Pentanedioic acid, compd. with (1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

01 April 1998 and 28 June 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The UK guidelines for the testing of chemicals for mutagenicity. Report of Health and Social Subjects, 35, UK Department of Health 1989.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The UKEMS Sub-Committee on Guidelines for Mutagenicity Testing Report Part 1 revised, Basic Mutagenicity Tests: UKEMS recommended procedures, Ed DJ Kirkland 1990, Cambridge University Press.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Guidelines for Testing Drugs for Toxicity. Pharmaceuticals Affairs Bureau, Notice No. 118. Ministry of Health and Welfare, Japan. JMHW, 1984.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Abacavir Succinate

IUPAC Name:

Abacavir Succinate

Constituent 2

Reference substance name:

168146-84-7

Cas Number:

168146-84-7

IUPAC Name:

168146-84-7

Details on test material:

- Name of test material (as cited in study report): 1592U89 Succinate
- Molecular formula (if other than submission substance): Please see Attachment 1.
- Molecular weight (if other than submission substance): Please see Attachment 1.
- Structural formula attached as image file (if other than submission substance): Please see Attachment 1.
- Purity: The purity factor initially used, 1.544, was corrected to 1.518 following further investigation.
- Batch number: 94/5977/045
- Stability: stable in DMSO up to 8 hours at room temperature.

Method

Target gene:

Tryptophan operon.

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli, other: WP2(pKM101)

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-fraction (Phenobarbitone and ß-naphthoflavone)

Test concentrations with justification for top dose:

with metabolic activation: 102, 339, 678, 1017, 3114*, 3387 and 5086µg/plate
without metabolic activation: 102, 339, 678, 1017, 3114*, 3387** and 5086µg/plate

* Only tested in Test 2: WP2uvrA(pKM101) - Absence of S9-mix
** Test 2: WP2uvrA(pKM101) - Absence of S9-mix not tested.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Stable in DMSO up to 8 hours at room temperature.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

1 µg/plate

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

WP2(pKM101 ) - without metabolic activation

Migrated to IUCLID6: (ENNG)

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

5 µg/mL

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

WP2uvrA(pKM101 ) - without metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates.

Negative solvent / vehicle controls:

yes

Remarks:

DSMO

True negative controls:

not specified

Positive controls:

yes

Remarks:

1000 µg/mL

Positive control substance:

other: 2-Aminofluorene (2-AF)

Remarks:

WP2(pKM101 ) - with metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Remarks:

5 µg/mL

Positive control substance:

other: 2-Aminoanthracene (2-AAn)

Remarks:

WP2uvrA(pKM101 ) - with metabolic activation

Details on test system and experimental conditions:

Culture media and reagents
Medium constituents
Nutrient Broth
Ampicillin
Tryptophan
NADP
Glucose-6-phosphate

Methods
Test systems
The genotype of each bacterial strain used in this study is outlined below in "any other information on materials and methods".
The cells are stored as frozen permanents and subsequently grown in nutrient broth for 10 hours at 37°C to yield a minimum of 1 x 10E9 cells/ml. The strains are grown in nutrient broth containing ampicillin to maintain the plasmid copy number.
Vogel Bonner E agar plates were supplied by Unipath Ltd, Wade Road, Basingstoke, Hampshire. RG24 8PW. England.

Metabolising system
A rat hepatic post-mitochondrial fraction (S9-fraction) was made by Genetic and Reproductive Toxicology Department, Glaxo Wellcome Research and Development, Ware, Herts, essentially as described by Maron and Ames, 1983. Phenobarbitone and ß-naphthoflavone were used to induce the synthesis of particular liver enzymes (Elliott et al, 1992) of Han Wistar male rats of within the age range of 6-9 weeks , as supplied by the Small Animal Breeding Unit, Glaxo Wellcome Research and Development, Ware, Herts. The batch reference ofthe S9-fraction used in this study was Sept 95 and April 96. For each batch, a selected range of cytochrome P450 enzymes were analysed by the Drug Metabolism Department, Glaxo Wellcome Research and Development, Ware, Herts. The S9-fraction was stored at approximately -70°C for up to eight months.
The S9-fraction was thawed immediately before required and used in conjunction with a NADPH-generating system, which included NADP (4mM) and glucose-6-phosphate (5mM), (Maron and Ames, 1983). This preparation, hereafter termed S9-mix, was sterilised by filtration (Millex-HV non pyrogenic membrane, pore size 0.45µm) and stored on ice until used. The S9-mix contained 100 µl S9-fraction per ml.

Selection of test concentrations
The maximum concentration used is one that reduces bacterial growth (as determined by a reduction in the bacterial lawn or by a reduction in the number of spontaneous revertants), or by the solubility of the test compound. If non-toxic and freely soluble then the maximum concentration would be 5000 µg per plate.The test concentrations were selected by reference to an earlier GLP study carried out on the test material at Hazleton Washington US (Lawlor, 1993). The maximum concentration was achieved using a purity factor of 1.544, giving a slightly higher maximum concentration of 5086 µg per plate.

