Benzoic acid, 4-[[[4-[(1-oxo-2-propenyl)oxy]butoxy]carbonyl]oxy]-, 2-methyl-1,4-phenylene ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not mutagenic in bacteria and in mammalian cells in vitro (OECD 471 and 476, GLP). A weak clastogenic effect was observed at precipitating concentrations in vitro (OECD 473, GLP) which may be an artefact related to the precipitates. The substances was not clastogenic in the micronucleus test vivo (OECD 474, GLP).

Link to relevant study records

Reference

Reference 1

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Physical state: powder / grey-white
- Analytical purity: 95.8% (per weight; HPLC)
- Lot/batch No.: ZD 1151/ 31 (production: 03-Dec-1997)
- Storage condition of test material: refrigerator, protected from light.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

supernatant of rat liver homogenates (5 male SD rats receive a single intrapentoneal injection of 500 mg Aroclor 1254 [as a solution in com oil with a concentration of 200 mg/ml] per kg body weight 5 days before sacrifice) mixed with cofactors

Test concentrations with justification for top dose:

Pretests for dose selection: 50 - 5000 µg/ml; first experiment: 1, 10, 100, 500 and 1000 µg/ml; second experiment: 1, 5, 10, 50 and 100 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the V79 in vitro cytogenetic test and for which historical control data are available.

Untreated negative controls:

other: historical control data exist, demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - without metabolic activation: 350 pg ethyl methanesulfonate (EMS)/mI culture medium added in a volume of 1.0 ml; - with metabolic activation: 0.5 pg cyclophosphamide (CPP)/mI culture medium added in a volume of 1.0 ml.

Remarks:

The stability of EMS and CPP is well-defined under the selected culture conditions, since both positive control articles are weII-established reference clastogens.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 18 or 28 hours
- Fixation time (start of exposure up to fixation or harvest of cells):

SPINDLE INHIBITOR (cytogenetic assays): 2 - 3 hours prior to harvesting the cells, 0.2 pg colcemid/mI culture medium (1 pg colcemid dissolved in 0.1 ml PBS/culture) was added to each chamber in order to arrest mitosis in the metaphase.
STAIN (for cytogenetic assays): 5 ml of fixative (methanol glacial acetic acial/3: 1, after hypotonic treatment using 5 ml of a 0.4% KCI solution) was added to the cells, kept for at least 15 minutes and then replaced. After about another 10 minutes, the fixative was replaced again and kept for at least 5 minutes at room temperature for complete fixation. The preparations were dried in the air and subsequently stained in a solution of Giemsa and Titrisol for 10 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

NUMBER OF REPLICATIONS: the expression time was set to cover 1-1.5 cell cycle or any delayed cell cycle

NUMBER OF CELLS EVALUATED: counted for all test groups, and if cells had 20 - 22 chromosomes, they were analyzed for structural chromosome aberrations. Numerical chromosome aberrations were also recorded. Structural chromosome aberrations and numerical chromosome aberrations (so-called heteroploldies, including aneuploidy and polyploidy) were then analyzed.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: a mitotic index based on 1000 cells/culture was determined for all test groups in both experiments. For the determination of cytotoxicity, additional cell cultures (using 25 cm plastic flasks) were treated in the same way as in the main experiment. Growth inhibition was estimated by counting the number of cells in the dose groups in comparison with the concurrent vehicle control at the end of the culture period using a counting chamber.

OTHER: About 3 hours after test substance treatment, cultures of all test groups were checked for ceII morphology, which is an indication of attachment of the cells to the slides.

Evaluation criteria:

The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-related and reproducible significant increase in the number of structural chromosomal aberrations.
- The proportion of aberrations exceeded both the concurrent negative control range and the negative historical control range.
A test substance is generally considered nonclastogenic in this test system if:
- There was no significant increase in the number of chromosomally damaged cells at any dose above concurrent control frequencies.
- The aberration frequencies were within the historical control range.

Statistics:

- The statistical evaluation of the data was carried out using the MUCHAN program System.
- The proportion of metaphases with aberrations was calculated for each group.
- A comparison of each dose group with the vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

40% cell count (relative to vehicle control) was observed at 500 µg/ml (without S-9 mix) during the first main experiment.

