Ethyl 4-dimethylaminobenzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the Ames test, EDB is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay. Under the conditions of Chromosome aberration assay, EDB induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in the presence of metabolic activation. Under the conditions of Mouse lymphoma cell mutation assay, EDB did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the L5178Y cell line in the presence or absence of metabolic activation. 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 January 2006 to 02 February 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC L1362000 Annex 4D

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine locus
- E. coli: Tryptophan locus

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Obtained from Trinova Biochem GmbH (35394 Gießen, Germany)
- Method of culture: The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

MEDIA USED
- Type and identity of media for pre-cultures: From the thawed ampoules of the strains, 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 ML ampicillin (25 µg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth and 5 g NaCl. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.
- Properly maintained: Yes. Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Obtained from Trinova Biochem GmbH (35394 Gießen, Germany)
- Method of culture: The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

MEDIA USED
- Type and identity of media for pre-cultures: From the thawed ampoules of the strains, 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. This nutrient medium contains per litre: 8 g Merck Nutrient Broth and 5 g NaCl. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.
- Properly maintained: Yes. Regular checking of the properties of the strains regarding the membrane permeability as well as spontaneous mutation rates is performed.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Pre-Experiment /Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Experiment II: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (experiment I) and pre-incubation (experiment II)
The following materials were mixed in a test tube and poured onto the selective agar plates: 100 µL test solution at each dose level, solvent or positive control), 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 µL bacteria suspension and 2000 µL overlay agar.
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

NUMBER OF REPLICATIONS: The study was performed in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Assessment of the background lawn
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to precipitation of the test material and reduced background growth, the revertant colonies were partly counted manually.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- Regular background growth in the negative and solvent control
- The spontaneous reversion rates in the negative and solvent control are in the range of historical data
- The positive control substances should produce a significant increase in mutant colony frequencies

EVALUATION OF RESULTS
A test material is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

A statistical analysis of the data was not carried out

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate +S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 and 5000 µg/plate -S9mix; 1000 to 5000 µg/plate +S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate -S9mix; 2500 and 5000 µg/plate +S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate -S9mix; 1000 to 5000 µg/plate +S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 to 5000 µg/plate +S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

The historical control range was slightly exceeded in the untreated WP2 uvrA with/without activation in experiment I. This deviation is based on biologically irrelevant fluctuations in the number of colonies and has no impact on the outcome of the study.

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No visible reduction of the background growth was observed with or without metabolic activation in experiment I. In experiment II, reduced background growth was observed at higher concentrations with and without metabolic activation in all strains used.
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed at the following concentrations (µg/plate):
TA 1535: 5000 µg/plate with S9 mix (Experiment II)
TA 1537: 5000 µg/plate with and without S9 mix (Experiment I); 2500 and 5000 µg/plate without S9 mix, 1000 - 5000 µg/plate with S9 mix (Experiment II)
TA 98: 5000 µg/plate with and without S9 mix (Experiment I); 2500, 5000 µg/plate with S9 mix (Experiment II)
TA 100: 5000 µg/plate without S9 mix, 1000 - 5000 µg/plate with S9 mix (Experiment II)
WP2 uvrA: 5000 µg/plate with S9 mix (Experiment I); 1000 - 5000 µg/plate with S9 mix (Experiment II).
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
The laboratory´s historical control range was slightly exceeded in the untreated control of strain WP2 uvrA with and without metabolic activation in experiment I. This deviation is judged to be based on biologically irrelevant fluctuations in the number of colonies and has no impact on the outcome of the study.
Under the experimental conditions reported, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Table 1: Summary of Pre-Experiment and Experiment I

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	DMSO Untreated 3 10 33 100 333 1000 2500 5000	114 121 110 123 112 113 95 112 101P 85P	21 16 19 19 23 19 16 22 23P 20P	47 50 56 51 55 61 45 41 39P 30P	27 28 27 25 30 26 28 29 28P 11P	13 14 8 12 11 13 10 6 7P 6P
+	DMSO Untreated 3 10 33 100 333 1000 2500 5000	128 123 115 125 131 130 111 110 98P 74P	26 21 25 18 25 26 17 24 20P 16P	63 68 69 64 64 69 61 45 38P 13P	30 29 28 31 29 28 25 28 26P 8P	15 16 19 15 12 13 19 20 18P 5P
Positive Controls
-	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentration (µg/plate)	10	10	4.0 µL	10	50
	Mean no. colonies/plate	2013	1236	1435	357	109
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2.5	2.5	10	2.5	2.5
	Mean no. colonies/plate	1749	286	294	1364	134

P = Precipitate

NaN3 = sodium azide

4-NOPD = 4-nitro-o-phenylene-diamine

MMS = methyl methane sulfonate

2AA = 2-aminoanthracene

Table 2: Summary of Experiment II

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	DMSO Untreated 10 33 100 333 1000 2500 5000	115 164 139 147 129 104 117 61P 44P	16 17 19 18 19 15 18 10P 9P	57 66 50 53 53 49 47 31P 28P	24 24 28 26 27 18 16 16P 11P	9 13 8 7 8 5 4 2P 2P
+	DMSO Untreated 10 33 100 333 1000 2500 5000	163 198 172 158 155 129 62 50P 53P	21 28 30 26 29 15 12 10P 9P	68 72 77 69 82 64 28 18P 10P	38 36 29 29 33 34 26 12P 12P	12 8 12 9 14 16 3 3P 2P
Positive Controls
-	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentration (µg/plate)	10	10	4.0 µL	10	50
	Mean no. colonies/plate	1865	1347	416	390	95
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2.5	2.5	10	2.5	2.5
	Mean no. colonies/plate	3476	521	272	4953	143

