabolic activation testing, 2 + 17.8h for metabolic activation testing
- Harvest time after the end of treatment (sampling/recovery times): 2.5h after washing step

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colcemide (colchicine analoga) with 0.1 µg/mL for 2.5h before harvesting of the cells
- Methods of slide preparation and staining technique used including the stain used: good morphology cells were selected and cells with number of centromers equal to modal number of 21 +/- 2 (range 19-23) were air dried, stained in pH 6.8 buffered 5% Giemsa
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 25-100 cells from each duplicate (for positive control at least 25 cells; 100 cells for test item and negative/solvent control)
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- not computed: chromatid gap, chromosome gap, uncoiled gap, polyploid cell, endoreduplication
- simple computed: chromatid break, chromosome break, double minute fragment
- complex computed: interstitial deletion, triradial, quadriradial, complex rearrangement, dicentric, tricentric, multicentric, ring, ringchromatid, chromosome interchange, translocation, pulverised chromsome
- Determination of polyploidy: A cell containing multiple copies of the number (n) of chromosomes. Only indexed if very common. Not counted in the cells, scored for aberrations.
- Determination of endoreplication: 4n cell in which separation of chromosome pairs has failed. Only indexed if very common. Not counted in the cells scored for aberrations.

Evaluation criteria:

1. The overall chromosomal aberration frequencies.
2. The percentage of cells with any aberrations.
3. The percentage of cells with more than one aberration.
4. Any evidence for increasing amounts of damage with increasing dose, i.e., a positive dose response.
5. The estimated number of breaks involved in the production of the different types of aberrations which were observed, i.e., complex aberrations may have more significance than simple breaks.

Statistics:

Fisher's exact test: percentage of cells with aberrations in each treatment group in comparison with the results from the pooled solvent and negative controls
- significance established: p<0.05

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- precipitation observed at 1.67 mg/mL and 5.0 mg/mL at the time of the harvest
- citotoxicity observed at 500 µg/mL through 5.0 mg/mL
- first mitotic division cells obtained at 167 µg/mL and 500 µg/mL; cell cycle delay obsorved through 16.7 µg/mL

The following abbreviations are used in the tables 1-4:

TG: Chromatid gap, SG: Chromosome gap, TB: Chromatide break, SB: Chromosome break, DM: "Souble Minute" fragment, ID: Interstitial deletion, TR: triradial (three-armed cofiguration of a chromosome), QR: Quadriradial, CR: Complex rearrangement, D: Diecentric, R: Ring, CI: Chromsome Interchange, PU: Pulverized chromosome, GT: Greater than 10 aberrations

Table 1: Results of the chromosome aberration without activation, cells fixed 20.1 hours after treatment

Treatment	Cells sorted	Number and type of aberration																No. of aberrations per cell	% cells with aberrations	% cells with >1 aberrations
		Not computed			Simple			Complex								Other
		TG	SG		TB	SB	DM	ID	TR	QR	CR	D	R	CI		PU	GT
Controls  Negative and solvent	200	5				2	1					2						0.03	2.5	0
Positive: Mitomycin C  80 ng/mL	25	1			1	1			4	2								0.32	24.0*	8.0*
Test compound
50 µg/mL	100	5			20	20		6	35	32	2		4	7		1	1	>1.37	77.0*	35.0*
125 µg/mL	50	2	2		33	12		7	31	15	9		2	7		2	7	>3.76	96.0*	78.0*
250 µg/mL	50	2	2		25	38		6	30	12	9			5			22	>6.90	100.0*	98.0*
375 µg/mL	50	14	1		30	33		4	31	20	14		2	1		6	17	>6.22	100.0*	100.0*
500 µg/mL (a)

(a) chromosome morphology too degenerate for accurate analysis.