Vehicle controls validity:

valid

Untreated negative controls validity:

other: historical control data exist, demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: precipitation was generally observed from 10 µg/ml upwards

- Other confounding effects: according to the results of the present in vitro cytogenetic study, under most of the experimental conditions the test substance led to a slight increase in the number of structural chromosomal aberrations including and excluding gaps both without S-9 mix and after the addition of a metabolizing System in two experiments performed independently of each other selecting different exposure times (4 hours treatment and continuous treatment) and harvest times (18 and 28 hours).
These slightly elevated figures were always observed only at the selected top doses at which evident test substance precipitation was found. These precipitated test substance particles may be phagocytized leading to high local concentrations within the cells with the possible result of lysosomal damage and a subsequent release of nucleases representing an overload phenomenon. Therefore, it is highly probable that the effect observed at the highest dose levels is the result of an indirect mechanism (chromosome damage by liberated nucleases) due to treatment conditions rather than a DNA-damaging activity of the test substance.
No increase in the number of cells containing numerical chromosomal aberrations was demonstrated.
The increase in the frequencies of chromosomal aberrations induced by the positive control agents EMS and CPP clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S-9 mix employed.

RANGE-FINDING/SCREENING STUDIES: the cell count was 77.3, 85.0, 69.6, 59.2, 71.5 and 61.5, respectively at 50, 100, 500 1000, 2500 and 5000 µg/ml during the pretest experiments (with precipitation generally observed from 50 µg/ml upwards; cell attachment and mitotic index not changed). Therefore, dose levels ranging from 1 to 1000 µg/ml were selected for the first and second main experiments.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity was observed in the second experiment (cell survival ranging from 80.8 to 121.8). Cell morphology (fibroblast-like) and attachment were not affected, in the pretest and in the 2 main experiments. Osmolality (range: 395 - 418) and pH (range: 7.5 - 7.7) were also not affected by the treatment.

1) Chromosome analysis

- First experiment

	Aberrations (%)			Exposure	Harvest
Treatment (µg/ml)	Metaphases including gaps	Metaphases including gaps	S-9 mix	time (hours)	time (hours)
DMSO	9 (4.5%)	6 (3.0%)	-	4	18
	4 (2.0%)	2 (1.0%)	+	4	18
Test substance (10)	4 (2.0%)	2 (1.0%)	-	4	18
	10 (5.0%	7 (3.5%)	+	4	18
Test substance (100)	13 (6.5%)	11 (5.5%)	-	4	18
	20 (10.0%)	8 (4.0%)	+	4	18
Test substance (500)	13 (6.5%)	13 (6.5%)	-	4	18
(from pretest)	28 (10.0%)	18 (9.0%)	-	4	18
	36 (18.0%)	13 (6.5%)	+	4	18
Positive control (350)	(20.0%)	(17.0%)	-	4	18
Positive control (0.5)	(26.0%)	(21.0%)	+	4	18

- Second experiment

	Aberrations (%)			Exposure	Harvest
Treatment (µg/ml)	Metaphases including gaps	Metaphases including gaps	S-9 mix	time (hours)	time (hours)
DMSO	17 (8.5%)	4 (2.0%)	-	4	18
	17 (8.5%)	9 (4.5%)	-	18	28
	7 (3.5%)	6 (3.0%)	+	4	28
Test substance (5)	11 (5.5%	6 (3.0%)	-	4	18
Test substance (10)	21 (10.5%)	11 (5.5%)	-	4	18
	6 (3.0%)	3 (1.5%)	+	4	28
Test substance (50)	28 (14.0%)	14 (7.0%)	-	4	18
	9 (4.5%)	6 (3.0%)	+	4	28
Test substance (100)	35 (17.5%)	13 (6.5%)	-	18	28
	7 (3.5%)	2 (1.0%)	+	4	28
Positive control (350)	(27.0%)	(22.0%)	+	4	18

Reference 2

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:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Physical state: powder / grey-white
- Analytical purity: 95.8% (per weight; HPLC)
- Lot/batch No.: ZD 1151/ 31 (production: 03-Dec-1997)
- Storage condition of test material: refrigerator, protected from light.