P = Precipitate

NaN3 = sodium azide

4-NOPD = 4-nitro-o-phenylene-diamine

MMS = methyl methane sulfonate

2AA = 2-aminoanthracene

Conclusions:

Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and Commission Directive 2000/32/EC L1362000 Annex 4D under GLP conditions using the bacterial reverse mutation assay.

The study was performed to investigate the potential of the test material to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test material was tested at the following concentrations: Pre-Experiment /Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate; Experiment II: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

No visible reduction of the background growth was observed with and without metabolic activation in experiment I. In experiment II, reduced background growth was observed at higher concentrations with and without metabolic activation in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed at 5000 µg/plate in strains TA 1537 and TA 98 with and without metabolic activation and in strain WP2 uvrA with metabolic activation in experiment I. In experiment II, toxic effects were observed at higher concentrations with metabolic activation in all strains and without metabolic activation in strains TA 1537 and TA 100.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Appropriate untreated and solvent controls were also used and the study was considered to be valid.

During the described mutagenicity test and under the experimental conditions reported, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Under the conditions of this study, the test material is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 April 2006 to 15 May 2006

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)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4A: ”Mutagenicity – In vitro Mammalian Chromosome Aberration Test“

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome aberration study in mammalian cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, D-64287 Darmstadt
- Cell cycle length, doubling time or proliferation index: Doubling time of clone V79/D3 in stock cultures: 12 hrs)
- Plating efficiency of untreated cells: More than 70 %
- Methods for maintenance in cell culture if applicable: Thawed stock cultures were propagated at 37 °C in 80 cm² plastic flasks. About 5 x 10^5 cells per flask were seeded into 15 mL of MEM (Minimal Essential Medium) supplemented with 10 % foetal calf serum (FCS). The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
- Modal number of chromosomes: The cells have a stable karyotype with a modal chromosome number of 22.

MEDIA USED
- Properly maintained: Yes, stocks were stored in liquid nitrogen
- Periodically checked for Mycoplasma contamination: Yes, before freezing
- Periodically checked for karyotype stability: Yes, before freezing

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

- Range-finding test (with and without S9 mix): 15.6, 31.3, 62.5, 125.0, 250.0, 500.0, 1000.0 and 2000.0 µg/mL
- Main study (without S9 mix): 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL
- Main study (with S9 mix): 31.3, 62.5, 125.0, 250.0, 500.0 and 1000.0 µg/mL

A pre-test on cell growth inhibition with 4 and 24 hours of treatment was performed in order to determine the toxicity of the test material. The highest concentration used in the cytogenetic experiments was chosen with regard to the OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of non-toxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible.
With respect to the molecular weight of the test material, in the pre-test 2000 µg/mL (approximately 10 mM) of test material was applied as top concentration for treatment of the cultures. Test material concentrations between 15.6 and 2000 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Precipitation of the test material after 4 hours of treatment was observed at 500 µg/mL and above.
Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after treatment with 125 µg/mL and above in the absence of S9 mix. In addition, 4 hours of treatment with 500 µg/mL and above in the presence of S9 mix induced strong toxic effects. Considering the toxicity data of the pre-test, 250 µg/mL (without S9 mix) and 1000 µg/mL (with S9 mix) were chosen as top concentrations in the main experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen for its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 0.5 % v/v in culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
The chromosomes were prepared 18 hours after start of treatment with the test material. The exposure period was 4 hrs with and without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

Exponentially growing stock cultures more than 50 % confluent were treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then the enzymatic treatment was stopped by adding complete culture medium and a single cell suspension prepared. The trypsin concentration for all sub-culturing steps is 0.5 % (w/v) in Ca-Mg-free salt solution.
Prior to the trypsin treatment the cells are rinsed with Ca-Mg-free salt solution. The Ca-Mg-free salt solution is composed as follows (per litre): NaCl 8000 mg, KCl 200 mg, KH2PO4 200 mg and Na2HPO4 150 mg.
The cells were seeded into Quadriperm dishes that contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 1 x 10^4 to 6 x 10^4 cells were seeded with regard to the preparation time. The medium was MEM with 10 % FCS (complete medium).
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test material. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used.
After 4 hours the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time. The "Saline G" solution was composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, Glucose x H2O 1100 mg, Na2HPO4 x 7H2O 290 mg and KH2PO4 150 mg; the pH was adjusted to 7.2. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
Colcemid was added (0.2 µg/mL culture medium) to the cultures 15.5 hrs after the start of the treatment. The cells on the slides were treated 2.5 hrs later, in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Per experiment two slides per group were prepared.

EXPOSURE DURATION: 4 hours

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Two slides per group were prepared. Since the test material was considered to be clastogenic after 4 hours of treatment a second experiment was not performed.

