Ethyl 2-methyl-1,3-dioxolane-2-acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Endpoint Conclusion:No adverse effect observed (negative)

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:

7 July 2004 - 19 July 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: "Commission Directive 2000/32/EC, L1362000, Annex 4D", dated May 19, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: TA 100, TA102 & TA1535 are sensitive to agents inducing base pair substitution. TA 1537 & TA98 are sensitive to agents inducing frame-shift mutations.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

33; 100; 333; 1000; 2500, and 5000 µg/plate

Vehicle / solvent:

On the day of the experiment, the test item FRUCTONE was dissolved in DMSO (purity > 99 %, MERCK, D-64293 Darmstadt). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: sodium azide, 4-nitro-o-phenylene-diamine and methyl methane sulfonate. With metabolic activation: 2-aminoanthracene.

Details on test system and experimental conditions:

Test System
-Characterisation of the Salmonella typhimurium Strains
The TA strains used in this study can be described as follows:
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA 1537 his C 3076; rfa ; uvrB- frame shift mutations
TA 98 his D 3052; rfa; uvrB-; R-factor frame shift mutations
TA 1535 his G 46; rfa ; uvrB- base-pair substitutions
TA 102 his G 428; rfa; uvrB+; R-factor base-pair substitutions
TA 100 his G 46; rfa ; uvrB-; R-factor base-pair substitutions

Regular checking of the properties of the strains regarding the membrane permeability, ampicillin- and tetracycline-resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to B. Ames et al. and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 were obtained from Trinova Biochem GmbH (35394 Gieffen, Germany).

-Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.
-Precultures
From the thawed ampoules of the strains 0.5 mL bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 µL ampicillin (25 µg/mL) was added to the strains TA 98, TA 100, and TA 102. Additionally 20 µL tetracycline (2 µg/mL) was added to strain TA 102. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt
5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.
-Selective Agar
The plates with the minimal agar were obtained from E. Merck, D-64293 Darmstadt.
- Overlay Agar
The overlay agar contains per litre:
6.0 g MERCK Agar Agar*
6.0 g NaCl*
10.5 mg L-Histidine x HCI x H2O*
12.2 mg Biotin*
* (MERCK, D-64293 Darmstadt)

Sterilisations were performed at 121° C in an autoclave.

- Mammalian Microsomal Fraction S9 Mix
The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
- S9 (Preparation by RCC - CCR)
Phenobarbital/(β-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and β-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 x gravity. Aliquots of the supernatant are frozen and stored in ampoules at -80° C. Small numbers of the ampoules can be kept at -20°C for up to one week.

The protein concentration in the S9 preparation was 30.8 mg/mL (lot no. R 060204) in the pre-experiment and in experiment I, and 29.0 mg/mL (lot no. R 230404) in experiment II.

-S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCI2
33 mM KCI
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

- Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.
- Dose Selection
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.

The concentration range included two logarithmic decades. The following concentrations were tested:

33; 100; 333; 1000; 2500, and 5000 µg/plate

- Experimental Performance

For each strain and dose level, including the controls three plates were used.

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 (negative control) or reference mutagen solution (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 (cf. test system, pre-culture of the strains),
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 incubated 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 minimal agar plates.

After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

- Data Recording
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben). The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates.

Evaluation criteria:

Acceptability of the Assay
The Salmonella typhimurium 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 our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation of Results
-A test item 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 TA 102) 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 is not required.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item FRUCTONE was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, and the controls, were tested in triplicate. The test item was tested at the following concentrations:
33; 100; 333; 1000; 2500, and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FRUCTONE at any concentration 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 in induced revertant colonies.

In experiment II with metabolic activation, the number of colonies did not quite reach the lower limit of our historical control data in the solvent control of strain TA 1535. Since this deviation is rather small, this effect is judged to be based upon biological fluctuations and has no detrimental impact on the outcome of the study.

In experiment I, the historical range of positive controls was exceeded in strains TA 102 without metabolic activation and TA 1537 with metabolic activation. This effect indicates the sensitivity of the strains rather than compromising the assay.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results :
negative

The substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

The substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay. The study followed the procedures indicated by the following internationally accepted guidelines and recommendations:

 

"Ninth Addendum to OECD Guidelines for Testing of Chemicals", Section 4, No. 471: "Bacterial Reverse Mutation Test", adopted July 21, 1997.

