2-{2-[(2R,4R,6R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl]ethyl}-1H-isoindole-1,3(2H)-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 January 2008 - 18 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Report includes a statement of GLP compliance and study was performed according to most recent OECD and EEC guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-{2-[(2R,4R,6R)-4,6-dihydroxyoxan-2-yl]ethyl}-2,3-dihydro-1H-isoindole-1,3-dione

EC Number:

700-188-0

Cas Number:

1333481-90-5

Molecular formula:

C15H17NO5

IUPAC Name:

2-{2-[(2R,4R,6R)-4,6-dihydroxyoxan-2-yl]ethyl}-2,3-dihydro-1H-isoindole-1,3-dione

Constituent 2

Reference substance name:

Lactol

IUPAC Name:

Lactol

Constituent 3

Reference substance name:

Phthalamido Lactol

IUPAC Name:

Phthalamido Lactol

Details on test material:

- Name of test material (as cited in study report): Lactol
- Physical state: Solid (Whte powder)
- Analytical purity: Not indicated by the sponsor; treated as 100% pure.
- Impurities (identity and concentrations): None specified.
- Purity test date: Not specified
- Lot/batch No.: PDC-010-194
- Expiration date of the lot/batch: 13 December 2008 (allocated by NOTOX, 1 year after receipt of the test substance)
- Stability under test conditions: Not indicated (stable under storage conditions)
- Storage condition of test material: At room temperature in the dark

Method

Target gene:

. gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains
. gene of tryptophan-requiring Escherichia coli bacterial strain resulting in a tryptophan-independent strain

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330, and 5000 µg/plate.
Mutation assay: At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. The highest concentration of Lactol used in the mutation assay was 5 mg/plate (based on dose range finding test). Concentrations used: 100, 333, 1000, 3330, and 5000 µg/plate.

Vehicle / solvent:

Dimethylsulphoxide (DMSO)

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activiation (-S9-mix)

Migrated to IUCLID6: Solvent : saline

Controls 3

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activiation (-S9-mix)

Migrated to IUCLID6: Solvent : Milli-Q water

Controls 4

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activiation (-S9-mix)

Migrated to IUCLID6: Solvent : DMSO

Controls 5

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activiation (-S9-mix)

Migrated to IUCLID6: Solvent : DMSO

Controls 6

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activiation (-S9-mix)

Migrated to IUCLID6: Solvent : DMSO

Controls 7

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA); Solvent : DMSO

Remarks:

With metabolic activiation (+S9-mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Incubation period: 48 hours at 37±1ºC


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (observation of bacterial background lawn and number of revertants)


OTHER EXAMINATIONS:


OTHER:

Evaluation criteria:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in
tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No formal hypothesis testing was done (refer to evaluation criteria, above).

Results and discussion

Test results

Test results 1

Species / strain:

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

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

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of Lactol on the plates was not observed at the start or at the end of the incubation period.


RANGE-FINDING/SCREENING STUDIES:
Lactol was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10,33,100,333,1000,3330 and 5000 µg/plate in the absence and presence of S9-mix.

Precipitate
Precipitation of Lactol on the plates was not observed at the start or at the end of the incubation period in both tester strains.

Toxicity
To determine the toxicity of Lactol, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of
the revertant colonies were examined.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutagenicity
In the dose range finding test, no increase in the number of revertants was observed upon treatment with Lactol under all conditions tested.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions
were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Any other information on results incl. tables