EVALUATION OF CELL NUMBERS
For evaluation of cytotoxicity indicated by reduced cell numbers two additional cultures per test material and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hours, in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.

ANALYSIS OF METAPHASE CELLS
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphase plates per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

ACCEPTABILITY OF THE TEST
The chromosome aberration test is considered acceptable if it meets the following criterion:
a) The number of structural aberrations found in the solvent controls falls within the range of historical laboratory control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory´s historical control data.

EVALUATION OF RESULTS
A test material is classified as non-clastogenic if: the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of historical control data and/or no significant increase of the number of structural chromosome aberrations is observed.
A test material is classified as clastogenic if: the number of induced structural chromosome aberrations is not in the range of historical control data and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistical significance was confirmed, however, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid: A test material can be classified as aneugenic if: the number of induced numerical aberrations is not in the range of historical control data (0.0 - 8.5 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the pre-experiment, precipitation of the test material in culture medium was observed after treatment with 500 µg/mL and above in the absence and presence of S9 mix. No relevant influence of the test material on the pH value or osmolarity was observed (solvent control 406 mOsm pH 7.6 versus 376 mOsm and pH 7.5 at 2000 µg/mL).

RANGE-FINDING/SCREENING STUDIES
In a range finding pre-test on toxicity cell numbers were scored 24 hours after start of treatment as an indicator for cytotoxicity. Concentrations between 15.6 and 2000 µg/mL were applied. Clear toxic effects were observed after treatment with 125 µg/mL and above in the absence of S9 mix and with 500 µg/mL and above in the presence of S9 mix.

CONTROL DATA
- Positive control data: EMS (400 µg/mL) and CPA (1.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

RESULTS
In accordance with the OECD guideline, only one experiment was performed since the test material was considered to be mutagenic after 4 hours of treatment.
In the presence of S9 mix, cytotoxicity indicated by reduced cell numbers (38.5 % of control) was observed at the highest evaluated concentration (125 µg/mL). In contrast, in the absence of S9 mix, the highest applied concentration showing clear cytotoxicity was not evaluable for cytogenetic damage.
In the absence of S9 mix, no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test material (1.0 - 3.0 % aberrant cells, exclusive gaps) were close to the corresponding solvent control value (1.0 % aberrant cells, exclusive gaps) and within the range of historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.
In contrast, in the presence of S9 mix, dose-dependent, statistically significant and biologically relevant increases in the numbers of cells carrying structural chromosomal aberrations were observed at the two highest evaluated concentrations 62.5 and 125 µg/mL (17.5 and 30.0 % aberrant cells, exclusive gaps, respectively). Moreover, the dose-dependent increase in the number of cells carrying exchanges (11.5 and 19.0 %) provides additional evidence for a clastogenic potential of the test material.
No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test material (1.3 - 2.7 %) as compared to the rates of the solvent controls (2.0 - 2.7 %).

Table 1: Summary of results of the chromosome aberration study with the test material

Preparation Interval	Test material concentration (µg/mL)	Polyploid cells (%)	Cell number‡	Mitotic index (%)	Aberrant cells
					including gaps†	excluding gaps†	with exchanges
Exposure period: 4 hours without S9 mix
18 hours	Solvent control	2.7	100.0	100.0	4.0	1.0	0.0
	EMS 400.0	1.4	-nt	87.2	14.0	13.0*	3.5
	7.8	nd	94.3	127.6	nd	nd	nd
	15.6	nd	117.7	135.0	nd	nd	nd
	31.3	2.7	76.4	93.3	4.0	3.0	0.0
	62.5	1.9	70.5	119.5	2.0	2.0	0.0
	125.0	1.8	66.1	89.2	1.0	1.0	0.5
	250.0	nd	11.3	0.0	ne	ne	ne
Exposure period: 4 hours with S9 mix
18 hours	Solvent control	2.0	100.0	100.0	3.5	2.5	1.0
	CPA 1.0	1.8	nt	73.0	12.0	11.5*	3.5
	31.3	1.6	83.8	93.3	4.0	3.5	0.5
	62.5	1.3	53.6	102.2	17.5	17.5*	11.5
	125.0	1.4	38.5	58.5	33.0	30.0*	19.0
	250.0	nd	41.1	45.7	nd	nd	nd
	500.0	nd	12.3	0.0	ne	ne	ne
	1000.0	nd	38.1	36.2	ne	ne	ne

‡The toxicity of the test material was examined using the determination of the cell number.

†Inclusive of cells carrying exchanges

nd = Not determined

nt = Not tested

ne = Not evaluated

*Aberration frequency statistically significantly higher than corresponding control values. Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the Fisher´s exact test. Evaluation was performed only for cells carrying aberrations exclusive of gaps.

Solvent control = DMSO 0.5 % (v/v)

Conclusions:

Under the conditions of this study, the test material induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in the presence of metabolic activation.

Executive summary:

A study was performed to investigate the potential of the test material to induce structural chromosome aberrations in V79 cells of the Chinese hamster line in vitro. The study was performed in accordance with the standardised guidelines OECD 473 and Commission Directive 2000/32/EC L1362000 Annex 4A under GLP conditions.