Study design
Plate incorporation test (Ames)
Plate incorporation tests were carried out essentially as described by Maron and Ames (1983). Late log phase broth cultures of the two E. coli WP2 tryptophan requiring mutants were used to inoculate soft agar overlays, (containing a trace of tryptophan) held at 45°C, to which one of six concentrations of the test material (1.02, 3.39, 6.78,10.17,33.87 (or 31.14) and 50.86mg base per ml) were added to give 102, 339, 678, 1017, 3387 (or 3114) and 5086 µg test material per plate.
After mixing, the overlays were poured onto dried Vogel-Bonner plates and allowed to set at room temperature. The plates were inverted and incubated for approximately 72 hours at 37°C (± 1°C) protected from light before scoring for revertant colonies. Positive controls using known mutagens and untreated and solvent controls were included in each experiment. For the untreated and solvent controls 6 plates per group were used, for the treated groups 3 plates per group, the positive controls 2 plates per group and for the sterility controls 1 plate per group were used.
Tests were carried out in the absence and presence of a rat liver S9-mix. The level of S9 fraction used was 50 µl per plate. At least two independent experiments were carried out on different occasions to demonstrate the reproducibility of the result.

Liquid pre-incubation test (Yahagi)
At least one pre-incubation test (Yahagi et ai, 1975) was carried out in which the test material (at concentrations to give 102, 339, 678,1017,3387 (or 3114) and 5086 µg test material per plate) was pre-incubated at 37°C for 1 hour with S9-mix and test bacteria in a final volume of 0.7ml, before the addition of the overlay agar. The level of the S9-fraction used was 50 µl per plate. This method of achieving metabolism in a liquid medium at 37°C avoids loss of enzyme activity that may occur in agar at 45°C, and allows the detection of any highly reactive metabolite which may bind irreversibly to constituents of the agar. Tests were also carried out in the absence of S9-mix. Positive and untreated controls were included in each experiment. The replicates used per group were as in the Ames.

Evaluation criteria:

The criteria for a positive response is a two fold increase in revertants for the E. coli strains. The effect should be reproducible in an independent assay, and ideally there should be evidence of a dose related response.
Where equivocal results are obtained then the results may be compared with the historical control range.

Statistics:

The data generated by the bacterial tests were analysed statistically using the computer program AMES. Dunnett's statistic (Dunnett, 1955), was used to produce confidence limits around the increase in the arithmetic mean of each treatment over the solvent control using both one-sided 5% and 1% significance levels. The positive control data were not included in the statistical analysis. This statistical analysis is used as an aid to interpretation of the data obtained, and the criteria for a positive response are as described above.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli, other: WP2(pKM101)

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

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

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 material showed no evidence of mutagenic activity in any of the test systems used in this study.

Plate Incorporation Test (Ames)
Negative results were obtained in the absence and presence of S9-mix, up to a maximum concentration of 5086 µg test material per plate. Statistically significant increases in the number of revertant colonies were obtained in the following tests :-

Test 1 and 2, with WP2uvrA(pKM101) in the absence of S9-mix at 3387 and 5086, and 678 and 1017 µg per plate, respectively.
Test 1, with WP2 (pKM101) in the presence of S9-mix at 1017 µg per plate.
Test 1, with WP2 uvrA(pKM101) in the presence of S9-mix at 6781 µg per plate.

However these increases showed no evidence of a dose related increase, were not reproducible in subsequent retests, and therefore are considered to be of no biological relevance.

Liquid Pre-incubation Test (Yahagi)
Negative results were obtained both in the absence and presence of S9-mix, up to a maximum concentration of 5086 µg per test material per plate. A statistical significant increase in the number of revertant colonies was obtained at 678 and 1017 µg test material per plate. However this increase showed no evidence of a two-fold increase, and not reproducible in a subsequent retest, therefore thought to be of no biological relevance.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material showed no evidence of mutagenic activity in the Ames and liquid pre-incubation (Yahagi) tests, when tested up to a maximum concentration of 5086 µg test material per plate.

Executive summary:

The test material was tested for microbial mutagenicity as part of a general screen to identify possible oncogenic and heritable genetic hazards that might be associated with the use of this compound. The test material was not mutagenic in the absence or presence of S9-mix towards any of the strains of Escherichia coli used in this study, when tested up to a concentration of 5086 µg test material per plate. Negative results were obtained in an earlier microbial mutagenicity screen with the Salmonella typhimurium tester strains (Lawlor, 1993).

All tests were carried out in the absence and presence of an in vitro metabolic activation system (rat liver S9-mix). The maximum test concentration used in both the plate incorporation (Ames) and liquid pre-incubation (Yahagi) tests was 5086 µg test material per plate.