 

"Commission Directive 2000/32/EC, L1362000, Annex 4D", dated May 19, 2000.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 June 2013 to 12 July 2013

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). The cell stocks were kept in a freezer at -80 ± 10°C. The stock was checked for mycoplasma infection. No infection of mycoplasma was noted.
Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution was used for cell detachment to subculture (cells were rinsed with 1X PBS before detachment). The laboratory cultures were maintained in 150 cm² plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium). During the treatments, the serum content of the medium was reduced to 5 (v/v) % (DMEM-5).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

0 (solvent control), 0.30, 0.91, 2.74, 8.23, 24.7, 74.1, 222.2, 666.7, 2000 µg/mL (all treatments)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (1% v/v)
- Justification for choice of solvent/vehicle: the test material is soluble in dimethyl sulfoxide (DMSO) at 500 mg/mL concentration. As this vehicle (solvent) is compatible with the test system, it was selected for vehicle (solvent) of the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

PRELIMINARY TOXICITY TEST
Two separate assays were performed. In Assay A, cells were treated for 3-hours in the presence and absence of S9-mix with a 20-hour harvesting time. In Assay B, cells were treated for 3 hours in the presence of S9-mix and for 20 hours in the absence of S9-mix with a 28-hour harvesting time.
The assays were performed with a range of test material concentrations to determine cytotoxicity. Treatment was performed as described for the main test. However, single cultures were used and positive controls were not included. Visual examination of the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed at the end of the treatment period.
At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer.

CHROMOSOME ABERRATION ASSAYS
The Chromosome Aberration Assays were conducted as two independent experiments (Assay 1 and Assay 2) in the presence and in the absence of metabolic activation.

- Treatment of the cells
1-3 day old cultures (more than 50 % confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 10^5 cells/dish concentration and incubated for approximately 24 hours at 37°C in 10 mL of culture medium (DMEM-10). Duplicate cultures were used for each test material concentration or controls. After the seeding period, the medium was replaced with 9.9 mL treatment medium (DMEM-5) in case of experiments without metabolic activation or with 9.4 mL treatment medium (DMEM-5) + 0.5 mL S9-mix in case of experiments with metabolic activation.
Cells were treated with different concentration test material solutions, negative (solvent) or positive control solution (treatment volume: 100 µL/dish in all cases) for the given period of time at 37°C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1 v/v% Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.
Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) or 28 hours (approximately 2 normal cell cycles) from the beginning of treatment.
Solubility of the test material in the final treatment medium was visually examined at the beginning and end of the treatment in each case. Measurement of pH and osmolality was also performed at the end of the treatment period in both main tests.
For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells (expressed as % relative survival) was determined using a haemocytometer. For an additional measurement of cytotoxicity, mitotic index (MI) was determined on the slides.

- Preparation of chromosomes
2-2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 µg/mL). The cells were swollen with 0.075 M KCl hypotonic solution, then were washed in fixative (methanol : acetic-acid 3 : 1 (v : v) mixture) until the preparation became plasma free (4 washes).
Then, a suspension of the fixed cells was dropped onto clean microscope slides and air-dried (at least three slides were prepared for each culture in the main tests). The slides were stained with 5 % Giemsa solution, air-dried and coverslips were mounted.

- Examination of slides
At least one hundred metaphases with 22 ± 2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. When the origin of a fragment was clear, it was recorded under that category (e.g. a dicentric chromosome with a fragment was recorded as one chromosome exchange event). When the origin of the fragment was not clear, it was recorded as a chromatid break. Metaphases with more than five aberrations (excluding gaps) were recorded as showing multiple damage.
Additionally, the number of polyploid and endoreduplicated cells was scored. Marked reductions in the numbers of cells on the slides were recorded if needed. The Vernier co-ordinates of at least five metaphases (with aberrations, where possible) were recorded for each culture. Mitotic index was also measured by counting a total of 1000 cells in interphase and mitosis for each code assessed for chromosome aberrations. For this step, two slides of a culture were used and 500 cells were evaluated from each slide.

Evaluation criteria:

The assay was considered valid, if the following criteria were met:
- The negative (solvent) control data were within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induced increases in the aberration frequency, which were significant.