Cultures were exposed to the test material with and without S9 mix for 4 hours, with a recovery of 14 hours and preparation interval of 18 hours. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (2000 µg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test material with respect to the OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data. Selected concentrations were: 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL without S9 mix and 31.3, 62.5, 125.0, 250.0, 500.0 and 1000.0 µg/mL with S9 mix.

In the presence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. In contrast, in the absence of S9 mix, the highest applied concentration showing clear cytotoxicity was not evaluable for cytogenetic damage.

In the absence of S9 mix, no clastogenicity was observed at the concentrations evaluated. In contrast, in the presence of S9 mix, dose-dependent, statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed at the two highest evaluated concentrations (17.5 and 30.0 % aberrant cells, exclusive gaps, respectively). Moreover, the dose-dependent increase in the number of cells carrying exchanges (11.5 and 19.0 %) provides additional evidence for a clastogenic potential of the test material. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test material as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Under the conditions of this study the test material induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in the presence of metabolic activation. Therefore the test material is considered to be clastogenic in this chromosome aberration test in the presence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 December 2005 to 27 February 2006

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)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000 Annex 4E

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell mutation assay

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Cell cycle length, doubling time or proliferation index: 10 to 12 hour doubling time in stock cultures
- Cloning efficiencies of untreated cells: Usually more than 50 %
- Methods for maintenance in cell culture if applicable: Cells are cultured in plastic flasks; they are sub-cultured at least three times a week and cells are incubated at 37 °C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % air.
- Modal number of chromosomes: The cells have a stable karyotype with a near diploid (40 ± 2) chromosome number.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with hypoxanthine 1.0 × 10^-4 M, aminopterin 2.0 × 10^-7 M and thymidine 1.6 × 10^-5 M. The incubation of the cells in HAT-medium is followed by a recovery period of 2 days in RPMI 1640 medium containing hypoxanthine 1.0 × 10^-4 M and thymidine 1.6 × 10^-5 M. After this incubation the cells are returned to normal RPMI 1640 medium (complete culture medium).
- Properly maintained: Yes; stocks of the cleansed cell line are stored in liquid nitrogen
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Pre-test: 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
- Experiment I: 30, 60, 120, 160, 180 and 200 µg/mL (without S9 mix) and 31.3, 62.5, 125, 250, 500 and 1000 µg/mL (with S9 mix)
- Experiment II: 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL (without S9 mix)

The concentrations chosen for the main experiments were based on the results of the pre-test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 1 x 10^7 cells /flask

DURATION
- Exposure duration: 4 hours in Experiment I, 24 hours in Experiment II
- Expression time (cells in growth medium): Total expression and growth period of 72 h in Experiment I, 48 hours for Experiment II.
In the mutation experiment 1 × 10^7 cells/flask (80 cm2 flasks) suspended in 10 mL RPMI medium with 3 % horse serum (15 % during 24 h treatment) were exposed to various concentrations of the test material either in the presence or absence of metabolic activation. After 4 h (after 24 h in the second experiment) the test material was removed by centrifugation (425 g, 10 min) and washing twice in "saline G". Subsequently the cells were re-suspended in 30 mL complete culture medium and incubated for a total expression and growth period of 72 h. In the second experiment the expression time without metabolic activation was 48 hours (RPMI medium with 15 % horse serum).
The cell density was determined each day and adjusted to 3 × 10^5 cells/mL, if necessary. Relative suspension and total growth (RSG and RTG) of the treated cell cultures were calculated after 48 h (72 h following continuous treatment) according to the method of Clive and Spector. One sample of the cells was taken at the end of the treatment (4 h and 24 h, respectively), diluted and seeded into microtiter plates (about 2.0 cells/well), to determine the survival of the cells after treatment (cloning efficiency 1).
After the expression period the cells were seeded into microtiter plates. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4 × 10^3 cells in selective medium with TFT. The viability (cloning efficiency 2) was determined by seeding about 2.0 cells per well into 2 microtiter plates (same medium without TFT). The plates were incubated at 37 °C in 4.5 % CO2 and 95.5 % humidified air for 10 - 15 days to determine the cloning efficiency and to evaluate mutagenicity. Then the plates were evaluated manually.

MEDIA
- Complete Culture Medium: RPMI 1640 medium supplemented with 15 % horse serum (HS), 100 U/100 µg/mL Penicillin/Streptomycin, 220 µg/mL Sodium-Pyruvate and 1.25 U/mL Amphotericin used as antifungal.
- Cloning Medium: RPMI 1640 (complete culture medium)
- Selective Medium: RPMI 1640 (complete culture medium) by addition of 5 µg/mL TFT.
- Saline G Solution: Composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1100 mg, Na2HPO4x7H2O 290 mg and KH2PO4 150 mg, pH 7.2.

SIZE DISTRIBUTION OF THE COLONIES
The numbers of colonies were counted manually. In accordance with their size the colonies were classified into two groups. The colony size distribution was determined in the controls and at all concentrations of the test material. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the large colonies).