* Significantly greater than the pooled negative and solvent controls, p<0.01

Table 2: Results of the chromosome aberration with activation, cells fixed 20.1 hours after treatment

Treatment	Cells sorted	Number and type of aberration																No. of aberrations per cell	% cells with aberrations	% cells with >1 aberrations
		Not computed			Simple			Complex								Other
		TG	SG		TB	SB	DM	ID	TR	QR	CR	D	R	CI		PU	GT
Controls  Negative and solvent	200	2																0	0	0
Positive: Cyclophosphamide  12.5 µg/mL	25	1			2	1			2	4	1		1					0.44	32.0*	12.0*
Test compound
50 µg/mL	200	12	5		4	18	10		2	1		3						0.19	13.0*	3.5
125 µg/mL	200	17	5		23	25		3	28	16	3	2	2	3				0.53	35.5*	12.5*
250 µg/mL	50	1	2		34	24		7	25	16	4			6		2	8	>3.96	100.0*	88.0*
375 µg/mL	50	5	1		34	44		11	26	18	9	1	5	8		4	7	>4.60	100.0*	90.0*
500 µg/mL (a)

(a) Toxic level

* Significantly greater than the pooled negative and solvent controls, p<0.01

Conclusions:

The test item is considered positive for inducing chromosomal aberrations in Chinese hamster ovary cells under both the metabolic activation and nonactivation conditions of the performed assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only strains TA1535 and TA100 tested, cell seeding concentration missing

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Cordova Chemical Company and Lot 12

Target gene:

his

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. Bruce Ames (University of California at Berkeley)

For cell lines:
- Methods for maintenance in cell culture: stock culture plates made every four to six weeks from single colony isolates, checked for their genotypic characterisation

MEDIA USED
- 4°C, minimal agar plate (with excess of bioltin and histidine)

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
Dr. Bruce Ames (University of California at Berkeley)


For cell lines:
- Methods for maintenance in cell culture: stock culture plates made every four to six weeks from single colony isolates, checked for their genotypic characterisation

MEDIA USED
- 4°C, minimal agar plate (with excess of bioltin and histidine)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : adult male rats (250-300 g)
- method of preparation of S9 mix:
- rats are given single 500-1118/kg intraperitoneal injection of Aroclor 1254 (a mixture of polychlorinated biphenyls)
- four days after injection: food removerd, water ad libitum
- 5th day liver homogenate prepared by liver washing in 0.15M KCl, homogenated with a Potter-Elvehjem apparatus and centrifuged for 10 min at 9,000xg
- supernatent used as S-9 fraction (frozen and stored at -80°C)
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL S-9 fraction in 10 mL metabolic activation mixture; 0.5 mL activation mixture in 2.6 mL final minimal agar plate

Test concentrations with justification for top dose:

100, 250, 500, 1,000, 2,500, 5,000, 10,000 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2- Anthramine: with and without S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY : counting and recording of number of his+-revertants

Rationale for test conditions:

- positive controls are known to revert each strain

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Ames test:
- Signs of toxicity : no signs observed
- Individual plate counts : Details are given in table 1.

Table 1: Results for the in vitro assay with Salmonella typhimurium

Compound	Metabolic activation	Micrograms of Compound	Histidine Revertants per Plate
		Added per Plate	TA1535		TA100
Negative Control (H2O)	-		12	5	88	99
	+		4	9	100	115
Positive controls
Sodium azide	-	1	341	250	387	331
2-Anthramine	-	1	16	3	73	86
	+	1	210	215	408	432
XAMA-7	-	100	24	16	88	110
	-	250	30	28	96	135
	-	500	62	55	128	138
	-	1000	116	76	178	175
	-	2500	211	189	224	237
	-	5000	330	306	299	397
	-	10000	478	452	370	532
	+	100	16	25	110	97
	+	250	26	26	126	141
	+	500	86	42	140	142
	+	1000	125	76	191	227
	+	2500	233	211	230	336
	+	5000	393	450	399	495
	+	10000	666	673	503	588

Conclusions:

Mutagenic activity with the test item was observed starting at 100 µg/plate in strain 1535 and starting at 1000 µg/plate in strain TA100; with both strains, the activity was seen both with and without activation.
No toxicity was observed with either compound.