The test material is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes were observed at one or more test concentrations (only data without gaps were considered).
- The increases were reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases were statistically significant.
- The increases were not associated with large changes in pH or osmolarity of the treated cultures.

The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.

The test material was concluded to have given a negative response if no reproducible, statistically significant increases were observed.

Statistics:

Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
No insolubility was observed during the preliminary experiment. Relative survival data showed no cytotoxicity of the test material with or without metabolic activation. Lower mitotic index values compared to the negative (solvent) control were observed in some test material treated samples and relative mitotic index values were variable; however, both effects did not follow a dose response. Hence there was no indication of a cytotoxic effect with or without metabolic activation.

CHROMOSOME ABERRATION ASSAYS
In Assay 1, no insolubility was detected at the end of the treatment period in the final treatment medium. There were no large changes in the pH and osmolality. There was no indication of cytotoxicity either measured by cell count (relative survival) or mitotic index. Therefore, concentrations of 2000, 666.7 and 222.2 µg/mL concentrations (a total of three) were chosen for evaluation in both experiments. None of those treatment doses caused a significant increase in the number of cells with structural chromosome aberrations with or without metabolic activation when compared with the appropriate negative (solvent) control values. This assay was considered as negative.

In Assay 2, similarly to the first experiment, no insolubility was detected at the end of the treatment period. No large changes in the pH and osmolality were observed. There was no indication of cytotoxicity either measured by cell count (relative survival) or mitotic index. Therefore, concentrations of 2000, 666.7 and 222.2 µg/mL concentrations (a total of three) were chosen for evaluation in both experiments. None of those treatment doses caused a significant increase in the number of cells with structural chromosome aberrations with or without metabolic activation when compared with the appropriate negative (solvent) control values. Therefore, this assay confirmed the negative results observed in the first main test.

Polyploid metaphases (1-3) and endoreduplicated metaphases (1-2) were found in some cases in the negative (solvent) control, positive control or treated samples in the performed experiments.

The negative (solvent) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; three test material treated concentrations were evaluated in each assay. The tests were considered to be valid.

Remarks on result:

other: all strains/cell types tested

Table 1: Summary of Results from Chromosome Aberration Assay 1

Treatment	Substance	Dose (µg/mL)	Cell no. (cells/mL)	Relative survival (%)#	Mitotic Index	Relative MI (%)#	Mean % aberrant cells~
3 hour treatment / 20 hour sampling time without metabolic activation	Negative (solvent) control		4.38^5	100	6.20	100	1.5
	Test material	2000	3.93^5	90	5.25	85	2.0
		666.7	3.93^5	90	5.60	90	3.0
		222.2	3.20^5	73	4.90	79	1.5
		74.1	3.90^5	89	5.00	81	NE
		24.7	4.53^5	103	5.90	95	NE
		8.23	4.20^5	96	5.45	88	NE
		2.74	4.55^5	104	5.35	86	NE
		0.91	4.28^5	98	4.40	71	NE
		0.30	4.50^5	103	4.00	65	NE
	Positive control (1µL/mL EMS)		3.55^5	81	5.55	90	5.5*
3 hour treatment / 20 hour sampling time with metabolic activation	Negative (solvent) control		4.18^5	100	5.80	100	2.0
	Test material	2000	3.03^5	72	5.30	91	1.5
		666.7	3.85^5	92	4.85	84	2.0
		222.2	3.98^5	95	3.40	59	1.0
		74.1	3.60^5	86	4.75	82	NE
		24.7	4.25^5	102	4.25	73	NE
		8.23	3.88^5	93	4.90	84	NE
		2.74	3.35^5	80	5.30	91	NE
		0.91	4.43^5	106	4.05	70	NE
		0.30	3.85^5	92	4.10	71	NE
	Positive control (6µg/mL CP)		2.70^5	65	3.45	59	62.5***