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay is considered acceptable if it meets the following criteria:
a) All plates, from either the cloning efficiency 2 or the TFT resistance-testing portion of the experiment are analysable.
b) The absolute cloning efficiency 2 of the negative and/or solvent controls is > 0.5 (50 %).
c) The spontaneous mutant frequency in the negative and/or solvent controls is in the range of historical control data
d) The positive controls (MMS and CPA) induce significant (at least 2-fold) increases in the mutant frequencies. The cloning efficiencies and the relative total growth are greater than 10 % of the concurrent vehicle control group.

EVALUATION OF RESULTS
A test material is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points. A test material producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
The test material is classified as mutagenic if it reproducibly induces a mutation frequency that is at least two times higher than the spontaneous mutation frequency (negative or solvent control) in the experiment. The test material is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.
A positive response is considered to be reproducible if it occurs in both parallel cultures. However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software. The number of mutant colonies obtained for the groups treated with the test material was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance should equally be considered.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDY:
In the pre-experiment precipitation was observed by the naked eye at 250 µg/mL and above in the absence and at 500 µg/mL in the presence of metabolic activation (4 h treatment). After 24 h treatment precipitation occurred at 500 µg/mL and above. In the main experiments precipitation was only noted at the two highest concentrations of the first experiment with metabolic activation.

EXPERIMENT I
The experimental part of the first experiment without metabolic activation was not considered valid since the positive controls did not respond properly in both parallel cultures. Therefore, a repeat experiment (IA) was performed with 4 h treatment in the absence of metabolic activation. The concentration range of experiment IA was adjusted to the toxicity observed in the first experiment.
Relevant toxic effects as indicated by a relative cloning efficiency 1 or a relative total growth of less than 50 % of survival in both parallel cultures occurred in the first experiment at 160 µg/mL and above without metabolic activation. The data generated at the maximum concentration without metabolic activation are not considered valid since both parameters of toxicity fell short of the 10 % threshold of survival at both cultures. The data generated at 160 µg/mL are considered acceptable since only one parameter of toxicity out of three was below the 10 % limit. In the presence of metabolic activation toxic effects were noted only in the second culture at 500 µg/mL and above indicated by relative total growth of less than 50 %. Thus, the concentration range in the presence of metabolic activation was limited by precipitation of the test material.
No substantial and reproducible dose dependent increase in mutant colony numbers was observed up to the maximum concentration of the first experiment with and without metabolic activation at acceptable levels of toxicity. In the absence of metabolic activation the total number of mutant colonies/10^6 cells exceeded the historical data range of solvent controls at 60 µg/mL in culture I. However, the threshold of twice the mutation frequency of the corresponding solvent control was not reached and no increase was observed in the parallel culture under identical conditions. Therefore, this isolated effect was judged as a biologically irrelevant fluctuation.
A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 only occurred in the second culture of the first experiment with metabolic activation. However, all absolute values of the mutation frequency remained well within the historical range of solvent controls so the putative trend observed in one of the parallel cultures is based on biologically irrelevant fluctuations.

EXPERIMENT II
In the second experiment toxic effects were observed at 250 µg/mL and above in both cultures. The data generated at 250 µg/mL were not considered acceptable. Even though the relative cloning efficiency 1 was still fairly high (34.9 to 49.9 %), the relative total growth was only 5.2 and 6.2 % of the corresponding solvent control. Since the relative total growth is derived from the cell cultures used to measure the mutagenicity data (mass cell cultures) the impact of very low relative total growth values is judged as biologically more relevant than the cloning efficiency 1 which is derived from separate cell cultures seeded right after treatment.
In the second experiment the mutation frequency slightly exceeded the historical control range of solvent controls at almost all concentrations but this increase was not dose dependent at all and the threshold of twice the mutation frequency of the corresponding solvent control was not reached by far.

CONTROL DATA
In this study the range of the solvent controls was from 43 up to 204 mutant colonies per 10^6 cells; the range of the acceptable groups treated with the test material was from 38 up to 294 mutant colonies per 10^6 cells.
MMS (13.0 and 19.5 µg/mL) and CPA (4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies.