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

supernatant of rat liver homogenates (5 male SD rats receive a single intrapentoneal injection of 500 mg Aroclor 1254 [as a solution in com oil with a concentration of 20 g/100 ml] per kg body weight 5 days before sacrifice) mixed with cofactors

Test concentrations with justification for top dose:

0; 20; 100; 500; 2,500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with S-9 mix: 2-aminoanthracene, 2.5 µg/plate (strains: TA1535, TA100, TA1537, TA98); 60 µg/plate (strain: E. coli WP2 uvrA).

Remarks:

positive control without S-9: N-methyl-N'-nitro-N-nitrosoguanidine, 5 µg/plate (TA1535, TA100); 4-nitro-o-phenytefldiamifle, 10 µg/plate (TA98), 9-aminoacridine, 100 µg/plate (TA1537), 4-Nitroquinoline-N-Oxide, 5 µg/plate ( E. coli WP2 uvrA).

Details on test system and experimental conditions:

1) METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
The test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.6% agar + 0.6% NaCI) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at 45°C, and the remaining components are added in the following order: 0.1 ml test solution or vehicle, 0.1 ml fresh bacterial culture and 0.5 ml 5-9 mix (in tests with metabolic activation or 0.5 ml phosphate buffer (in tests without metabolic activation). After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
- 0.1 ml of the overnight cultures is diluted to 10E-6 in each case. Test tubes containing 2-mI portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order: 0.1 ml vehicle (without and with test substance), 0.1 ml fresh bacterial culture (dilution: 10E6) and 0.5 ml S-9 mix. After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48-72 hours in the dark, the bacterial colonies are counted.

2) METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 0.1 ml of the overnight cultures is diluted to 10E-6 in each case. 0.1 ml vehicle (with and without test substance), 0.1 ml bacterial suspension and 0.5 ml 5-9 mix are incubated at 37°C for about 20 minutes using a shaker.
- Expression time (cells in growth medium): Subsequently, 2 ml of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 370C for 48 -72 hours in the dark, the bacterial colonies are counted.


SELECTION AGENT (mutation assays): Individual plate counts ( triplicate plating used for all test groups at least in the Ist experiment), the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as weil as for the positive and negative (vehicle) controls in all experiments. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the Ist experiment.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: determined by detecting decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his or trp background growth) and reduction in the titer

- The titer is generally determined only in the experimental parts with 5-9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

OTHER:
1) Acceptance criteria:
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
- The sterility controls reverted no indication of bacterial contamination.
- The positive control articles both with and without 5-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.
- The titer of viable bacteria was >=10E9 /ml.

Evaluation criteria:

- The test chemical is considered positive in this assay if the following criteria are met: a dose-related and reproducible increase in the number of revertant colonies (increase in the number of his+ or trp+ revertants), i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing System.
- The test substance is generally considered non-mutagenic in this test if: the number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: - No bacteriotoxic effect (reduced background growth, decrease in the number of revertants, reduction in the titer) was observed in the standard plate test. - In the preincubation assay a slight reduction in the titer was occasionally observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

other: historical control data exist, demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test substance precipitation was found from about 100 pg/plate onward.
COMPARISON WITH HISTORICAL CONTROL DATA:
Positive and negative control results were within the range of the historical control data from the testing laboratory

The maximum revertants /and the corresponding concentration observed with/without S-9 mix are resumed below:

1) Standard plate test:

Strain	Treatment (µg)	S-9 mix	Revertants/plate	Factor
TA1535	DMSO	without	18±1	1.0
	DMSO	with	21±2	1.0
	Test substance (100)	with	22±1	1.1
	Test substance (500)	without	17±1	0.9
	Positive control (2.5)	with	313±9	14.9
	Positive Control (5.0)	without	1036±75	58.6
TA100	DMSO	without	102±2	1.0
	DMSO	with	108±3	1.0
	Test substance (20)	without	106±3	1.0
	Test substance (100)	with	113±14	1.0
	Positive control (2.5)	with	1529±86	14.1
	Positive control (5.0)	without	1160±242	11.4
TA1537	DMSO	without	8±1	1.0
	DMSO	with	11±1	1.0
	Test substance (20)	without	9±1	1.1
	Test substance (100)	with	12±2	1.0
	Positive control (2.5)	with	183±12	16.1
	Positive control (100)	without	984±26	95.2
TA98	DMSO	without	24±1	1.0
	DMSO	with	41±3	1.0
	Test substance (20)	with	42±3	1.0
	Test substance (5000)	without	23±2	0.9
	Positive control (2.5)	with	1069±49	26.1
	Positive control (10)	without	672±39	28.0
E. coli WP uvrA	DMSO	without	33±2	1.0
	DMSO	with	38±3	1.0
	Test substance (100)	without	36±1	1.1
	Test substance (500, 2500)	with	38±2	1.0
	Positive control (5)	without	1059±23	32.1
	Positive control (60)	with	236±29	6.2

2) Preincubation test:

Strain	Treatment (µg)	S-9 mix	Revertants/plate	Factor
TA1535	DMSO	without	18±1	1.0
	DMSO	with	18±1	1.0
	Test substance (20)	with	17±1	0.9
	Test substance (500)	without	16±1	0.9
	Positive control (2.5)	with	321±9	17.9
	Positive Control (5.0)	without	1109±103	62.8
TA100	DMSO	without	107±1	1.0
	DMSO	with	111±2	1.0
	Test substance (20)	without	106±5	1.0
	Test substance (5000)	with	111±10	1.0
	Positive control (2.5)	with	863±139	7.8
	Positive control (5.0)	without	1033±38	9.7
TA1537	DMSO	without	8±1	1.0
	DMSO	with	10±1	1.0
	Test substance (20, 2500, 5000)	without	8±2	1.0
	Test substance (20, 5000)	with	10±1	1.0
	Positive control (2.5)	with	121±12	12.5
	Positive control (100)	without	783±22	93.9
TA98	DMSO	without	23±	1.0
	DMSO	with	33±4	1.0
	Test substance (2500)	with	22±2	1.0
	Test substance (5000)	without	33±3	1.0
	Positive control (2.5)	with	739±7	22.2
	Positive control (10)	without	844±100	36.2
E. coli WP uvrA	DMSO	without	26±4	1.0
	DMSO	with	31±1	1.0
	Test substance (20)	with	29±4	0.9
	Test substance (2500)	without	28±3	1.1
	Positive control (5)	without	738±41	28.4
	Positive control (60)	with	155±7	5.0

CONCLUSION

Under the experimental conditions in this study, the test substance is not a mutagenic agent in a bacterial reverse mutation test in vitro

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test concentrations with justification for top dose:

6.3 - 250 microgramm/ml (see tables 1 and 2), limited by precipitation

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

DURATION
- Preincubation period:
- Exposure duration: 4h
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 6-TG

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Table 1: Results without metabolic activation

Experiment	Exposure period [h]	Concentration	S9 mix	Precipitation	Genotoxicity** MFcorr. [per 10exp6 cells]	Cytotox, CE1 [%]	Cytotox, CE2 [%]
1	4	Vehicle control1	-	n.d.	3.77	100.0	100.0
		7.8 µg/mL	-	-	n.c.	198.3	n.c.
		15.6 µg/mL	-	-	4.21	137.6	104.0
		31.3 µg/mL	-	-	3.38	97.5	106.0
		62.5 µg/mL	-	+	2.28	114.7	104.8
		125.0 µg/mL	-	+	1.12	90.1	106.4
		250.0 µg/mL	-	+	2.51	82.2	111.2
		Positive control2	-	n.d.	118.81S	59.6	133.5
2	4	Vehicle control 1	-	n.d.	0.35	100.0	100.0
		6.3 µg/mL	-	-	n.c.	99.2	n.c.
		12.5 µg/mL	-	-	0.41	136.9	85.9
		25.0 µg/mL	-	-	3.80	109.2	83.5
		50.0 µg/mL	-	+	2.30	126.2	76.4
		100.0 µg/mL	-	+	1.21	126.5	87.3
		200.0 µg/mL	-	+	0.39	95.4	90.8
		Positive control 2	-	n.d.	87.87S	83.5	84.2

