Mutagenesis vol. 20 no. 5 pp. 345–350, 2005 doi:10.1093/mutage/gei045
Advance Access publication 12 July 2005
Modified bacterial mutation test procedures for evaluation of peptides
and amino acid-containing material
Crista Thompson, Paul Morley1, David Kirkland2 and on testing of biological molecules including hormones, growth
Raymond Proudlock factors and proteins. ICH S6 states that genotoxicity studies
Charles River Laboratories, Preclinical Services, CTBR, 87 Senneville
normally conducted for pharmaceuticals are not applicable or
Road, Senneville, Quebec, Canada H9X 3R3, 1Zelos Therapeutics, needed for biologicals, such as peptides, which are considered
Building M-54, 1200 Montreal Road, Ottawa, Ontario, Canada K1A 0R6 unlikely to react directly with DNA. It indicates that genotox-
and 2Covance Laboratories Ltd, Otley Road, Harrogate, North Yorkshire icity studies may be required on a case-by-case basis, e.g.
HG3 1PY, UK where a chemical linking agent is used to modify the molecule
Biological materials can release amino acids during the or when potentially mutagenic impurities or degradants are
course of bacterial mutation testing. Low levels of released present. Although the guidance indicates that the standard
amino acids from soluble materials can cause moderate genotoxicity studies are inappropriate for assessing biologicals
increases in the number of revertant colonies on the plate, in such cases, it does not give specific guidance on which
whereas higher levels lead to overgrowth of the background studies would be appropriate.
lawn, making counting of revertant colonies impossible. In practice, the bacterial mutation test is the most widely
For poorly soluble material, the released amino acids can used and best validated bioassay for assessment of potentially
be present at high levels in localized spots on the plate, mutagenic contaminants. Since it is relatively inexpensive and
leading to the growth of ‘pseudorevertant’ colonies. The sensitive, it seems an appropriate assay to use to confirm the
‘treat and wash’ modified preincubation method employed absence of genotoxic contaminants produced during synthesis,
here is an adaptation of the treat and plate method (used for or remaining after purification of biological molecules (1–3).
evaluation of antibiotics) and involves washing the bacteria Although the bacterial mutation assay may be the first choice
free of test compound after a 90 min exposure prior to for routine genetic toxicity screening of chemicals, it has
plating out on minimal plates. The MC overlay method is long been recognized that materials containing, or capable
a modified version of the standard plate incorporation of releasing amino acids can interfere with the assay. For
assay, in which a top overlay containing 4% high viscosity example, biological samples (urine, feces, food), proteins,
methylcellulose is used in place of agar to stabilize the test peptides and histidine itself can cause additional growth of
compound in solution, preventing precipitation and sub- Salmonella on minimal medium plates and result in additional
sequent localized amino acid release. Both modified meth- spontaneous mutations and/or overgrowth of the background
ods produce the expected results for negative and positive lawn of non-revertant bacteria which obscures revertant
controls. Peptides [synthetic curtailed analogs of human colonies (4–9).
parathyroid hormone, PTH(1–34) and Ostabolin-CTM] To avoid misinterpretations when testing histidine/
that produced false positive results or could not be evalu- tryptophan containing compounds, it has been proposed that a
ated owing to overgrowth of the background lawn using modified preincubation method with extensive washing prior to
standard methods, showed no artifacts and no evidence of plating could be employed (4,10). Although various complex
genotoxicity using the modified methods. It is concluded washing methods have been demonstrated by other groups
that the treat and wash and MC overlay methods are (11–13), the present study verifies that a simple modification
valid versions of the bacterial mutation test for avoiding of the ‘treat and plate’ method (14), i.e. washing the bacteria
complications associated with released amino acids. free of test compound after a 90 min exposure prior to plating
out on minimal plates (‘treat and wash’ method), avoids false
positive responses and overgrowth of the background bacterial
lawn resulting from free amino acids, while producing clear
Introduction positive responses with appropriate positive controls.
The International Conference on Harmonization of Technical Precipitation of a peptide in the top agar with subsequent
Requirements for Registration of Pharmaceuticals for Human release of amino acids can also lead to confounding results.