Table 1: Summary of results

Treatment	Conc. µg/mL	S9 mix	Relative cloning efficiency 1	Relative total growth	Mutant colonies/ 10^6 cells	Induction factor	Relative cloning efficiency 1	Relative total growth	Mutant colonies/ 10^6 cells	Induction factor
Experiment I/ 4 h treatment			Culture I				Culture II
Medium	-	-	100.0	100.0	200	-	100.0	100.0	145	-
DMSO	-	-	100.0	100.0	204	1.0	100.0	100.0	168	1.0
MMS	19.5	-	50.9	45.2	638	3.2	64.7	26.8	777	5.4
Test material	30.0	-	56.7	109.1	102	0.5	95.4	138.8	140	0.8
Test material	60.0	-	55.1	105.5	293	1.4	114.4	137.2	140	0.8
Test material	120.0	-	42.7	60.1	150	0.7	64.7	70.0	178	1.1
Test material	160.0	-	10.3	6.2	164	0.8	13.6	10.9	140	0.8
Test material	180.0	-	4.2	0.5	764	3.7	2.2	3.7	68	0.4
Test material	200.0	-	2.0	culture was not continued*			2.0	culture was not continued*
Medium	-	+	100.0	100.0	43	-	100.0	100.0	70	-
DMSO	-	+	100.0	100.0	48	1.0	100.0	100.0	86	1.0
CPA	4.5	+	37.7	73.2	228	5.2	27.6	41.3	379	5.4
Test material	31.3	+	culture was not continued**				culture was not continued**
Test material	62.5	+	81.0	110.4	48	1.0	83.5	65.8	80	0.9
Test material	125.0	+	82.2	112.1	38	0.8	96.6	43.3	139	1.6
Test material	250.0	+	84.8	60.6	66	1.4	93.4	54.7	109	1.3
Test material	500 (p)	+	95.0	60.0	82	1.7	98.3	40.5	155	1.8
Test material	1000 (p)	+	77.4	55.3	60	1.3	84.8	39.6	149	1.7
Experiment II/ 24 h treatment			Culture I				Culture II
Medium	-	-	100.0	100.0	130	-	100.0	100.0	144	-
DMSO	-	-	100.0	100.0	147	1.0	100.0	100.0	181	1.0
MMS	13.0	-	34.3	29.0	362	2.8	38.8	20.2	503	3.5
Test material	15.6	-	97.4	108.8	215	1.5	73.5	82.6	245	1.3
Test material	31.3	-	121.4	101.1	155	1.1	100.0	81.2	294	1.6
Test material	62.5	-	100.0	74.3	134	0.9	69.6	71.8	290	1.6
Test material	125.0	-	84.5	50.3	233	1.6	69.6	89.5	236	1.3
Test material	250.0	-	34.9	5.2	2998	20.4	49.9	6.2	1631	9.0
Test material	500.0	-	4.2	culture was not continued*			2.3	culture was not continued*

* not determined, culture not continued due to exceedingly strong toxic effects

** culture was not continued since a minimum of four concentrations is required by the guidelines

(p) precipitation visible to the unaided eye

The values printed in bold are judged as invalid, since the acceptance criteria are not met.

Conclusions:

Under the conditions of this study the test material did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the L5178Y cell line in the presence or absence of metabolic activation. The test material is considered to be non-mutagenic.

Executive summary:

A study was performed to investigate the potential of the test material to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The study was conducted in accordance with the standardised guidelines OECD 476 and Commission Directive 2000/32/EC L1362000 Annex 4E under GLP conditions.

The assay was performed in three independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. Since the positive control in the absence of metabolic activation did not meet the acceptance criteria at both parallel cultures, this experimental part was repeated (experiment IA). The concentration range of experiment IA was adjusted to the toxicity observed in the first experiment. The second experiment was solely performed in the absence of metabolic activation with a treatment time of 24 hours.

Following the pre-test, the doses in Experiment I were 30, 60, 120, 160, 180 and 200 µg/mL (without S9 mix) and 31.3, 62.5, 125, 250, 500 and 1000 µg/mL (with S9 mix). In Experiment II the doses were 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL (without S9 mix).

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments at acceptable levels of toxicity.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

Under the conditions of this study the test material did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the L5178Y cell line in the presence or absence of metabolic activation. The test material is considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Under the conditions of the in vivo micronucleus study, the test material is considered to be non-mutagenic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 August 2006 to 20 October 2006

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, Annex 4C

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vivo mouse micronucleus

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 to 10 weeks prior to acclimatisation
- Mean weight at study initiation: Females: 28.6 ± 1.8 g, Males: 35.1 ± 1.8 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Singly housed in wire mesh top cages with granulated soft wood bedding
- Diet: Pelleted standard diet ad libitum
- Water: Tap water ad libitum
- Acclimation period: Minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 to 90 % (relative)
- Photoperiod: Artificial light from 6.00 am to 6.00 pm

IN-LIFE DATES: Not reported

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 30 % DMSO, 70 % PEG 400
- Justification for choice of solvent/vehicle: The vehicle was chosen for its relative non-toxicity for the animals.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment the test material was formulated in the vehicle

- Dose volume: 10 mL/kg bodyweight

Duration of treatment / exposure:

24 h and 48 h after a single administration of the test material the bone marrow cells were collected for micronuclei analysis.

Frequency of treatment:

A single dose administration

Post exposure period:

Groups were sacrificed after 24 hours (500, 1000 and 2000 mg/kg bodyweight) or 48 hours (2000 mg/kg bodyweight only).

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 males and 6 females were assigned per group; 5 of each sex were evaluated for micronuclei

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 40 mg/kg bodyweight at a dose volume of 10 mL/kg bodyweight
- Stability: The stability of Cyclophosphamide at room temperature is sufficient. At 25 °C only 3.5 % of its potency is lost after 24 hours. The solution was prepared on the day of administration.

Tissues and cell types examined:

Slides were prepared from the bone marrow of the femora and stained. The numbers of micronucleated polychromatic erythrocytes and normochromatic erythrocytes were recorded.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study with respect to animal strain, vehicle, route, frequency and volume of administration. The animals were treated orally with the test material at doses of 100, 500, 1000 and 2000 mg/kg bw and examined for acute toxic symptoms at intervals of around 1, 2-4, 6, 24, 30 and 48 h after administration of the test material.
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test materials. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. The volume to be administered should be compatible with physiological space available. Three adequately spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment. On the basis of the data from the preliminary study, 2000 mg/kg bodyweight was estimated to be suitable.