* Precipitation occured at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 10exp6 cells corrected with the CE2 value

n.c. Culture was not continued since a minimum of at least four analysable concentrations are required

n.d. Not determined

s Statistically significant p ≤ 0.05

1 Acetone 1% (v/v)

2 EMS 400 μg/mL

Table 2: Results with metabolic activation

Experiment	Exposure period [h]	Concentration	S9 mix	Precipitation	Genotoxicity** MFcorr. [per 10exp6 cells]	Cytotox, CE1 [%]	Cytotox, CE2 [%]
1	4	Vehicle control 1	+	n.d.	5.62	100.0	100.0
		7.8 µg/mL	+	-	n.c.	105.1	n.c.
		15.6 µg/mL	+	-	0.37	137.1	101.1
		31.3 µg/mL	+	-	3.40	107.9	99.3
		62.5 µg/mL	+	+	5.64	97.5	99.6
		125.0 µg/mL	+	+	4.45	77.3	92.5
		250.0 µg/mL	+	+	1.69	78.5	110.5
		Positive control 3	+	n.d.	141.58S	59.5	71.2
2	4	Vehicle control1	+	n.d.	2.22	100.0	100.0
		6.3 µg/mL	+	-	n.c.	108.2	n.c.
		12.5 µg/mL	+	-	2.79	111.5	79.6
		25.0 µg/mL	+	-	3.52	111.9	84.1
		50.0 µg/mL	+	+	2.44	110.4	75.9
		100.0 µg/mL	+	+	0.34	81.4	109.6
		200.0 µg/mL	+	+	3.02	92.6	85.9
		Positive control 3	+	n.d.	97.49S	100.0	73.7

* Precipitation occured at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 10exp6 cells corrected with the CE2 value

n.c. Culture was not continued since a minimum of at least four analysable concentrations are required

n.d. Not determined

s Statistically significant p ≤ 0.05

1 Acetone 1% (v/v)

3 DMBA 1.25 μg/mL

Conclusions:

The substance is not mutagenic in the hprt test.

Executive summary:

The 1st and 2nd Experiment in the absence and presence of metabolic activation fulfilled the requirements of OECD Guideline 476. No cytotoxicity was observed when tested up to the highest applied concentration of about 250 μg/mL showing clear test substance precipitation in culture medium. No biologically relevant increase in the mutation frequency exceeding the historical negative control data range was observed in any experimental part of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Physical state: powder / grey-white
- Analytical purity: 95.8% (per weight; HPLC)
- Lot/batch No.: ZD 1151/ 31 (production: 03-Dec-1997)
- Storage condition of test material: refrigerator, protected from light.

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: young adult animals (male animals approx. 8- 12 weeks, female animals approx. 14-18 weeks)
- Weight at study initiation: mean weight 28.47 g
- Fasting period before study: the animals were given no feed at least 16 hours before administration, but water was available ad libitum.
- Housing: Makrolon cages type MIII, in groups of 5. The animais were identified using cage cards. No bedding in the cages
- Diet, ad libitum: standardized pelleted feed (Kliba Haltungsdiät, Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water, ad libitum: drinking water from bottles.
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Central air-conditioning guaranteed a range of 20 - 24 degrees
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12 hours (6.00 am - 600 pm/ 6.00 pm - 6.00 am)

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to the insolubility of the test substance in water; DMSO had been demonstrated to be suitable in the in vivo micronucleus test (historical data available).
- Concentration of test material in vehicle: 12.5 g/100 ml, 25 g/100 ml and 50 g/100 ml, respectiveliy for the 3 dose groups

Duration of treatment / exposure:

2 days

Frequency of treatment:

once daily

Post exposure period:

- animals of the vehicle control and the dose groups: samples of bone marrow were collected 24 hours after the last treatment.
- animals of the positive control groups: samples of bone marrow were collected after 24 hours after the unique treatment.