Executive summary:

In summary, it is concluded that the test item contains a mutagen that induced base-pair substitution mutations S. typhimurium strains TA1535 and TA100 in the Ames Salmonella/microsimes assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-08-07 until 1986-11-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

04 April 1984

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Celanese Corporation
- Date received: 11 July 1986

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in the dark
- Stability under test conditions: responsibility of the sponsor, given
- Solubility and stability of the test substance in the solvent/vehicle: miscible in water, solubility tested in F12 culture medium at 20 mg/mL and additional dilutions in fetal bovine serum (FBS)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: 1:2 into culture medium (15% FBS for nonactivation; 5% FBS for metabolic activation studies)

FORM AS APPLIED IN THE TEST solution within F12 culture medium

OTHER SPECIFICS:
- measurement of pH in the culture medium to which the test chemical is added:
- specific gravity: 1.185

Target gene:

HGPRT locus, located on the X chromosome

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1-BH4

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: BH4 subclone of CHO-K1 cells, Dr. A.W. Hsie (Oak Ridge National Laboratory, Oak Ridge, Tennessee)
- Suitability of cells: demonstrated to be sensitive to many chemical mutagens
- Normal cell cycle time (negative control): 1-15*E-6

For cell lines:
- Absence of Mycoplasma contamination: checked regularily
- Methods for maintenance in cell culture: cultured in cleansing medium for 2-3 days, placed in recovery medium for one day, returned to culture medium
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
- maintenance medium: Ham's Nutrient Mixture F12, supplemented with L-glutamine, antibiotics, and FBS (10% v/v)
- cleansing medium: (reduction of spontaneous freequency) culture medium (5% serum), supplemented with 5.0*E-6 M thymidine, 1.0*E-5 ; hypoxanthine, 1.0*E-4 glycine, and 3.2*E-4 aminopterin or methotrexate
- recovery medium: cleansing medium minus aminopterin or methotrexate, + 10% FBS
- selection medium: hypoxanthine-free F12 medium with 10 µg/mL 6-thioguanine (TG) and 5% FBS v/v
- temperature: 37 ± 1°C
- CO2 concentration: 4.5 - 6%

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Sprague Dawley rats treated with Arocolor-1254
- method of preparation of S9 mix: 500 mg/kg Arocolor was given to rats 5 days before sacrifice; purchased
- concentration or volume of S9 mix and S9 in the final culture medium: 20 µL/mL
- quality controls of S9: mutational activity tested against reference chemicals, i.e.: benzo(a)pyrene or 3-methylcholanthrene

Test concentrations with justification for top dose:

- rangefinding cytotoxicity study was performed to select seven doses for the mutation assay with 0% - 90% reduction in colony-forming ability
- cytotoxicity tested: 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5. 10 mg/mL
- nonactivation: 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 mg/mL
- activation: 0.01, 0.03, 0.06, 0.10, 0.12, 0.16, 0.20 mg/mL

Vehicle / solvent:

- Vehicle used: - F12 culture medium at 20 mg/mL and serial dilutions performed in F12 culture medium

- Justification for choice of vehicle: - solubility tested up to 20 mg/mL, no pH alterations measured: the culture medium does not interferes with the test system

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

other: 5-bromo-2'-deoxyuridine: without metabolic activation, 50 µg/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single
- Number of independent experiments: 2 - one with and one without metabolic activation

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: cleansed cells plated at about 2*E6 cells per T75 (75 cm2)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: four hours
- Harvest time after the end of treatment (sampling/recovery times): directly after treatment, cells were washed, trypsinized and suspended in culture medium, replated at two 1.5*E6 cells/150 mm dishes and 200 cells into three 60-mm dishes for 7-8 days

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time: 7-10 days
- Fixation time (start of exposure up to fixation): 14-17 days
- Method used: agar dishes
- If a selective agent is used 6-thioguanine: 10 µg/mL, cells (2*E5 cells) were seeded onto 100 mm dishes for 7 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 200 cells seeded into each of the two 60 mm dishes for 7-8 days to determine cytotoxicity
- Criteria for small (slow growing) and large (fast growing) colonies:
- absolute cloning efficiency (ACE) = ability of cells to form colonies (number of mutant colonies) / 200 cells * 100%
- ACE of negative controls: 70 - 115%; low cloning efficiencies = 50 - 70%; unacceptable if <= 50%