# compared to the 1% (v/v) DMSO solvent control

~ excluding gaps

* p < 0.05

*** p < 0.001

NE: not evaluated

Table 2: Summary of Results from Chromosome Aberration Assay 2

Treatment	Substance	Dose (µg/mL)	Cell no. (cells/mL)	Relative survival (%)#	Mitotic Index	Relative MI (%)#	Mean % aberrant cells~
20 hour treatment / 28 hour sampling time without metabolic activation	Negative (solvent) control		8.63^5	100	6.55	100	0.0
	Test material	2000	7.25^5	84	4.90	75	0.5
		666.7	8.25^5	96	4.80	73	0.0
		222.2	9.68^5	112	5.90	90	1.5
		74.1	9.33^5	108	5.15	79	NE
		24.7	9.63^5	112	5.25	80	NE
		8.23	8.90^5	103	5.90	90	NE
		2.74	8.93^5	103	6.20	95	NE
		0.91	9.30^5	108	6.75	103	NE
		0.30	9.53^5	110	6.65	102	NE
	Positive control (0.4µL/mL EMS)		5.68^5	66	3.95	60	29.7***
3 hour treatment / 28 hour sampling time with metabolic activation	Negative (solvent) control		8.40^5	100	6.75	100	2.0
	Test material	2000	7.75^5	92	4.90	73	3.0
		666.7	8.90^5	106	4.85	72	2.5
		222.2	9.00^5	107	5.35	79	0.0
		74.1	8.73^5	104	5.65	84	NE
		24.7	9.13^5	109	6.20	92	NE
		8.23	8.93^5	106	5.95	88	NE
		2.74	9.25^5	110	5.45	81	NE
		0.91	7.88^5	94	4.90	73	NE
		0.30	8.08^5	96	6.45	96	NE
	Positive control (6µg/mL CP)		5.55^5	66	6.85	101	27.5***

# compared to the 1% (v/v) DMSO solvent control

~ excluding gaps

*** p < 0.001

NE: not evaluated

Table 3: Summary of Polyploid abd Endoreduplicated Cells in Assays 1 and 2

Concentration (µg/mL)	Treatment/Harvest Time (hours)	No. of Cells observed
		Polyploid	Endoreduplicated
Assay 1 without metabolic activation (-S9)
Solvent control	3/20	0	0
222.2 µg/mL	3/20	0	0
666.7 µg/mL	3/20	0	0
2000 µg/mL	3/20	0	0
Positive control (400)	3/20	1	1
Assay 1 with metabolic activation (+S9)
Solvent control	3/20	0	0
222.2 µg/mL	3/20	0	0
666.7 µg/mL	3/20	0	2
2000 µg/mL	3/20	0	0
Positive control (48)	3/20	0	1
Assay 2 without metabolic activation (-S9)
Solvent control	20/28	3	0
222.2 µg/mL	20/28	2	0
666.7 µg/mL	20/28	1	0
2000 µg/mL	20/28	1	0
Positive control (101)	20/28	0	0
Assay 2 with metabolic activation (+S9)
Solvent control	3/28	0	0
222.2 µg/mL	3/28	0	0
666.7 µg/mL	3/28	0	0
2000 µg/mL	3/28	0	0
Positive control (111)	3/28	0	0

The number of polyploidy and endoreduplicated calls were determined in 200 cells of each test group except as shown in brackets for the controls.

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Under the conditions of the study, the test material did not induce any statistically and biologically significant, repeatable, dose-dependent increase in the frequency of chromosome aberrations either with or without metabolic activation. The test material is therefore considered not clastogenic in this test system.

Executive summary:

The clastogenic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 473, EU Method B.10 and EPA OPPTS 870.5375. The study consisted of two independent chromosome aberration assays.

In the first chromosome aberration assay (3-hour treatment with and without metabolic activation with sampling performed 20 hours after the beginning of the treatment in both cases) the examined concentrations of test material were 2000, 666.7, 222.2, 74.1, 24.7, 8.23, 2.74, 0.91 and 0.30 µg/mL. No insolubility was detected at the end of the treatment period in the final treatment medium. There were no large changes in the pH and osmolality. There was no indication of cytotoxicity either measured by cell count (relative survival) or mitotic index. Therefore, concentrations of 2000, 666.7 and 222.2 µg/mL were chosen for evaluation in both experiments. None of the treatment doses evaluated caused a significant increase in the number of cells with structural chromosome aberrations with or without metabolic activation when compared with the appropriate negative (solvent) control values. This assay was considered as negative.