Remarks:

Doses / Concentrations:
484, 964, and 1912 mg/kg bw (actually injected in 4 ml DMSO: 500, 1000 and 2000 mg/kg bw)
Basis:
analytical conc.
HPLC

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPP) and vincristine (VCR)
- Route of administration: ip
- Doses / concentrations: 20 mg CPP in 10 ml solution and 0.15 mg VCR in10 ml solution

Tissues and cell types examined:

bone marrow (from femora)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity, all animals (male and female) survived following two treatments with the highest recommended dose according to the OECD Guideline. No symptomatic differences were observed between the male and female animals. Thus, only male animals were used for the cytogenetic investigations. Therefore, dose of 500, 1000 and 2,000 mg/kg body weight were selected for the present study.

DETAILS OF SLIDE PREPARATION:
- 2 femora were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 mI/femur).
- The Suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µl fresh FCS.
- 1 drop of this Suspension was dropped onto clean microscopic sildes. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
- The sildes 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 7.5% Giemsa solution for 15 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS (Microscopic):
In general, 2000 polychromatic erythrocytes (PCE) from each of the male 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. An alteration of this ratio indicates that the test substance actually reached the target Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
- Number of small micronuclei (d < D/4) and of large micronuclei (d >= D/4) (d = diameter of micronucleus, D = cell diameter)
- Slides were coded before microscopic analysis.

OTHER:
Clinical examinations: the animals were examined for any evident clinical signs of toxicity in each treatment group.

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.
- An alteration of the polychromatic to normochromatic erythrocytes ratio indicates that the test substance actually reached the target. Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
- The size of micronuclei may give an indication on the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect.
- The test chemical is to be considered positive in this assay if the following criteria are met: 1) a dose-related and significant increase in the number of micronucleated polychromatic erythrocytes was observed; 2) the proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
- A test substance is generally considered negative in this test system if: 1) there was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies; 2) the frequencies of cells containing micronuclei were within the historical control range.

Statistics:

- The statistical evaluation of the data was carried out using the program system MUKERN.
- A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

piloerection and irregular respiration were observed in all tested animals during the 4 subsequent hours to each application, but were completely reversible afterwards.

Vehicle controls validity:

valid

Negative controls validity:

other: historical control data exist, demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: limit test; 2000 mg/kg bw
- Solubility: yes, in DMSO
- Clinical signs of toxicity in test animals: as clinical signs only piloerection and irregular respiration were observed

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable
- Induction of micronuclei (for Micronucleus assay): within the vehicle control range
- Ratio of PCE/NCE (for Micronucleus assay): within the vehicle control range

Genotoxic effects:

- Vehicle control treatment led to 0.5‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval.

- The highest dose administration (2000 mg/kg bw) led to 0.9‰ polychromatic erythrocytes containing micronuclei.

- In the two lower dose groups, rates of micronuclei of about 0.9‰ (1,000 mg/kg group) and 0.6‰ (500 mg/kg group) were detected.

- With 18.6‰ the positive control substance CPP (clastogenicity) led to the expected increase in the number of polychromatic erythrocytes containing exclusively small micronuclei.

- With 76.3‰, the positive control VCR (induction of spindle poison) also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with an amount of large micronuclei (9.4‰).

- The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups.

- Thus, the test substance did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the vehicle control value and was within the historical control range. Nor were large micronuclei (d >= D1/4) observed either in the negative control group or in the 3 dose groups.

- No inhibition of erythropoiesis, induced by the treatment of mice with the test substance was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

Clinical examinations:

- The administrations of the vehicle was tolerated by all animals without any signs or symptoms.

- The administration of the test substance led irregular respiration and piloerection.

- No evident signs of toxicity were observed in the positive control animals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The Ames-test was conducted using strains TA1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The test substance did not induce an increase in revertants in any strain tested. An increase in chromosomal aberrations was observed in chinese hamster lung cells only with concomitant substance precipitation. The effect is therefore very likely secondary to substance precipitation without biological relevance. The in vivo micronucleus assay with intraperitoneal application in mice revealed an incidence in micronuclei in the same range as in the vehicle control even at a dose of 2000 mg/kg bw. An inhibition of erythropoeisis was not observed.

Justification for classification or non-classification

The bacterial mutagenicity, mammalian cell as well as chromosomal aberration assay in vitro revealed no biologically relevant mutagenic effect of the test material. This was confirmed by an in vivo micronucleus study. Therefore, classification regarding this endpoint is not warranted according to UN GHS criteria.