TREATMENT AND SAMPLING TIMES: A single dose was administered with sacrifice after 24 (500, 1000 and 2000 mg/kg bodyweight) or 48 hours (2000 mg/kg bodyweight only).

DETAILS OF SLIDE PREPARATION: The animals were sacrificed using CO2 following by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT . At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining 6th animal of each sex in the respective test group test group is usually evaluated in case an animal dies in its test group spontaneously.

Evaluation criteria:

ACCEPTANCE CRITERIA
The study is considered valid if the following criteria are met:
- The negative controls are in the range of historical control data.
- The positive controls are in the range of historical control data.
- At least 4 animals per group and sex can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.

EVALUATION OF RESULTS
A test material is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
A test material that fails to produce a biologically relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

The nonparametric Mann-Whitney test was used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

minor clinical signs observed at 2000 mg/kg bw; no cytotoxicity

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT FOR TOXICITY
- Clinical signs of toxicity in test animals: Reduction of spontaneous activity and ruffled fur was observed in some or all animals at all concentrations but no symptoms were seen after 48 hours. Eyelid closure was observed at 2000 mg/kg bodyweight in two animals but this was not seen after 24 hours.
- Rationale for exposure: On the basis of the results from the pre-experiment for toxicity, 2000 mg/kg bodyweight was selected as the highest dose for the main study.

RESULTS OF DEFINITIVE STUDY
A summary of the micronucleus test results can be seen in Table 1. The mean number of polychromatic erythrocytes was not decreased after treatment with the test material as compared to the mean value of PCEs of the vehicle control indicating that the test material did not have any cytotoxic properties in the bone marrow.
Animals treated with the low (500 mg/kg) and mid (1000 mg/kg) dose levels of test material did not express any toxic reactions. Animals treated at the high dose level (2000 mg/kg) showed some minor signs. Two to 4 hours after treatment with the test material, 5 males and 6 females exhibited a reduction of spontaneous activity and ruffled fur. Six hours after treatment, the reduction of spontaneous activity was still evident in 3 males and 4 females. Ruffled fur was recorded in three animals of each sex. By the 24 h observation, all animals appeared normal.
40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

DISCUSSION
In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test material. The mean values of micronuclei observed after treatment with the test material were below or near to the value of the vehicle control group. The mean frequency of micronucleated PCEs in the low dose group (0.180 %) is slightly above the upper historical control range (0.16 %). The number of micronucleated PCEs per 2000 PCEs (1-8 microncuelated PCEs per 2000 PCEs) of the individual animals within this group, however, does not show any relevant increase and is within the historical range (up to 8 per 2000 PCEs). This increase is not observed at higher doses and the observed increase is also not statistically significant. Therefore, it is concluded that the observed increase in the low dose group is biologically irrelevant.

Table 1: Summary of Micronucleus Test Results

Test group	Dose mg/kg b.w	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythocytes
Vehicle	0	24	0.150	0-7	1199
Test material	500	24	0.180	1-8	1215
Test material	1000	24	0.130	0-5	1212
Test material	2000	24	0.145	1-6	1212
Positive control	40	24	2.765	17-138	1109
Test material	2000	48	0.105	1-4	1222

Conclusions:

Under the conditions of the study, the test material is considered to be non-mutagenic.

Executive summary:

The potential of the test material to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse was investigated in accordance with the standardised guidelines OECD 474 and Commission Directive 2000/32/EC Annex 4C under GLP conditions.

The test material was formulated in 30 % DMSO/ 70 % PEG 400, which was also used as vehicle control. The volume administered orally was 10 mL/kg bodyweight. 24 and 48 hours after a single administration of the test material the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test material the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test material were investigated: 24 hour preparation interval: 500, 1000 and 2000 mg/kg bodyweight; 48 hour preparation interval: 2000 mg/kg bodyweight. The highest dose (2000 mg/kg, maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.

After treatment with the test material the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test material did not exert any cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test material and with any dose level used. 40 mg/kg bodyweight cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

The test material did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse, therefore under the conditions of this study the test material is considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Three key and two supporting studies are available. The key studies were all awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997) while the supporting studies were awarded scored of 2.

Key study 1: Ames test (Sokolowski, 2006)

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and Commission Directive 2000/32/EC L1362000 Annex 4D under GLP conditions using the bacterial reverse mutation assay.

The study was performed to investigate the potential of the test material to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test material was tested at the following concentrations: Pre-Experiment /Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate; Experiment II: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

No visible reduction of the background growth was observed with and without metabolic activation in experiment I. In experiment II, reduced background growth was observed at higher concentrations with and without metabolic activation in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed at 5000 µg/plate in strains TA 1537 and TA 98 with and without metabolic activation and in strain WP2 uvrA with metabolic activation in experiment I. In experiment II, toxic effects were observed at higher concentrations with metabolic activation in all strains and without metabolic activation in strains TA 1537 and TA 100.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Appropriate untreated and solvent controls were also used and the study was considered to be valid.