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, i.e.: cloning efficiency (average colony counts in treated cultures versus control cultures)

Statistics:

Statistical tables by Kastenbaum and Bowman (1970) used to determine if the results are of statistical significance, compared to the negative controls. The confidence limits used were 95% and 99%.
The statistical poisson distribution was used by the test to compare the variables by summing up the probabilities in the tails of two binomial distributions.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDIES:
In the preliminary cytotoxicity test with and without activation, ten dose levels between 0.01 mg/mL and 10 mg/mL were applied to the cells. Toxicity was observed without metabolic activation at >0.01 mg/mL in an increasing matter up to 0.05 mg/mL and completely toxic at >=0.1 mg/mL. With activation, incresing toxicity was observed from 0.05 - 0.2 mg/mL with total toxicity at >=0.5 mg/mL.

HISTORICAL CONTROL DATA

A) activation studies
- Positive historical control data: 411.4 ± 174.7*E-6
- Negative (solvent/vehicle) historical control data: 3.2 ± 2.4*E-6
- Number of experiments: 47
- Number of controls: 58-67

B) nonactivation studies
- Positive historical control data: 110.9 ± 47.1*E-6
- Negative (solvent/vehicle) historical control data: 3.6 ± 3.2*E-6
- Number of experiments: 47
- Number of controls: 57-65

Table 1: Mutation assay without metabolic activation in F12 culture medium, with 10 µg/mL 6 -thioguanine.

Nonactivation test condition	Survival to treatment		Relative population growth (% of control)	Mutant colonies in dish number												Total mutant colonies	Absolute C.E. ± S.D. (%)	Mutant frequencies in 10E-4 units (a)
	Mean colony numbers ± S.D.	Percent neg. control		1	2	3	4	5	6	7	8	9	10	11	12
Negative control	179.0 ± 17.8	98.5	91.5	1	1	0	0	0	0	0	0	0	0	0	0	2	99.5 ± 3.9	0.8
Negative control	184.3 ± 5.7	101.5	108.5	0	0	0	1	0	0	2	0	1	0	C (b)	0	4	89.2 ± 5.7	2
Positive control (50 µg/mL BrdU, c)	112.0 ± 5.3	61.7	34.9	22	29	29	37	20	28	29	26	30	32	23	0	305	90.0 ± 7.6	154.0**
Test Article
0.005 mg/mL	171.7 ± 4.2	94.5	75.6	0	0	0	2	0	1	0	0	0	0	2	0	5	99.7 ± 9.8	2.8
0.01 mg/mL	161.0 ± 7.5	88.6	70.9	1	1	0	0	0	0	0	3	0	0	1	1	7	90.0 ± 5.7	3.2
0.02 mg/mL	148.3 ± 4.9	81.6	50.6	2	1	2	1	2	1	2	1	1	2	2	1	18	87.7 ± 5.6	8.6**
0.03 mg/mL	131.3 ± 11.9	72.3	44.5	1	3	0	6	2	2	5	7	4	0	3	2	35	85.2 ± 2.0	18.7**
0.04 mg/mL	102.3 ± 8.5	56.3	33.9	0	0	4	2	2	4	2	2	2	5	3	4	30	81.2 ± 3.5	18.5**
0.05 mg/mL	76.3 ± 4.7	42	19.1	0	0	9	6	6	3	6	7	7	4	4	4	56	75.9 ± 5.1	36.4**
0.06 mg/mL	51.7 ± 10.0	28.5	11.9	2	2	4	4	3	4	2	2	0	5	3	2	33	80.5 ± 9.9	17.1**

a) Mutant frequency = total mutant colonies/(No. of dishes*2 *E5*absolute C.E.)

b) C = lost due to contamination

c) BrdU = 5 -bromo-2'-deoxyuridine

** Significant increase, p<=0.01

Table 2: Mutation assay with metabolic activation in F12 culture medium, with 10 µg/mL 6 -thioguanine.