Use (ICH) consists of a series of committees, mainly concerned A modification of the standard plate incorporation method
with standardizing the range of toxicology tests needed for new (‘MC overlay’ method) is presented which has been used to
pharmaceuticals. ICH S2B guidance was finalized in 1997 and stabilize a peptide in solution and avoid confounding artifacts.
adopted by the US, European and Japanese regulatory author-
ities shortly after. The guidance indicates that the expected Materials and methods
standard battery of genotoxicity tests normally required for
Chemicals
small molecule pharmaceuticals should consist of a bacterial
9-Aminoacridine (9AC), 2-aminoanthracene (2AA), benzo[a]pyrene (BaP),
mutation test, an in vitro chromosome aberration test or a histidine, methylcellulose (MC), reagent or pharmaceutical grade is acceptable,
mouse lymphoma assay, and a rodent bone marrow micronuc- 2-nitrofluorene (2NF), 4-nitroquinoline N-oxide (NQO), sodium azide (NaAz)
leus test. ICH document S6 (1997) gives additional guidance and tryptophan were obtained from Sigma-Aldrich (Oakville, Canada). 9AC,
To whom correspondence should be addressed. Tel: 11 514 630 8254; Fax: 11 514 630 8230; Email:
Ó The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
All rights reserved. For permissions, please email: 345
, C.Thompson et al.
2AA, BaP, 2NF and NQO were formulated in DMSO; histidine, MC, chloride/MC) and the mixture was stirred at 50–60 C throughout use. A 0.5 ml
tryptophan and NaAz were formulated in water. aliquot of S9 mix or phosphate buffer 0.2 M pH 7.4 was combined with 0.1 ml
overnight bacterial culture in a sterile container. A 2 ml aliquot of the MC
Peptides overlay suspension was added to the tube, and a 0.1 ml aliquot of the treatment
PTH(1–34) and Ostabolin-CTM were independently developed as stable solution was added immediately afterward. The mixture was overlaid on a
synthetic curtailed analogs of human parathyroid hormone for potential treat- prewarmed (37 C) minimal glucose plate. The plates were held at 4 C for 1 h
ment of osteoporosis. after plating to ensure gelling of the MC overlay. The plates were incubated
Peptide PTH(1–34) corresponds to the first 34 amino acids of the human agar side down (not inverted) to maintain the position of the MC layer.
parathyroid hormone. The peptide was manufactured by Bachem, Torrance, Typically, plates are inverted during incubation to prevent condensation drop-
CA, USA (86.84% pure; all concentrations are expressed in terms of active lets from falling onto the surface of the agar. Incubating the MC overlay plates
ingredient). PTH(1–34) has a molecular weight of 4118 Da, and contains one agar side down has the potential drawback of condensation droplets causing the
tryptophan and three histidine residues in the sequence H2N-Ser-Val-Ser-Glu- spread of colonies resulting in artificially high colony counts or indistinguish-
Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val- able colonies (unscorable plate); however, this was not observed in our
Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH. PTH(1–34) was laboratory.
formulated as a solution in water on each occasion of use, just prior to use.
Ostabolin-CTM corresponds to the first 31 amino acids of the human Evaluation and interpretation of results
parathyroid hormone with a Leu27, cyclo(Glu22-Lys26) modification (15). The Sterility, negative/vehicle and positive controls were included in each experi-
peptide was supplied by Zelos Therapeutics, Ottawa, Canada (85% pure; all ment. Triplicate plates were evaluated at each experimental point. Revertant
concentrations expressed as material as supplied). The peptide has a molecular colonies were routinely counted using an automated colony counter (ProtoCOL
weight of 3685 Da, and contains one tryptophan and two histidine residues by Synbiosis, Cambridge, UK). If precipitate interfered with the operation of
in the sequence H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys- the system, counts were enumerated manually. The background lawn was
His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Leu-Leu-Gln-Asp- evaluated using an inverted microscope. Toxicity was identified by a substan-
Val-NH2. Ostabolin-CTM has limited solubility in water; therefore, it was tial reduction in the integrity of the lawn, or a marked reduction in the number
formulated as an apparent solution in aqueous 1% (w/v) MC (400 cps) on of revertants compared with the vehicle control (fold response <0.6). A posi-
each occasion of use, just prior to use. tive mutagenic response was defined as a dose-related increase in revertant
colony numbers to at least twice the concurrent untreated control levels
Bacterial strains
(1.53 for strain TA100).
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and
Escherichia coli strain WP2 uvrA were originally supplied by Moltox (NC,
USA) and were characterized and maintained as previously recommended Results
(14,16).
