1,4-Butanediyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

December 14th, 2010 to January 11th, 2011

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH): S2A,1996 and S2B,1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-{[3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoyl]oxy}butyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate

EC Number:

700-652-2

Cas Number:

1259300-69-0

Molecular formula:

C20H34O8

IUPAC Name:

4-{[3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoyl]oxy}butyl 3-(2,4-dimethyl-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:

S9-mix

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.
Confirmatory mutation assay (Experiment B2): 50, 150, 500, 1500 and 5000 µg/plate
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: DMSO
- Justification for choice of solvent/vehicle: soluble in DMSO and compatibility with target cells.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene for all the strains

Remarks:

with metabolic activation

Controls 2

Untreated negative controls:

yes

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:

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 (Vogel and Bonner, 1956) 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.

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 50 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.

DETERMINATION OF CYTOTOXICITY:
Method: A reduction in the number of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators.

Evaluation criteria:

A dose level is considered toxic if one or both of the following criteria are met:
(a) >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value.
(b) At least a moderate reduction in the background lawn If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility results: the test substance formed a soluble and clear solution in DMSO at a concentration of approximately 500 mg/ml, the maximum concentration tested in the solubility test.
- Sterility results: no contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions and the S9 and Sham mixes.
- Neither precipitate nor appreciable toxicity was observed

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

INITIAL TOXICITY MUTATION ASSAY RESULTS
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Neither precipitate nor appreciable toxicity was observed.

CONFIRMATORY MUTAGENICITY ASSAY RESULTS
In Experiment B2 (Confirmatory Mutagenicity Assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Under the conditions of this study, the test item did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

DOSING FORMULATION ANALYSIS
Concentration analysis indicates that the actual mean concentrations of the analyzed dose level (nominally 100 mg/ml) were 102 and 99 % of target with < 5% RSD. This indicates that the regulatory-required top dose level was achieved in each case and the results support the validity of the study conclusion. No test substance was detected in the vehicle control samples.
Stability data were not provided for the neat test substance. In the absence of stability data, it was concluded that the test substance, as supplied, was negative with the caveat that the maximum required concentration may not have been achieved due to instability of the test substance. Also, the stability of the test substance in the solvent was not determined. In the absence of formal formulation stability data, it was concluded that there was no adverse impact since the analyzed formulations were within protocol specification.

Any other information on results incl. tables

Tester strain titer results

Experiment	Tester Strains
	TA 98	TA100	TA1535	TA1537	E.coli WP2 uvrA
	Titer value(X 10^9cells/ml)
B1	1.6	1.1	1.0	0.7	2.6
B2	1.3	0.5	0.9	0.8	2.4

Validity of the test: all criteria for a valid study were met as described in the protocol.

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, ICH genotoxicity Guideline 1996 and S2B1997 in compliance with GLP. Tester strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and strain WP2uvrA of Escherichia 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 in the initial experiment. 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 found to be non-mutagenic in the bacterial reverse mutation assay.