Activation test condition	Survival to treatment		Relative population growth (% of control)	Mutant colonies in dish number												Total mutant colonies	Absolute C.E. ± S.D. (%)	Mutant frequencies in 10E-4 units (a)
	Mean colony numbers ± S.D.	Percent neg. control		1	2	3	4	5	6	7	8	9	10	11	12
Negative control	173.0 ± 9.3	92.9	110.4	0	1	0	0	0	0	0	0	0	0	0	0	1	86.0 ± 7.0	0.5
Negative control	199.7 ± 19.7	107.1	89.6	0	0	1	1	3	2	0	0	0	3	1	1	12	92.2 ± 5.4	5.4
Positive control (5 µg/mL 3-MCA, c)	172.7 ± 15.4	92.6	58.2	48	45	46	51	49	53	53	43	45	40	57	60	590	83.2 ± 5.3	295.5**
Test Article
0.01 mg/mL	196.7 ± 3.5	105.5	81.2	0	0	1	C ('c)	1	0	0	0	0	C	C	0	2	87.4 ± 5.4	1.3
0.03 mg/mL	186.0 ± 10.8	99.7	84.2	2	3	1	2	1	2	0	4	1	C	2	0	18	84.2 ± 1.1	9.7**
0.06 mg/mL	153.7 ± 11.9	82.4	45	C	2	C	3	0	4	0	3	3	4	C	0	19	83.4 ± 7.4	12.7**
0.10 mg/mL	108.3 ± 4.5	58.1	36.4	6	2	2	2	5	4	1	8	2	5	C	C	37	79.5 ± 0.9	23.3**
0.12 mg/mL	92.3 ± 11.6	49.5	18.6	1	6	2	7	4	7	7	6	7	8	C	C	55	74.2 ± 0.3	37.1**
0.16 mg/mL	46.7 ± 4.9	25	5.5	C	C	16	8	11	12	9	4	9	9	11	8	97	72.4 ± 6.0	67.0**
0.20 mg/mL	13.7 ± 6.1	7.3	1.9	2	7	6	6	11	12	3	3	7	10	10	5	82	64.2 ± 2.8	53**

a) Mutant frequency = total mutant colonies/(No. of dishes*2 *E5*absolute C.E.)

b) 3 -MCA = 3 -Methylcholanthrene

c) C = lost due to contamination

** Significant increase, p<=0.01

Conclusions:

The test material is considered positive for inducing forward mutations at the HGPRT locus in Chinese hamster ovary cells under both the S9 metabolic activation and nonactivation conditions of the assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test substance is considered to be a chromosome-damaging (clastogenic) agent in bone marrow cells following an in vivo micronucleus study in NMRI mice according to OECD 474.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

21 July 1997

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Pflanzenbau und Pflanzenschutz, Essenheimer Str. 144, D-55128 Mainz

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF and 33-0988
- Expiration date of the lot/batch: 15 August 2002
- Purity test date: 15 August 2000

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator (4-10 °C)
- Stability under test conditions: confirmed for two years
- Solubility and stability of the test substance in the solvent/vehicle: stability in propylene carbonate verified analytically

FORM AS APPLIED IN THE TEST solution in vehicle

Species:

mouse

Strain:

NMRI

Remarks:

Crl:NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: mean = 27 g
- Assigned to test groups randomly: no, based on a randomization plan (computer program)
- Housing: at least 5 days the animals were housed in Makrolon cages; before the start of study: Makrolon cages, type MI
- Diet: ad libitum, Kliba Haltungsdiät, Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum, drinking water from bottles
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours)

IN-LIFE DATES: From: 09 March 2001 To: 28 August 2001

Route of administration:

oral: gavage

Vehicle:

Due to the hydrolytical sensitivity of the test substance in water, propylene carbonate was selected as the vehicle as requested by the sponsor.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: substance to be administered per kg body weight was dissolved in propylene carbonate