Treat and wash assay
S9 mix The treat and wash bacterial mutation test was employed to
Phenobarbital/5,6-benzoflavone-induced male Sprague–Dawley rat liver frac- evaluate the mutagenicity of soluble biological material cap-
tion (S9 fraction) was supplied by Moltox (NC, USA). S9 mix contained 10% able of releasing histidine and tryptophan. Histidine and trypto-
(by volume) S9 fraction unless otherwise indicated and the following sterile
co-factors: 8 mM magnesium chloride, 33 mM potassium chloride, 100 mM phan can cause overgrown background bacterial lawns and
sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM variable/sporadic increases in revertants in the plate incorpora-
nicotinamide adenine diphosphate. tion and preincubation versions of the bacterial mutation test
Bacterial mutation tests (Table I). Modification of the procedures to include a longer
Tests were based on previously recommended procedures (16). In addition to
preincubation followed by a washing step to remove the amino
histidine and biotin, the top agar contained minimal tryptophan (0.05 mM) to acids prior to plating (‘treat and wash’ method) prevented
support E.coli growth (17). Histidine, tryptophan and synthetic PTH(1–34) overgrown lawns and significant increases in revertant colony
were evaluated in the standard plate incorporation, pre-incubation and treat counts (Table II).
and wash assays. Ostabolin-CÔ was evaluated in the standard plate incorpora- A soluble peptide, PTH(1–34), caused overgrowth of the
tion and MC overlay assays.
background bacterial lawn for all strains at levels of 500 mg/
Standard plate incorporation assay plate in the presence of S9 using the plate incorporation
A 0.5 ml aliquot of S9 mix or phosphate buffer 0.2 M pH 7.4 was combined method, presumably as a result of S9-mediated release of
with 0.1 ml late log bacterial culture in a sterile container. A 0.1 ml aliquot of histidine and tryptophan (Table III). The overgrown lawns
the treatment solution was added, and 2 ml of molten top agar was added
immediately afterward. The solution was overlaid onto a minimal glucose plate prevented any assessment of revertant colony counts. Using
(1.5% agar, Vogel–Bonner medium E, 2% glucose). The plates were inverted the treat and wash method in the presence of S9 in a confirm-
and incubated at 37 C for 48 to 72 h. atory assay, no overgrowth of the background lawn was
Preincubation assay
observed for any strain at any concentration of the peptide
Procedures were as per the standard plate incorporation assay with the excep-
(Table III). Thus, revertant colony counts were available for
tion that the S9 mix/buffer, bacteria and treatment were incubated for 30 min the peptide up to the highest dose level recommended by
with shaking (180 r.p.m.) at 37 C before the addition of molten top agar. regulatory guidelines (5000 mg/plate). PTH(1–34) did not
cause any substantial increases in revertant colony counts
Treat and wash assay
either in the absence or in the presence of S9 mix using plate
Procedures were as per the preincubation assay with the exception that the
preincubation time was increased from 30 to 90 min. The extended duration of incorporation, preincubation or treat and wash methods.
bacterial exposure during the preincubation compensated for the absence of
bacterial exposure on plates (as the test compound was washed away prior to MC overlay assay
plating). After the 90 min preincubation, 15 ml of a wash solution of Oxoid The development of the MC overlay bacterial mutation test
No. 2 nutrient broth in phosphate buffered saline (1:7 v/v) was added and the was required because of problems in assessing a peptide,
washed bacteria were collected by centrifugation at 2000 g for 30 min. All
but 0.7 ml of the supernatant was removed and discarded, and the bacteria Ostabolin-CÔ, that was not freely soluble at the higher con-
were resuspended in the residual supernatant prior to plating via top agar. centrations typically used for bacterial mutation testing (i.e.
levels up to 5000 mg/plate).
