Tricyclo[3.3.1.13,7]decane-1-acetic acid, .alpha.-[[(1,1-dimethylethoxy)carbonyl]amino]-3-hydroxy-, (.alpha.S)-

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:

24 June 2002 to 11 October 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted in 2002 according to EU and OECD and in accordance with GLP. The study material is well characterized.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

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:

(2S)-2-{[(tert-butoxy)carbonyl]amino}-2-(3-hydroxyadamantan-1-yl)acetic acid

EC Number:

700-361-0

Cas Number:

361442-00-4

Molecular formula:

C17H27NO5

IUPAC Name:

(2S)-2-{[(tert-butoxy)carbonyl]amino}-2-(3-hydroxyadamantan-1-yl)acetic acid

Constituent 2

Reference substance name:

1-Hydroxyadamantanyl-3-(S)-Boc-glycine

IUPAC Name:

1-Hydroxyadamantanyl-3-(S)-Boc-glycine

Details on test material:

off-white powder; stored at room temperature in the dark; received at testing laboratory on 7 May 2002

Method

Target gene:

four histidine-requiring strains and one tryptophan-requiring 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:

Aroclor 1254-induced rat liver post mitochondrial fraction (rat-liver S-9)

Test concentrations with justification for top dose:

Range finding experiment and experiment #1 carried out at concentrations of 1.6, 8 , 40, 200, 1000 and 5000 ug/plate .
For experiment 2: doses were 51.2, 128, 320, 800, 2000 and 5000 ug/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

used as negative control

Remarks:

at same addition volumes per plate as the test article treatments

Controls 2

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

final concentration 5.0 ug/plate

Migrated to IUCLID6: use strain TA98

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

final concentration 2.0 ug/plate

Migrated to IUCLID6: use strain TA100,TA1535

Controls 4

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

final concentration 50.0 ug/plate

Migrated to IUCLID6: use strain TA 1537

Controls 5

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

final concentration 2.0 ug/plate

Migrated to IUCLID6: use strain WP2uvrA

Controls 6

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

final concentration 10.0 ug/plate with S-9

Migrated to IUCLID6: use strain TA98

Controls 7

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Use strain TA100,TA1535,TA1537 at concentration of 5.0 ug/plate and WP2uvrA at concentration of 10.0 ug/plate both with S-9

Details on test system and experimental conditions:

A toxicity range finding experiment was conducted in strain TA100, at the concentrations detailed previously. Triplicate plates without and with S-9 mix were used. Negative (solvent) and positive controls were included in quintuplicate and triplicate respectively, without and with
S-9 mix.

No evidence of toxicity was observed following this treatment. This test was acceptable and data from TA 100 strain supplemented experiment 1 where the remaining 4 strains were treated at the same concentrations as range finding study and with and without metabolic activation. These platings were achieved by the following sequence of additions to

2.5 mL molten agar at 46±1°C:
• 0.1 mL bacterial culture
• 0.1 mL test article solution or control
• 0.5 mL 10% S-9 mix or buffer solution

followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C in the dark for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted (see Colony counting).

As the results of the first experiment were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article or control solution (reduced to 0.05 mL), bacteria and S-9 mix detailed above, were mixed together and incubated for 1 hour at 37±1°C, with shaking, before the addition of 2.5 mL molten agar at 46±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure.

Colonies were counted electronically using a Seescan Colony Counter (Seescan plc) or manually where confounding factors such as split agar affected the accuracy of the automated counter. The background lawn was inspected for signs of toxicity.

Evaluation criteria:

Acceptance criteria for validity of assay : the mean negative control counts fell within normal ranges, the positive control chemical induced clear increases in revertant numbers confirming discrimination between different strains and an active S-9 preparation, and no more than 5 % of the plates were lost through contamination or some other unforeseen event. Evaluation criteria: test article would be considered mutagenic if: the assay was valid, Dunnett's test gave significant response ( p<= 0.01) and the data set(s) showed a significant dose correlation and the positive responses described above were reproducible.

Statistics:

Mean and standard deviation of the plate counts for each treatment were determined. For test data and positive control the m-statistic was calculated to check data were Poisson- distributed and the Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked using linear regression analysis.

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

Vehicle controls validity:

valid

Untreated negative 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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of BMS 528233-01 at 1.6, 8, 40, 200, 1000 and 5000 µg/plate, plus negative (solvent) and positive controls. Following these treatments, no evidence of toxicity was observed, as would normally be manifest by a thinning of the background bacterial lawn and/or a marked reduction in revertant numbers. These results were therefore considered acceptable for mutation assessment and are presented in this report as the TA100 mutagenicity data for Experiment 1.

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

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 with and without metabolic activation.

It was concluded that BMS 528233-01 did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2 uvrA) when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

BMS 528233-01 was assayed for mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA) of Escherichia coli, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

 

An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of BMS 528233-01 at 1.6, 8, 40, 200, 1000 and 5000 µg/plate,plus negative (solvent) and positive controls. Following these treatments, and those of the remaining test strains in Experiment 1, which employed the same test doses, no

evidence of toxicity was observed.

 

Experiment 2 treatments of all the tester strains retained 5000 µg/plate as the maximum test dose. A narrowed dose range was used for treatment of all strains (51.2-5000 µg/plate), in order to more closely examine those concentrations of BMS 528233-01 approaching the maximum test dose, and therefore considered most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way it was hoped to increase the range of mutagenic chemicals that could be detected

using this assay system. No clear evidence of toxicity was observed following any of the treatments performed in Experiment 2.

 

The test article was completely soluble in the aqueous assay system at all concentrations tested, in each of the experiments performed.

 

Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.

 

No BMS 528233-01 treatments of any of the tester strains resulted in any increases in revertant numbers sufficient to be considered as indicative of mutagenic activity.

 

It was concluded that BMS 528233-01 did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophan-requiring strain (WP2 uvrA) of Escherichia coli when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate, in the absence and in the presence of a rat liver metabolic activation system (S-9).