- Volume administered: 10 mL/kg bw

Duration of treatment / exposure:

single oral administration

Frequency of treatment:

once

Dose / conc.:

50 mg/kg bw/day

Remarks:

1st experiment

Dose / conc.:

100 mg/kg bw/day

Remarks:

1st experiment

Dose / conc.:

200 mg/kg bw/day

Remarks:

1st experiment

Dose / conc.:

100 mg/kg bw/day

Remarks:

2nd experiment

Dose / conc.:

150 mg/kg bw/day

Remarks:

2nd experiment

Dose / conc.:

200 mg/kg bw/day

Remarks:

2nd experiment

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

Once orally each in a volume of 10 mI/kg body weight: 20 mg cyclophosphamide and 0.15 mg vincristine sulphate.
The stability of both chemicals is welI-defined under the selected conditions, since both positive control articles are welI-defined clastogens and aneugens respectively.

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

In a pretest for the determination of the acute oral toxicity, deaths were observed down to a dose of 200 mg/kg body weight (male and femnale). At this dose, at which one animal died 48 hours after administration of the test substance, evident signs of toxicity were observed, such as gasping respiration, abdominal position and squatting posture, and the general state of the animais was poor. However, there were no symptomatic differences between the male and female animais. Thus, only male animals were used for the cytogenetic investigations.
Therefore, a dose of 200 mg/kg body weight was selected as the highest dose in the 1st cytogenetic study. 100 mg/kg and 50 mg/kg body weight were administered as further doses. In the 2nd experiment, doses of 200 mg/kg, 150 mg/kg and 100 mg/kg body weight were evaluated.

Evaluation criteria:

- test chemical considered positive:
- dose-related and significant increase in the number of micronucleated polychromatic erythrocytes at any of the intervals
- proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range
- test chemical considered negative:
- no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies and at any time
- frequencies of cells containing micronuclei were within the historical control range

Statistics:

- Wilcoxon hypothesis of equal medians: test for the number of micronuclei in polychromatic erythrocytes, comparison of dose group with the vehicle control
- Labels of significance: * for p 0.05, ** for p 0.01

Sex:

male

Genotoxicity:

positive

Remarks:

1st experiment: statistically significant increase in number of micronuclei at 200 mg/kg bw; 2nd experiment: slightly pronounced dose-dependent and statistically significant increase of small micronuclei at 100 mg/kg up to 200 mg/kg body

Toxicity:

yes

Remarks:

test substance led to signs of toxicity; no signs after administration of cyclophosphamide or vincristine

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY (acute oral toxicity study)
- Dose range:
- Solubility:
- Clinical signs of toxicity in test animals: evident signs of toxicity observed, such as gasping respiration, abdominal position and squatting posture and general poor state of animals;
- Evidence of cytotoxicity in tissue analyzed:
- Rationale for exposure: 200 mg/kg bw selected as high dose in 1st cytogenetic study
- Other: no symptomatic differences between male and female; therefore, only male animais were used for the cytogenetic investigations

For table 1-4:

PCE: polychromatic erythrocytes, NCE: normochromatic erythrocytes, CPP: Cyclophosphamide, VCR: Vincristine Sulphate

Wilcoxon test (one-sided): *: p<= 0.05, **: p<=0.01; a pairwise comparison of each dose group with the vehicle control group

Table 1: Summary table of the results of the 1st experiment for polychromatic and normochromatic erythrocytes

	Interval: 24 hours				Interval: 48 hours
	Total No. of		MN (o/oo) in		Total No. of		MN (o/oo) in
	PCE’s	NCE’s	PCE’s	NCE’s	PCE’s	NCE’s	PCE’s	NCE’s
untreated control:	10000	3227	1.1	0.6	10000	2862	1.7	0.0
propylene carbonate:	10000	3583	1.9	0.8	10000	2661	1.2	0.0
50 mg/kg	10000	2756	2.1	1.1
100 mg/kg	10000	3985	2.3	0.8
200 mg/kg	10000	3678	3.9*	0.5	10000	4617	3.9	0.9
CPP 20 mg/kg	10000	3710	21.1**	1.6
VCR 0.15 mg/kg	10000	4680	49.6**	1.1