In the second chromosome aberration assay (3-hour treatment with metabolic activation and a 20-hour treatment without metabolic activation with sampling performed 28 hours after the beginning of the treatment in both cases) the examined concentrations of test material were 2000, 666.7, 222.2, 74.1, 24.7, 8.23, 2.74, 0.91 and 0.30 µg/mL. As in the first experiment, no insolubility was detected at the end of the treatment period. No large changes in the pH and osmolality were observed. There was no indication of cytotoxicity either measured by cell count (relative survival) or mitotic index. Therefore, concentrations of 2000, 666.7 and 222.2 µg/mL concentrations were chosen for evaluation. None of the treatment doses evaluated caused a significant increase in the number of cells with structural chromosome aberrations with or without metabolic activation when compared with the appropriate negative (solvent) control values. Therefore, this assay confirmed the negative results observed in the first main test.

The negative (solvent) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; three test material treated concentrations were evaluated in each assay. The tests were considered to be valid.

In conclusion, under the conditions of the study, the test material did not induce a significant level of chromosome aberrations either with or without metabolic activation. The test material is therefore considered not clastogenic in this test system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 May 2013 to 3 June 2013

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

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

tk locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI-10 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

ASSAY 1
- 3 hour treatment with metabolic activation
31.25, 62.5, 125, 250, 500, 1000, 1250, 1500, 1750, 2000 µg/mL

- 3 hour treatment without metabolic activation
15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000 µg/mL

ASSAY 2
- 3 hour treatment with metabolic activation
31.25, 62.5, 125, 250, 500, 1000, 1250, 1500, 1750, 2000 µg/mL

- 24 hour treatment without metabolic activation
15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: results from a short solubility test revealed that DMSO was a suitable vehicle for use in the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

PRELIMINARY TOXICITY TEST
A 3 hour treatment in the presence and absence of S9-mix, and a 24 hour treatment in the absence of S9-mix, were performed at a range of test material concentrations to determine toxicity. The procedures were performed as described for the main test; however, the test used single cultures and positive controls were not included. Following treatments, cell concentrations were determined using a haemocytometer. Visual examinations for insolubility in the final culture medium were conducted at the beginning and end of treatment. Measurements of pH and osmolality were performed at the end of the treatment period. To check for late phase cytotoxicity, cells were transferred for the expression period for two additional days and repeat cell counting was performed.

MAIN MUTATION ASSAYS
In Assay 1, cells were treated for 3 hours in the presence and absence of S9-mix. In Assay 2, cells were treated for 3 hours in the presence of S9-mix and for 24 hours in the absence of S9-mix. Duplicate cultures were used for each treatment.

METHOD OF APPLICATION: in medium
0.2 mL of RPMI-5 medium, solvent, test material formulation or positive control solution, and 0.1 mL of S9-mix (in experiments with metabolic activation) or of 150 mM KCl (in case of 3 hour treatment without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3 hour treatments, at least 10^7 cells were plated in each series of 75 cm² sterile flasks. For the 24 hour treatment, at least 4 x 10^6 cells were plated in each of a series of 25 cm² sterile flasks. The treatment medium contained a reduced serum level of 5% v/v RPMI-5.
During the treatment period the cultures were incubated at 37 ± 1 °C (approximately 5% CO2 in air). Gentle shaking was used during the 3 hour treatments. After the treatment period the cultures were centrifuged at 2000 rpm for 5 minutes, washed with tissue culture medium and suspended in 20 mL RPMI-10. The number of viable cells was counted manually using a haemocytometer. Measurement of pH and osmolality was also performed after the treatment period. Where sufficient cells survived, cell density was adjusted to a concentration of 2 x 10^5 cells/mL. Cells were transferred to flasks for growth through the expression period or diluted to be plated for survival.

PLATING FOR SURVIVAL
Cultures of cell density 2 x 10^5 cells/mL, were further diluted to 8 cells/mL and 0.2 mL of the final concentration of each culture were placed into each well of two, 96-well microplates averaging 1.6 cells per well. Microplates were incubated at 37 ± 0.5 °C containing approximately 5% v/v CO2 in air for about 2 weeks. Wells containing viable clones were identified by eye using background illumination and counted.

EXPRESSION PERIOD
Cultures were maintained in flasks for approximately 3 days. During the expression period, subculturing was performed daily. On each day, cell density was adjusted to a concentration of 2 x 10^5 cells/mL and transferred to flasks for further growth. On completion of the expression period, at least eight concentrations, untreated controls and positive controls were plated for determination of viability and 5-trifluorothymidine (TFT) resistance.