MC overlay assay
Ostabolin-CÔ caused apparent increases in colony numbers
An MC overlay was prepared on the day of the test by combining 5 g sodium
chloride and 40 g MC (4000 cps) in 900 ml of water heated to 80 C. The of strains TA98 and WP2 uvrA in the presence of S9 using the
suspension was kept stirring and cooled to 50 C. A solution of histidine, plate incorporation method (occasion 1 in Table IV). No
tryptophan and biotin (each 0.5 mM) was added (100 ml per 900 ml sodium increases in colony counts were observed with any other strain
346
Advance Access publication 12 July 2005
Modified bacterial mutation test procedures for evaluation of peptides
and amino acid-containing material
Crista Thompson, Paul Morley1, David Kirkland2 and on testing of biological molecules including hormones, growth
Raymond Proudlock factors and proteins. ICH S6 states that genotoxicity studies
Charles River Laboratories, Preclinical Services, CTBR, 87 Senneville
normally conducted for pharmaceuticals are not applicable or
Road, Senneville, Quebec, Canada H9X 3R3, 1Zelos Therapeutics, needed for biologicals, such as peptides, which are considered
Building M-54, 1200 Montreal Road, Ottawa, Ontario, Canada K1A 0R6 unlikely to react directly with DNA. It indicates that genotox-
and 2Covance Laboratories Ltd, Otley Road, Harrogate, North Yorkshire icity studies may be required on a case-by-case basis, e.g.
HG3 1PY, UK where a chemical linking agent is used to modify the molecule
Biological materials can release amino acids during the or when potentially mutagenic impurities or degradants are
course of bacterial mutation testing. Low levels of released present. Although the guidance indicates that the standard
amino acids from soluble materials can cause moderate genotoxicity studies are inappropriate for assessing biologicals
increases in the number of revertant colonies on the plate, in such cases, it does not give specific guidance on which
whereas higher levels lead to overgrowth of the background studies would be appropriate.
lawn, making counting of revertant colonies impossible. In practice, the bacterial mutation test is the most widely
For poorly soluble material, the released amino acids can used and best validated bioassay for assessment of potentially
be present at high levels in localized spots on the plate, mutagenic contaminants. Since it is relatively inexpensive and
leading to the growth of ‘pseudorevertant’ colonies. The sensitive, it seems an appropriate assay to use to confirm the
‘treat and wash’ modified preincubation method employed absence of genotoxic contaminants produced during synthesis,
here is an adaptation of the treat and plate method (used for or remaining after purification of biological molecules (1–3).
evaluation of antibiotics) and involves washing the bacteria Although the bacterial mutation assay may be the first choice
free of test compound after a 90 min exposure prior to for routine genetic toxicity screening of chemicals, it has
plating out on minimal plates. The MC overlay method is long been recognized that materials containing, or capable
a modified version of the standard plate incorporation of releasing amino acids can interfere with the assay. For
assay, in which a top overlay containing 4% high viscosity example, biological samples (urine, feces, food), proteins,
methylcellulose is used in place of agar to stabilize the test peptides and histidine itself can cause additional growth of
compound in solution, preventing precipitation and sub- Salmonella on minimal medium plates and result in additional
sequent localized amino acid release. Both modified meth- spontaneous mutations and/or overgrowth of the background
ods produce the expected results for negative and positive lawn of non-revertant bacteria which obscures revertant
controls. Peptides [synthetic curtailed analogs of human colonies (4–9).
parathyroid hormone, PTH(1–34) and Ostabolin-CTM] To avoid misinterpretations when testing histidine/
that produced false positive results or could not be evalu- tryptophan containing compounds, it has been proposed that a
ated owing to overgrowth of the background lawn using modified preincubation method with extensive washing prior to
standard methods, showed no artifacts and no evidence of plating could be employed (4,10). Although various complex
genotoxicity using the modified methods. It is concluded washing methods have been demonstrated by other groups
that the treat and wash and MC overlay methods are (11–13), the present study verifies that a simple modification
valid versions of the bacterial mutation test for avoiding of the ‘treat and plate’ method (14), i.e. washing the bacteria
complications associated with released amino acids. free of test compound after a 90 min exposure prior to plating
out on minimal plates (‘treat and wash’ method), avoids false
positive responses and overgrowth of the background bacterial
lawn resulting from free amino acids, while producing clear
Introduction positive responses with appropriate positive controls.
The International Conference on Harmonization of Technical Precipitation of a peptide in the top agar with subsequent
Requirements for Registration of Pharmaceuticals for Human release of amino acids can also lead to confounding results.