Table 2: Summary table of the results of the 1st experiment for polychromatic erythrocytes, differentiation between small and large micronuclei

	Interval: 24 hours			Interval: 48 hours
	Total No. of	Cells (o/oo) with		Total No. of	Cells (o/oo) with
	PCE’s	MN. d<D/4	MN. d>=D/4	PCE’s	MN. d<D/4	MN. d>=D/4
untreated control:	10000	1.1	0.0	10000	1.7	0.0
propylene carbonate:	10000	1.9	0.0	10000	1.2	0.0
50 mg/kg	10000	2.1	0.0
100 mg/kg	10000	2.1	0.2
200 mg/kg	10000	3.9*	0.0	10000	3.9	0.0
CPP 20 mg/kg	10000	21.0**	0.1
VCR 0.15 mg/kg	10000	42.3**	7.3**

Table 3: Summary table (summary of 1st, 2nd and 3rd evaluation) of the results of the 2nd experiment for polychromatic and normochromatic erythrocytes

	Interval: 24 hours
	Total No. of		MN (o/oo) in
	PCE’s	NCE’s	PCE’s	NCE’s
propylene carbonate:	30000	9207	1.9	1.2
100 mg/kg	30000	12231	4.5**	1.4
150 mg/kg	30000	13246	4.5**	1.0
200 mg/kg	30000	15903	5.0**	1.1

Table 4: Summary table (summary of 1st, 2nd and 3rd evaluation) of the results of the 2nd experiment for polychromatic erythrocytes, differentiation between small and large micronuclei

	Interval: 24 hours
	Total No. of	Cells (o/oo) with
	PCE’s	MN. d<D/4	MN. d>=D/4
propylene carbonate:	30000	1.9	0.0
100 mg/kg	30000	4.4**	0.1
150 mg/kg	30000	4.5**	0.0
200 mg/kg	30000	4.9**	0.1

Conclusions:

According to the results of the present study, the single oral administration of the test substance led to a slight but relevant and statistically significant increase in the number of polychromatic erythrocytes containing small micronuclei in two experiments carried out independently of each other.
A slight and dose-dependent inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected only in the 2nd experiment.

Under the experimental conditions chosen here, the test substance considered to be a chromosome-damaging (clastogenic) agent in bone marrow cells in vivo.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Genetic toxicity in vitro:

There are no data on genetic toxicity in vitro available. However, a structural analogue substance (CAS 57116-45-7) was assessed for this toxicological endpoint:

Reverse bacterial muation, Ames:

Mutagenic activity with the test item was observed in a reverse bacterial mutation assay (Ames test) according to OECD 471.

The assay was conducted once in duplicate. The compound was tested over a wide range of concentrations, drom 100 to 10,000 µg/plate, both with and without metabolic activation.

The compound was tested in the strains Salmonella typhimurium TA1535 and TA100 with test concentrations from 100 µg/mL to 10,000 µg/mL. Positive controls used were sodium azide and 2 -Anthramine.

In summary, it was conculded that the test item contained a mutagen that induced base-pair substitution mutations in S. typhimurium strains TA1535 and TA100 in the Ames assay.

Cytogenicity in mammalian cells, Chromosomal aberration test:

The performed in vitro cytogenetic assay measuring chrosomal aberration frequencies was performed in Chinese Hamster Ovary (CHO) cells, according to OECD 473 with and without metabolic activation.

The test liquid was dissolved in McCoy's 5a culture medium at a stock concentration of 500 mg/mL. The test liquid was converted to weight/volume units using the sponsor provided specific gravity of 1.185. Toxicity was observed at 1.67 mg/mL and 5.0 mg/mL and severe cell cycle delay was present at at 167 µg/mL and 500 µg/mL in the nonactivation and activation rangefinding assays.

