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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
August 17th to September 8th, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: International Conference on Harmonisation (ICH): Genotoxicity S2A and S2B
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl 3-[4-(hydroxymethyl)-2-methyl-1,3-dioxolan-2-yl]propanoate
EC Number:
700-637-0
Cas Number:
902272-78-0
Molecular formula:
C10H18O5
IUPAC Name:
Ethyl 3-[4-(hydroxymethyl)-2-methyl-1,3-dioxolan-2-yl]propanoate

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9.
Test concentrations with justification for top dose:
Initial toxicity-mutation assay (Experiment B1): with and without metabolic activation - 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Retest of the initial toxicity-mutation assay (Experiment B1): with and without metabolic activation - 50, 150, 500, 1500 and 5000 μg/plate
Confirmatory mutation assay (Experiment B3): 50, 150, 500, 1500 and 5000 µg/plate.
The test substance formed a soluble and clear solution in water at a concentration of approximately 50 mg/ml, the maximum concentration tested in the solubility test. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility of the test substance and compatibility with the target cells
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene for all the strains at 1.0 (TA98, TA1535, TA1537), 2.0 (TA100) and 10 (WP2) μg/plate
Remarks:
with metabolic activation
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Remarks:
without metabolic activation
Details on test system and experimental conditions:
ANALYSIS OF DOSING FORMULATIONS: triplicate dosing formulation samples (1 ml from the most concentrated dose level and vehicle) were collected from each assay. Two sets of samples were analysed.

STERILITY OF TEST SUBSTANCE
To confirm the sterility of the test substance, the highest test substance dose levels used in the initial toxicity-mutation and confirmatory mutagenicity assays were plated on selective agar with an aliquot volume equal to that used in the assay. These plates were incubated under the same conditions as the assay.

TEST SYSTEM: overnight cultures were prepared by inoculating from the appropriate master plate, appropriate frozen permanent stock or with a lyophilized pellet into a vessel, containing ~30 to 50 ml of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at approximately 125 to 175 rpm at 37 ± 2 °C approximately 12 to 14 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x10^9 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

METABOLIC ACTIVATION SYSTEM
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 was prepared by and purchased from Moltox (Boone, NC). Upon arrival at the laboratory, the S9 was stored at -60 °C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize at least two promutagens to forms mutagenic to Salmonella typhimurium TA100.
The S9 mix was prepared immediately before its use and contained 10 % S9, 5 mM glucose-6-phosphate, 4 mM ß-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the S9 and Sham mixes, a 0.5 ml aliquot of each was plated on selective agar.

MINIMAL TOP AGAR
On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with 25 ml of water for each 100 ml of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water.

BOTTOM AGAR
Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).

INITIAL TOXICITY MUTATION ASSAY
Vehicle control, positive controls and a minimum of eight dose levels of the test substance were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. In the retest a minimum of five dose levels of test substance were used based on precipitation and toxicity profiles observed.

CONFIRMATORY MUTAGENICITY ASSAY
- Method of application: in agar (plate incorporation).
- A minimum of five dose levels of test substance along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test substance, vehicle control and positive controls were plated in triplicate.
- One-half (0.5) milliliter of S9 or Sham mix, 100 μl of tester strain (cells seeded) and 250 μl of vehicle or test substance dilution were added to 2.0 ml of molten selective top agar at 45 ± 2 °C. After vortexing, the mixture was overlaid onto the surface of 25 ml of minimal bottom agar. When plating the positive controls, the test substance aliquot was replaced by a 50 μl aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37 ± 2 °C. Plates that were not counted immediately following the incubation period were stored at 2 - 8 °C until colony counting could be conducted.
- Scoring Procedures: the condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. Precipitate was evaluated after the incubation period by visual examination without magnification.
Evaluation criteria:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance.
Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Sterility test
One contaminant colony was observed on the post-assay sterility plate for the vehicle control in Experiment B2. No contaminant colonies were observed on the remaining sterility plates for the vehicle control, the test substance dilutions and the S9 and Sham mixes.

Initial Toxicity Mutation assay results
No positive mutagenic responses were observed with any of the tester strains in the absence of S9 activation and with the Salmonella strains in the presence of S9 activation. Neither precipitate nor appreciable toxicity was observed. Due to an unacceptable (low) positive control value, tester strain WP2 uvrA in the presence of S9 was not evaluated but was retested based on the toxicity and precipitation profiles observed. In Experiment B2 (retest of the initial toxicity-mutation assay), the dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor appreciable toxicity was observed.

Confirmatory Mutagenicity Assay
Neither precipitate nor appreciable toxicity was observed.

Dosing Formulation analysis
Concentration analysis indicates that the actual mean concentrations of the analyzed dose level (nominally 20 mg/ml) were 84 and 97 % of target. Although the actual concentration of the high dose in Experiment B1 was lower than expected, the critical top dose level in Experiment B2 was within 85 to 115 % of target. This indicates that the regulatory-required top dose level was achieved in the confirmatory assay. The observed deviation from nominal concentration had minimal adverse impact on the assessment of the test substance’s mutagenicity and the results support the validity of the study conclusion. Since the most concentrated dosing formulation in Experiment B2 was within 85 to 115 % of target, the dosing formulations were considered stable for the purpose of this study.

Applicant's summary and conclusion

Conclusions:
negative with and without metabolic activation
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and ICH genotoxicity Guideline S2A and S2B. Strains TA98, TA100, TA1535 and TA1537 of S.typhimurium and strain WP2uvrA of E.coli in the presence and absence of S9 were exposed to the test substance. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. In the initial toxicity-mutation assay, the dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate. In the retest of the initial toxicity-mutation assay, the dose levels tested were 50, 150, 500, 1500 and 5000 μg/plate. No positive mutagenic responses were observed with any of the tester strains in the absence and presence of S9. Neither precipitate nor appreciable toxicity was observed.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed. The dose levels tested were 50, 150, 500, 1500 and 5000 μg/plate. Neither precipitate nor appreciable toxicity was observed.

 

Under the test conditions, the test substance was negative in the bacterial reverse mutation assay with and without metabolic activation.