Use (ICH) consists of a series of committees, mainly concerned A modification of the standard plate incorporation method
with standardizing the range of toxicology tests needed for new (‘MC overlay’ method) is presented which has been used to
pharmaceuticals. ICH S2B guidance was finalized in 1997 and stabilize a peptide in solution and avoid confounding artifacts.
adopted by the US, European and Japanese regulatory author-
ities shortly after. The guidance indicates that the expected Materials and methods
standard battery of genotoxicity tests normally required for
Chemicals
small molecule pharmaceuticals should consist of a bacterial
9-Aminoacridine (9AC), 2-aminoanthracene (2AA), benzo[a]pyrene (BaP),
mutation test, an in vitro chromosome aberration test or a histidine, methylcellulose (MC), reagent or pharmaceutical grade is acceptable,
mouse lymphoma assay, and a rodent bone marrow micronuc- 2-nitrofluorene (2NF), 4-nitroquinoline N-oxide (NQO), sodium azide (NaAz)
leus test. ICH document S6 (1997) gives additional guidance and tryptophan were obtained from Sigma-Aldrich (Oakville, Canada). 9AC,
To whom correspondence should be addressed. Tel: 11 514 630 8254; Fax: 11 514 630 8230; Email:
Ó The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
All rights reserved. For permissions, please email: 345
, C.Thompson et al.
2AA, BaP, 2NF and NQO were formulated in DMSO; histidine, MC, chloride/MC) and the mixture was stirred at 50–60 C throughout use. A 0.5 ml
tryptophan and NaAz were formulated in water. aliquot of S9 mix or phosphate buffer 0.2 M pH 7.4 was combined with 0.1 ml
overnight bacterial culture in a sterile container. A 2 ml aliquot of the MC
Peptides overlay suspension was added to the tube, and a 0.1 ml aliquot of the treatment
PTH(1–34) and Ostabolin-CTM were independently developed as stable solution was added immediately afterward. The mixture was overlaid on a
synthetic curtailed analogs of human parathyroid hormone for potential treat- prewarmed (37 C) minimal glucose plate. The plates were held at 4 C for 1 h
ment of osteoporosis. after plating to ensure gelling of the MC overlay. The plates were incubated
Peptide PTH(1–34) corresponds to the first 34 amino acids of the human agar side down (not inverted) to maintain the position of the MC layer.
parathyroid hormone. The peptide was manufactured by Bachem, Torrance, Typically, plates are inverted during incubation to prevent condensation drop-
CA, USA (86.84% pure; all concentrations are expressed in terms of active lets from falling onto the surface of the agar. Incubating the MC overlay plates
ingredient). PTH(1–34) has a molecular weight of 4118 Da, and contains one agar side down has the potential drawback of condensation droplets causing the
tryptophan and three histidine residues in the sequence H2N-Ser-Val-Ser-Glu- spread of colonies resulting in artificially high colony counts or indistinguish-
Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val- able colonies (unscorable plate); however, this was not observed in our
Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH. PTH(1–34) was laboratory.
formulated as a solution in water on each occasion of use, just prior to use.
Ostabolin-CTM corresponds to the first 31 amino acids of the human Evaluation and interpretation of results
parathyroid hormone with a Leu27, cyclo(Glu22-Lys26) modification (15). The Sterility, negative/vehicle and positive controls were included in each experi-
peptide was supplied by Zelos Therapeutics, Ottawa, Canada (85% pure; all ment. Triplicate plates were evaluated at each experimental point. Revertant
concentrations expressed as material as supplied). The peptide has a molecular colonies were routinely counted using an automated colony counter (ProtoCOL
weight of 3685 Da, and contains one tryptophan and two histidine residues by Synbiosis, Cambridge, UK). If precipitate interfered with the operation of
in the sequence H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys- the system, counts were enumerated manually. The background lawn was
His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Leu-Leu-Gln-Asp- evaluated using an inverted microscope. Toxicity was identified by a substan-
Val-NH2. Ostabolin-CTM has limited solubility in water; therefore, it was tial reduction in the integrity of the lawn, or a marked reduction in the number
formulated as an apparent solution in aqueous 1% (w/v) MC (400 cps) on of revertants compared with the vehicle control (fold response <0.6). A posi-
each occasion of use, just prior to use. tive mutagenic response was defined as a dose-related increase in revertant
colony numbers to at least twice the concurrent untreated control levels
Bacterial strains
(1.53 for strain TA100).
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and
Escherichia coli strain WP2 uvrA were originally supplied by Moltox (NC,
USA) and were characterized and maintained as previously recommended Results
(14,16).