Table 1: Experiment 1: Mutagenic response of Lactol in the Salmonella typhimurium reverse

mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/ 3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
	Without S9-mix
Positive control	1043 ± 34	325 ± 36	961 ± 56	956 ± 45	1256 ± 47
Solvent control	8 ± 1	3 ± 1	13 ± 4	130 ± 3	20 ± 3
3				132 ± 12	16 ± 3
10				142 ± 4	21 ± 2
33				123 ± 19	13 ± 3
100	8 ± 2	5 ± 1	21 ± 9	123 ± 15	16 ± 3
333	12 ± 1	4 ± 0	20 ± 3	111 ± 6	16 ± 3
1000	12 ± 0	8 ± 3	23 ± 3	116 ± 2	16 ± 5
3330	14 ± 3	4 ± 1	24 ± 3	120 ± 7	18 ± 1
5000	21 ± 4	3 ± 1	25 ± 1	129 ± 18	18 ± 4
	With S9-mix^1
Positive control	279 ± 18	387 ± 61	736 ± 11	1016 ± 64	324 ± 21
Solvent control	13 ± 1	5 ± 2	22 ± 3	104 ± 14	21 ± 2
3				99 ± 12	17 ± 1
10				100 ± 10	17 ± 3
33				95 ± 5	21 ± 1
100	10 ± 4	4 ± 2	26 ± 4	103 ± 4	18 ± 1
333	10 ± 5	4 ± 1	24 ± 6	100 ± 5	17 ± 2
1000	8 ± 4	7 ± 3	23 ± 3	94 ± 2	19 ± 4
3330	8 ± 1	4 ± 2	22 ± 4	93 ± 7	17 ± 2
5000	12 ± 4	3 ± 2	24 ± 5	95 ± 16	21 ± 8

Solvent control: 0.1 mL Dimethylsulphoxide

¹The S9-mix contained 5% (v/v) S9 fraction

n.b. Dose-finding range tests for TA100 and WP₂uvrA used for first mutation assay test.

 

Table 2: Experiment 2: Mutagenic response of Lactol in the Salmonellatyphimurium reversemutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/ 3 replicate plates (± S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain
	TA1535	TA1537	TA98	TA100	WP2uvrA
	Without S9-mix
Positive control	929 ± 15	292 ± 37	1009 ± 35	1009 ± 22	996 ± 29
Solvent control	11 ± 2	8 ± 3	15 ± 2	115 ± 12	21 ± 2
100	9 ± 4	10 ± 2	15 ± 3	88 ± 4	21 ± 2
333	8 ± 3	7 ± 2	18 ± 2	90 ± 9	23 ± 2
1000	11 ± 1	7 ± 5	13 ± 2	109 ± 8	19 ± 2
3330	12 ± 2	9 ± 2	16 ± 2	97 ± 6	19 ± 7
5000	16 ± 3	6 ± 2	16 ± 2	101 ± 9	27 ± 6
	With S9-mix^1
Positive control	114 ± 8	280 ± 4	433 ± 30	626 ± 70	269 ± 25
Solvent control	5 ± 2	6 ± 3	20 ± 4	66 ± 7	20 ± 3
100	5 ± 3	4 ± 1	22 ± 3	63 ± 3	22 ± 6
333	5 ± 3	6 ± 2	25 ± 2	69 ± 3	20 ± 2
1000	6 ± 3	8 ± 2	24 ± 3	62 ± 3	20 ± 6
3330	5 ± 2	4 ± 1	18 ± 3	63 ± 2	19 ± 6
5000	9 ± 4	6 ± 3	23 ± 7	65 ± 5	15 ± 5

¹The S9-mix contained 5% (v/v) S9 fraction

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative With and without metabolic activation.

Based on the results of this study it is concluded that Lactol is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

A bacterial reverse mutation test was performed by NOTOX B.V., the Netherlands on behalf of Pfizer Ireland Pharmaceuticals, Ltd, to assess the genotoxic potential of the test substance, Lactol. Four strains of Salmonella typhimurium (TA1535, TA1537, TA98, and TA100) and one strain of Escherichia coli (WP₂uvrA) were tested against at least five concentrations of the test substance both in the presence and in the absence of metabolic activation (S9-mix). The negative and strain-specific positive control values were within the laboratory historic control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. All bacterial strains showed negative responses over the entire dose range, i.e, no significant dose-related increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that Lactol is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.