PLATING FOR VIABILITY
At the end of the expression period the cell density in the selected cultures was adjusted to 1 x 10^4 cells/mL with RPMI-20. Samples from these cultures were diluted to 8 cells/mL and 0.2 mL of the final concentration of each culture was placed into each well of two 96-well microplates averaging 1.6 cells per well. Microplates were incubated at 37 ± 0.5 °C containing approximately 5% v/v CO2 in air for approximately 2 weeks. Wells containing viable clones were identified by eye using background illumination and counted.

PLATING FOR TFT RESISTANCE
At the end of the expression period, the cell concentration was adjusted to 1 x 10^4 cells/mL. TFT (300 µg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 µg/mL. 0.2 mL of each suspension was placed into each well of four 96-well microplates at 2 x 10^3 cells per well. Microplates were incubated at 37 ± 0.5 °C containing approximately 5% v/v CO2 in air for approximately 2 weeks and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed.

Evaluation criteria:

ASSAY ACCEPTANCE CRITERIA
The assay was considered valid if all of the following criteria were met:
- The mutant frequency in the negative control cultures fell within the normal range (50-170 mutants per 10^6 viable cells)
- The positive control chemicals induced a statistically significant increase in mutant frequency
- The plating efficiency of the negative controls was within the range of 65-120 % at the end of the expression period
- At least four test concentrations were present, where the highest concentration produced approximately 80-90 % toxicity, resulted in precipitation, or was at the highest (practical) concentration.

EVALUATION CRITERIA
The test material was considered to be mutagenic if all the following criteria were met:
- The assay was valid
- Statistically significant and biologically relevant increases in mutation frequency were observed in treated cultures compared to the corresponding negative control values at one or more concentrations
- The increases in mutation frequency were reproducible between replicate cultures and/or between tests
- There was a significant dose-response relationship
- The mutation frequency at the test concentrations showing the largest increase was at least 126 mutants per 10^6 viable cells higher than the corresponding negative control value (GEF value)

In this case the negative control = the solvent (vehicle) control.

Statistics:

Statistical significance of mutant frequencies was performed using Microsoft Excel software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELMINARY EXPERIMENT
No insolubility or cytotoxicity was observed in the preliminary experiment. Therefore, the highest selected concentration was the maximum concentration in each case. Lower test concentrations were separated by factor of two, but more closely spaced concentrations were selected in the experiments with metabolic activation due to the large pH decreases observed during treatment.

MUTATION ASSAYS
- Assay 1
No insolubility was detected in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation.

In the presence of S9-mix (3-hour treatment), no cytotoxicity of the test material was observed. There were no large changes in osmolality after treatment, but a larger than usual change was observed in pH (> 0.5) at 2000 and 1750 μg/mL concentrations. An evaluation was made using data of the eight highest concentrations (2000-125 μg/mL). No biologically relevant or statistically significant increase in the mutation frequency was observed at any of the evaluated concentrations. Slight dose-response to the treatment was indicated by the linear trend analysis, but based on the individual mutation frequency values (all of the observed mutation frequency values were within the historical control range, and the difference between the individual values and the corresponding negative (solvent) control value did not exceed the global evaluation factor of 126 mutants per 10^6 viable cells in any of those cases); this fact had no biological relevance.

In the absence of S9-mix (3-hour treatment), no cytotoxicity of the test material was observed. There were no large changes in pH or osmolality after treatment. An evaluation was made using data of the eight examined concentrations (2000-15.63 μg/mL). No biologically relevant or statistically significant increase in the mutation frequency was observed at any of the evaluated concentrations. No dose response to the treatment was indicated by the linear trend analysis.

- Assay 2
No insolubility was detected in the final treatment medium at the end of the treatment in the experiments with and without metabolic activation.

In the presence of S9-mix, no cytotoxicity of the test material was observed. There were no large changes in osmolality after treatment, but similarly to Assay 1, a larger than usual change in pH (> 0.5) was observed at 2000 and 1750 µg/mL concentrations. An evaluation was made using data of the eight highest concentrations (200 – 125 µg/mL).
Statistically significant increases in the mutation frequency were observed at the 2000 and 1750 µg/mL concentrations. However, the differences between the mutation frequencies of the test material treated samples and the corresponding solvent control value did not exceed the GEF value. Therefore, they were considered as biologically non relevant increases. Dose response to the treatment was indicated by the linear trend analysis, but based on the individual mutation frequency values; this fact had no biological relevance.

