6,8-Dioxabicyclo[3.2.1]octan-4-one, (1S,5R)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 May - 12 Jun 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

yes

Remarks:

Only 2-Aminoanthracene was used as positive control in the presence of S9 mix.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

Only 2-Aminoanthracene was used as positive control in the presence of S9 mix.

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1S,5R)-6,8-dioxabicyclo[3.2.1]octan-4-one

EC Number:

807-130-4

Cas Number:

53716-82-8

Molecular formula:

C6H8O3

IUPAC Name:

(1S,5R)-6,8-dioxabicyclo[3.2.1]octan-4-one

Details on test material:

- Name of test material: Cyrene™- Analytical purity: 99%- Expiration date of the lot/batch: 30 Sep 2015

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

nitroreductase deficient

Remarks:

for uvrB- strains TA 1537, TA 98, TA 1535, TA 100

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from phenobarbital/β-naphtoflavone induced rat liver

Test concentrations with justification for top dose:

1st experiment: 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate with and without metabolic activation.
2nd experiment: 33, 100, 333, 1000, 2500, and 5000 μg/plate with and without metabolic activation.

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)

Remarks:

+S9: 2-aminoanthracene (2-AA; 2.5 or 10 µg/plate, in DMSO, all strains); -S9: 4-NOPD (10 or 50 µg/plate, in DMSO, TA1537/TA98), sodium azide (NaN3, 10 µg/plate, in water, TA1535/TA100); methylmethanesulfonate (MMS, 2 µg/plate, in water, TA102)

Details on test system and experimental conditions:

Experiment I: in agar (plate incorporation)
Experiment II: pre-incubation
DURATION Experiment I - Exposure duration: 48 h
DURATION Experiment II - Preincubation period: 60 min
NUMBER OF REPLICATIONS: each concentration was tested in triplicates in two independent experiments both with and without S9 mix

DETERMINATION OF CYTOTOXICITY:
Method: inspection of the bacterial background growth and reduction in the number of revertant colonies

Evaluation criteria:

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.

Results and discussion

Test results

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:

TEST-SPECIFIC CONFOUNDING FACTORS:
Water solubility: The test item is dissolvable in water.
Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
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 (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
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 5000 μg/plate were chosen as maximal concentration.

Any other information on results incl. tables

Pre-Experiment for Toxicity

To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. 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 (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.

Table 1: Test Results of Experiment 1

EXPERIMENT 1
S9-Mix	Without
Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	TA 102
Verhicle control	17.7	10.7	24.7	175.0	443.3
Untreated control	2.0	1.2	8.7	166.7	468.7
Cyrene™ (3)	18.3	12.7	25.0	168.3	453.0
Cyrene™ (10)	18.0	11.7	27.3	183.7	447.0
Cyrene™ (33)	17.7	9.3	28.0	183.0	435.7
Cyrene™ (100)	19.7	9.0	23.0	174.7	440.7
Cyrene™ (333)	15.7	9.0	27.7	175.0	455.0
Cyrene™ (1000)	17.7	9.7	22.7	172.0	424.7
Cyrene™ (2500)	15.0	11.0	24.0	188.0	467.3
Cyrene™ (5000)	14.3	13.7	30.3	178.0	465.0
NaN3 (10)	2940.3			1906.7
4-NOPD (50)		58.3
4-NOPD (10)			273.7
MMS (2.0 µL/plate)					5586.7
S9-Mix	With
Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	TA 102
Vehicle control	16.7	24.0	38.3	177.0	595.3
Untreated control	15.3	20.0	36.7	170.0	618.3
Cyrene™ (3)	14.7	21.3	42.7	181.0	573.3
Cyrene™ (10)	15.3	24.0	41.3	184.7	587.7
Cyrene™ (33)	13.3	20.3	38.7	187.7	651.0
Cyrene™ (100)	12.0	20.0	34.3	185.3	690.7
Cyrene™ (333)	14.7	19.7	41.3	179.7	603.0
Cyrene™ (1000)	13.7	20.3	46.3	177.0	595.7
Cyrene™ (2500)	15.3	20.7	35.7	174.3	589.3
Cyrene™ (5000)	17.0	15.0	35.7	167.3	645.7
2-AA (2.5)	548.3	376.0	3912.0	3957.7
2-AA (10.0)					1384.7
2 -AA: 2 -aminoanthracene; 4 -NOPD: 4 -nitro-o-phenylene-diamine; NaN3: sodium azide; MMS: methylmethanesulfonate

Table 2: Test Results of Experiment 2

EXPERIMENT 2
S9-Mix	Without
Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	TA 102
Vehicle control	12.0	11.3	26.7	152.0	362.3
Untreated control	9.7	7.7	31.0	161.3	407.7
Cyrene™ (33)	12.7	11.0	25.3	159.7	366.3
Cyrene™ (100)	12.3	12.7	29.3	164.0	394.0
Cyrene™ (333)	13.3	9.0	29.7	165.3	351.7
Cyrene™ (1000)	17.3	8.7	29.7	165.3	391.7
Cyrene™ (2500)	13.0	12.7	33.3	163.0	395.7
Cyrene™ (5000)	14.0	11.3	29.7	152.3	425.3
NaN3 (10)	2758.7			1706.3
4-NOPD (50)		53.3
4-NOPD (10)			242.0
MMS (2.0 µL/plate)					1776.7
S9-Mix	With
Test item (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	TA 102
Vehicle control	11.0	16.0	43.0	200.0	526.7
Untreated control	15.3	11.7	42.7	160.7	534.3
Cyrene™ (33)	14.7	14.0	44.3	158.7	550.3
Cyrene™ (100)	11.7	19.7	42.7	156.3	447.3
Cyrene™ (333)	10.7	20.0	41.3	178.7	577.0
Cyrene™ (1000)	10.0	16.0	35.0	180.3	530.7
Cyrene™ (2500)	13.0	15.0	41.3	176.3	517.3
Cyrene™ (5000)	14.7	12.7	43.7	188.0	593.3
2-AA (2.5)	505.3	131.0	3724.3	2360.7
2-AA (10.0)					5146.7
2 -AA: 2 -aminoanthracene; 4 -NOPD: 4 -nitro-o-phenylene-diamine; NaN3: sodium azide; MMS: methylmethanesulfonate

Applicant's summary and conclusion

Conclusions:

Cyrene™ has been tested in a valid bacterial reverse mutation assay, conducted according to OECD Test Guideline 471 (1997) and in compliance with GLP, using Salmonella typhimurium strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to limit concentration. Appropriate positive, negative and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.