During the described mutagenicity test and under the experimental conditions reported, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Under the conditions of this study, the test material is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Key study 2: Chromosome aberration (Höpker, 2006)

A study was performed to investigate the potential of the test material to induce structural chromosome aberrations in V79 cells of the Chinese hamster line in vitro. The study was performed in accordance with the standardised guidelines OECD 473 and Commission Directive 2000/32/EC L1362000 Annex 4A under GLP conditions.

Cultures were exposed to the test material with and without S9 mix for 4 hours, with a recovery of 14 hours and preparation interval of 18 hours. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (2000 µg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test material with respect to the OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data. Selected concentrations were: 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL without S9 mix and 31.3, 62.5, 125.0, 250.0, 500.0 and 1000.0 µg/mL with S9 mix.

In the presence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. In contrast, in the absence of S9 mix, the highest applied concentration showing clear cytotoxicity was not evaluable for cytogenetic damage.

In the absence of S9 mix, no clastogenicity was observed at the concentrations evaluated. In contrast, in the presence of S9 mix, dose-dependent, statistically significant and biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed at the two highest evaluated concentrations (17.5 and 30.0 % aberrant cells, exclusive gaps, respectively). Moreover, the dose-dependent increase in the number of cells carrying exchanges (11.5 and 19.0 %) provides additional evidence for a clastogenic potential of the test material. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test material as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Under the conditions of this study the test material induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in the presence of metabolic activation. Therefore the test material is considered to be clastogenic in this chromosome aberration test in the presence of metabolic activation.

Key study 3: Mouse lymphoma cell mutation assay (Wollny, 2006)

A study was performed to investigate the potential of the test material to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The study was conducted in accordance with the standardised guidelines OECD 476 and Commission Directive 2000/32/EC L1362000 Annex 4E under GLP conditions.

The assay was performed in three independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. Since the positive control in the absence of metabolic activation did not meet the acceptance criteria at both parallel cultures, this experimental part was repeated (experiment IA). The concentration range of experiment IA was adjusted to the toxicity observed in the first experiment. The second experiment was solely performed in the absence of metabolic activation with a treatment time of 24 hours.

Following the pre-test, the doses in Experiment I were 30, 60, 120, 160, 180 and 200 µg/mL (without S9 mix) and 31.3, 62.5, 125, 250, 500 and 1000 µg/mL (with S9 mix). In Experiment II the doses were 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL (without S9 mix).

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments at acceptable levels of toxicity.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

Under the conditions of this study the test material did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the L5178Y cell line in the presence or absence of metabolic activation. The test material is considered to be non-mutagenic.

Supporting study 1: Ames Test (Davis, 1980)

The potential of the test material to cause genotoxicity was investigated in an Ames test carried out using methodology equivalent to the standardised guideline OECD 471 using Salmonella typhimurium.

S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to the test material in duplicate assays at dose levels up to 100 µg/plate in both the presence and absence of metabolic activation. DMSO was used as solvent and concurrent solvent and positive controls were carried out. 

The highest concentration selected was well tolerated by the bacteria as indicated by a good lawn of background growth. Revertant colonies were counted.

The mean numbers of revertant colony counts obtained with the test material did not indicate an increase. 

Under the conditions of this study the test material was considered to be non-mutagenic.

Supporting study 2: Ames Test (Davis, 1991)

The potential of the test material to cause genotoxicity was investigated in an Ames test carried out using methodology equivalent to the standardised guideline OECD 471 using Salmonella typhimurium.

S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to the test material in duplicate at dose levels up to 500 µg/plate using the plate incorporation method in both the presence and absence of metabolic activation. DMSO was used as solvent and concurrent solvent and positive controls were carried out. 

The highest concentration selected was well tolerated by the bacteria as indicated by a good lawn of background growth. Revertant colonies were counted and the mean number of revertant colonies per treatment group determined. The mean number of revertant colonies for all treatment groups was compared with those obtained for negative and positive control groups. The test material was deemed to provide evidence of mutagenic potential if the increase in the number of revertant colonies was at least twice the concurrent negative control values. The effect of metabolic activation was assessed by comparing the results obtained both in the presence and absence of the liver homogenate S-9 mix for each treatment group.

The mean numbers of revertant colony counts obtained with the test material did not indicate an increase. 

Under the conditions of this study the test material was considered to be non-mutagenic.

In vivo

Micronucleus test (Honarvar, 2007)

The potential of the test material to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse was investigated in accordance with the standardised guidelines OECD 474 and Commission Directive 2000/32/EC Annex 4C under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was formulated in 30 % DMSO/ 70 % PEG 400, which was also used as vehicle control. The volume administered orally was 10 mL/kg bodyweight. 24 and 48 hours after a single administration of the test material the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test material the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test material were investigated: 24 hour preparation interval: 500, 1000 and 2000 mg/kg bodyweight; 48 hour preparation interval: 2000 mg/kg bodyweight. The highest dose (2000 mg/kg, maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.

After treatment with the test material the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test material did not exert any cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test material and with any dose level used. 40 mg/kg bodyweight cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

The test material did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse, therefore under the conditions of this study the test material is considered to be non-mutagenic.

Justification for classification or non-classification

Based on the available data, no classification of Ethyl 4-dimethylaminobenzoate is required for genetic toxicity according to the Regulation (EC) No 1272/2008.