In the absence of S9-mix, no cytotoxicity was observed. An evaluation was made using data of the eight highest concentrations. No biologically relevant or statistically significant increase in the mutation frequency observed at any of the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.

The observed increases in the mutation frequency were examined for consistency throughout the study. The statistically significant increases observed at some concentrations in Assay 2 in the experiment with metabolic activation were biologically non-relevant, not repeatable and associated with large pH changes. Therefore, they were considered as not being above the limit of biological relevance (the mutation frequency at the test concentration showing the largest increase was less than 126 mutants per 10^6 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (solvent) control value) thus no mutagenic activity of the test material was observed in the performed experiments.

Remarks on result:

other: all strains/cell types tested

Table 1: Summary of Mutagenicity Results - Assay 1

Metabolic activation	Treatment period	Treatment (µg/mL)			No of empty wells out of the total no of wells (768)	No of large colonies out of the total no of wells (768)	No of small colonies out of the total no of wells (768)	Mutation frequency
with	3 hours	2000			646	67	55	113.1
		1750			655	63	50	107.9
		1500			665	46	57	101.2
		1250			658	44	66	96.8
		1000			667	55	46	87.6
		500			692	39	37	77.5
		250			689	44	35	73.6
		125			685	47	36	79.2
		62.5			673	48	47	88.9
		31.25			680	45	43	92.5
			DMSO		673	52	43	95.5
			Untreated		685	41	42	75.3
		CP: 4 µg/mL			210	268	290	1389.4*
without	3 hours	2000			697	36	35	67.2
		1000			706	23	39	57.9
		500			695	30	43	76.4
		250			699	38	31	66.6
		125			702	18	48	63.6
		62.5			696	30	42	73.7
		31.25			696	20	52	73.7
		15.63			702	24	42	63.1
		DMSO			701	30	37	66.0
		Untreated			698	36	34	64.3
		NQO: 0.15 µg/mL			345	107	316	966.5*

* Statistically significant

Table 2: Summary of Mutagenicity Results - Assay 2

Metabolic activation	Treatment period	Treatment (µg/mL)	No of empty wells out of the total no of wells (768)	No of large colonies out of the total no of wells (768)	No of small colonies out of the total no of wells (768)	Mutation frequency
with	3 hours	2000	317	74	77	154.9*
		1750	605	89	74	152.7*
		1500	651	71	46	113.7
		1250	642	68	58	109.8
		1000	665	66	37	116.6
		500	674	55	39	95.1
		250	677	53	38	91.8
		125	693	46	29	67.2
		62.5	683	52	33	86.6
		31.25	694	52	22	72.7
		DMSO	683	50	35	74.0
		Untreated	698	45	25	64.8
		CP: 4 µg/mL	200	220	348	1311.5*
without	24 hours	2000	389	44	35	71.0
		1000	670	56	42	98.0
		500	683	56	29	92.3
		250	686	55	27	80.5
		125	695	45	28	77.5
		62.5	689	50	29	82.5
		31.25	678	57	33	85.7
		15.63	684	59	25	79.1
		DMSO	680	56	32	84.3
		Untreated	667	65	36	104.1
		NQO: 0.15 µg/mL	241	344	183	1179.9*

* Statistically significant

Conclusions:

Interpretation of results:
negative with and without metabolic activation

No genotoxic effect of the test material was observed either with or without metabolic activation under the conditions of this assay.

Executive summary:

The genotoxicity of the test material was investigated in an in vitro mouse lymphoma assay which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 476 and EU Method B.17. Treatment was performed for 3 hours with and without metabolic activation and for 24 hours without metabolic activation.

No insolubility of the test material was observed in the final treatment mediums at the end of the treatment. There were no large changes in osmolality after treatment but a larger than normal change in pH was observed in treatments with metabolic activation. In each case an evaluation was made using data of the eight highest concentrations in each assay. In each case no biologically relevant or statistically significant increase in the mutation frequency was observed at any of the evaluated concentrations.

Therefore, under the conditions of the study the test material was not considered to be genotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In Vitro Data

Bacterial reverse mutation

The potential of the test material to cause gene mutation in bacterial strains was determined in two separate GLP-compliant studies. The study was conducted in accordance with standardised guidelines OECD 471 and “Commission Directive 2000/32/EC, L1362000, Annex 4D", dated May 19, 2000.