Duplicate CHO cultures were incubated in the aberration assay with 25 µg/mL through 500 µg/mL of test soution. Significant dose responsive increases in chromosomal aberrations were observed at 50 µg/mL, 125 µg/mL, 250 µg/mL and 375 µg/mL in the nonactivation and metabolic activation assays. The test item is considered positive for inducing chrosomal aberrations in Chinese hamster ovary cells under the conditions of metabolic activation and nonactivation.

Forward mammalian cell mutation, HGPRT assay:

In this in vitro assay the ability of the test substance to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary cells under conditions with and without metabolic activation, was evaluated. The test material was mixed in the F12 culture medium at different concentrations. After performing a preliminary cytotoxicity assay, concentration ranges of 0.005 mg/mL to 0.06 mg/mL and 0.01 mg/mL to 0.2 mg/mL for the mutational assay without and with metabolic activation respectively were used.

The test material produced dose related increases in mutant frequency that were statistically significant in both, the activation and nonactivation assays. Therefore, the test material was considered positive for inducing HGPRT mutations in CHO cells under both assay conditions.

Cell transformation assay in BALB/c-3T3 cells

The test substance was tested in vitro for the potential to transform the BALB/c-3T3 cell line. The endpoint is discussed for its scientific and regulatory potential to predict the genotoxic and carcinogenicity potential in vitro. However, a guideline is not achieved to date. The assay evaluated the morphological potential of the test material in cell culture. The objective of the semi-quantitative assay was to evaluate the test material for its ability to induce foci of transformed cells, recognized by dense, piled-up colonies on a monolayer of normal cells.

Cells were seeded at a density of 1*E4 - 3*E4 cells/vessel and preincubated with the test substance, the positive control (3 -methylcholanthrene at 2.5 µg/mL) or the negative control (culture medium only) for 72h. After washing the cells, the treatment period was 4 weeks and surviving colonies were stained, counted microscopically and scored for the foci.

Cytotoxicity was observed for the test material with a dose-related increase in cytotoxic activity over the range of concentrations from 0.75 to 13 nL/mL, and at concentrations >= 15.6 nL/mL the test material treatments also induced a dose-related increase in transforming activity. Test material treatments of 5, 7.5 and 13 nL/mL resulted in significant increases in transforming activity relative to spontaneous level of activity. Therefore, the test material was considered to be active in the BALB/c-3T3 cell transformation assay.

Genetic toxicity in vivo:

The test substance was tested for chromosomal damage (clastogenicity) and for the ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method according to OECD 474. For this purpose, the test substance, dissolved in propylene carbonate, was administered once orally to male animals at the following dose levels.

1st experiment: 50; 100 and 200 mg/kg body weight

2nd experiment: 100; 150 and 200 mg/kg body weight

The volume administered was 10 mL/kg body weight. 

As a negative control, male mice were administered merely the vehicle, propylene carbonate, by the same route. An untreated control was carried out in parallel, since no historical control data exist for the selected vehicle propylene carbonate. Both control groups gave frequencies of micronucleated polychromatic erythrocytes within the historical control range.

Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity (both experiments) and vincristine (only 1st experiment) for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity.

The administration of the test substance led to evident signs of toxicity.

In the 1st experiment, 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 200 mg/kg body weight and in the vehicle controls. In the test groups of 100 mg/kg and 50 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining the preparations, 2,000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2,000 polychromatic erythrocytes were also registered.

In the 2nd experiment, the animals were sacrificed and the bone marrow of the two femora was prepared only 24 hours after administration in all test groups. After staining of the preparations, 6,000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurringper

6,000 polychromatic erythrocytes were also recorded.

According to the results of the present study, the single oral administration of the test substance led to a slight but relevant and statistically significant increase in the number of polychromatic erythrocytes containing small micronuclei in two experiments carried out independently of each other. A slight and dose-dependent inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected only in the 2nd experiment.

Thus, under the experimental conditions chosen here, the test substance is considered to be a chromosome-damaging (clastogenic) agent in bone marrow cells in vivo.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

As a result the substance is considered to be classified for genotoxicity as Category 2, Suspected of causing genetic defects under Regulation (EC) No. 1272/2008.