Treat and wash assay
S9 mix The treat and wash bacterial mutation test was employed to
Phenobarbital/5,6-benzoflavone-induced male Sprague–Dawley rat liver frac- evaluate the mutagenicity of soluble biological material cap-
tion (S9 fraction) was supplied by Moltox (NC, USA). S9 mix contained 10% able of releasing histidine and tryptophan. Histidine and trypto-
(by volume) S9 fraction unless otherwise indicated and the following sterile
co-factors: 8 mM magnesium chloride, 33 mM potassium chloride, 100 mM phan can cause overgrown background bacterial lawns and
sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM variable/sporadic increases in revertants in the plate incorpora-
nicotinamide adenine diphosphate. tion and preincubation versions of the bacterial mutation test
Bacterial mutation tests (Table I). Modification of the procedures to include a longer
Tests were based on previously recommended procedures (16). In addition to
preincubation followed by a washing step to remove the amino
histidine and biotin, the top agar contained minimal tryptophan (0.05 mM) to acids prior to plating (‘treat and wash’ method) prevented
support E.coli growth (17). Histidine, tryptophan and synthetic PTH(1–34) overgrown lawns and significant increases in revertant colony
were evaluated in the standard plate incorporation, pre-incubation and treat counts (Table II).
and wash assays. Ostabolin-CÔ was evaluated in the standard plate incorpora- A soluble peptide, PTH(1–34), caused overgrowth of the
tion and MC overlay assays.
background bacterial lawn for all strains at levels of 500 mg/
Standard plate incorporation assay plate in the presence of S9 using the plate incorporation
A 0.5 ml aliquot of S9 mix or phosphate buffer 0.2 M pH 7.4 was combined method, presumably as a result of S9-mediated release of
with 0.1 ml late log bacterial culture in a sterile container. A 0.1 ml aliquot of histidine and tryptophan (Table III). The overgrown lawns
the treatment solution was added, and 2 ml of molten top agar was added
immediately afterward. The solution was overlaid onto a minimal glucose plate prevented any assessment of revertant colony counts. Using
(1.5% agar, Vogel–Bonner medium E, 2% glucose). The plates were inverted the treat and wash method in the presence of S9 in a confirm-
and incubated at 37 C for 48 to 72 h. atory assay, no overgrowth of the background lawn was
Preincubation assay
observed for any strain at any concentration of the peptide
Procedures were as per the standard plate incorporation assay with the excep-
(Table III). Thus, revertant colony counts were available for
tion that the S9 mix/buffer, bacteria and treatment were incubated for 30 min the peptide up to the highest dose level recommended by
with shaking (180 r.p.m.) at 37 C before the addition of molten top agar. regulatory guidelines (5000 mg/plate). PTH(1–34) did not
cause any substantial increases in revertant colony counts
Treat and wash assay
either in the absence or in the presence of S9 mix using plate
Procedures were as per the preincubation assay with the exception that the
preincubation time was increased from 30 to 90 min. The extended duration of incorporation, preincubation or treat and wash methods.
bacterial exposure during the preincubation compensated for the absence of
bacterial exposure on plates (as the test compound was washed away prior to MC overlay assay
plating). After the 90 min preincubation, 15 ml of a wash solution of Oxoid The development of the MC overlay bacterial mutation test
No. 2 nutrient broth in phosphate buffered saline (1:7 v/v) was added and the was required because of problems in assessing a peptide,
washed bacteria were collected by centrifugation at 2000 g for 30 min. All
but 0.7 ml of the supernatant was removed and discarded, and the bacteria Ostabolin-CÔ, that was not freely soluble at the higher con-
were resuspended in the residual supernatant prior to plating via top agar. centrations typically used for bacterial mutation testing (i.e.
levels up to 5000 mg/plate).
MC overlay assay
Ostabolin-CÔ caused apparent increases in colony numbers
An MC overlay was prepared on the day of the test by combining 5 g sodium
chloride and 40 g MC (4000 cps) in 900 ml of water heated to 80 C. The of strains TA98 and WP2 uvrA in the presence of S9 using the
suspension was kept stirring and cooled to 50 C. A solution of histidine, plate incorporation method (occasion 1 in Table IV). No
tryptophan and biotin (each 0.5 mM) was added (100 ml per 900 ml sodium increases in colony counts were observed with any other strain
346