 

In the study five strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were treated in the presence and absence of at rat liver derived metabolic activation system (S9 mix). In the study the test material was determined to be non-mutagenic under the conditions tested.

 

Chromosome aberration

The clastogenic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 473, EU Method B.10 and EPA OPPTS 870.5375. The study consisted of two independent chromosome aberration assays.

In the first chromosome aberration assay (3-hour treatment with and without metabolic activation with sampling performed 20 hours after the beginning of the treatment in both cases) the examined concentrations of test material were 2000, 666.7, 222.2, 74.1, 24.7, 8.23, 2.74, 0.91 and 0.30 µg/mL. No insolubility was detected at the end of the treatment period in the final treatment medium. There were no large changes in the pH and osmolality. There was no indication of cytotoxicity either measured by cell count (relative survival) or mitotic index. Therefore, concentrations of 2000, 666.7 and 222.2 µg/mL were chosen for evaluation in both experiments. None of the treatment doses evaluated caused a significant increase in the number of cells with structural chromosome aberrations with or without metabolic activation when compared with the appropriate negative (solvent) control values. This assay was considered as negative.

In the second chromosome aberration assay (3-hour treatment with metabolic activation and a 20-hour treatment without metabolic activation with sampling performed 28 hours after the beginning of the treatment in both cases) the examined concentrations of test material were 2000, 666.7, 222.2, 74.1, 24.7, 8.23, 2.74, 0.91 and 0.30 µg/mL. As in the first experiment, no insolubility was detected at the end of the treatment period. No large changes in the pH and osmolality were observed. There was no indication of cytotoxicity either measured by cell count (relative survival) or mitotic index. Therefore, concentrations of 2000, 666.7 and 222.2 µg/mL concentrations were chosen for evaluation. None of the treatment doses evaluated caused a significant increase in the number of cells with structural chromosome aberrations with or without metabolic activation when compared with the appropriate negative (solvent) control values. Therefore, this assay confirmed the negative results observed in the first main test.

The negative (solvent) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; three test material treated concentrations were evaluated in each assay. The tests were considered to be valid.

In conclusion, under the conditions of the study, the test material did not induce a significant level of chromosome aberrations either with or without metabolic activation. The test material is therefore considered not clastogenic in this test system.

 

In vitro gene mutation in mammalian cells

The genotoxicity of the test material was investigated in an in vitro mouse lymphoma assay which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 476 and EU Method B.17. Treatment was performed for 3 hours with and without metabolic activation and for 24 hours without metabolic activation.

No insolubility of the test material was observed in the final treatment mediums at the end of the treatment. There were no large changes in osmolality after treatment but a larger than normal change in pH was observed in treatments with metabolic activation. In each case an evaluation was made using data of the eight highest concentrations in each assay. In each case no biologically relevant or statistically significant increase in the mutation frequency was observed at any of the evaluated concentrations.

Therefore, under the conditions of the study the test material was not considered to be genotoxic.

 

All three studies were performed under GLP conditions and in line with accepted test guidelines, accordingly the studies were assigned a reliability score of 1 in line with the principles for assessing data quality defined by Klimisch (1997).

Justification for selection of genetic toxicity endpoint

Three studies have been selected as key in order to address the 3 different genetic toxicity endpoints under REACH. All three studies have been conducted in GLP certified laboratories, in line with standardised guidelines and are considered sufficiently reliable in accordance with Klimisch (1997) to be key studies.

Short description of key information:

IN VITRO DATA

Reverse mutation in bacteria: Negative (S. typhimurium strains TA 1537, TA 1535, TA 100, TA98 and TA 102 in the presence and absence of metabolic activation) OECD 471, Sokolowski (2004)

In vitro chromosome aberration: Negative (Chinese hamster V79 lung cells in the presence and absence of metabolic activation) OECD 473, EU Method B.10, EPA OPPTS 870.5375, Hargitai (2013)

Gene mutation in mammalian cells: Negative (mouse lymphoma L5178Y cells in the presence and absence of metabolic activation) OECD 476, EU Method B.17, Hargitai (2013)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for genetic toxicity based on the overall negative response noted in the available genetic toxicity studies.