ethynyl cyclopropane

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:

The experimental phase of the study was conducted between 4 March and 2 June 1997.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The substance SD957 was examined for mutagenic activity in four histidine dependent auxotrophy of Salmonella typhimurium, strains TA98, TA100, TA1535 and TA1537, and one tryptophan dependent auxotroph of Escherichia coli, strain CM891 (WP2uvrA/pKM 101), using a procedure in which agar plates, seeded with the tester strains, were exposed to the test material in vapour phase.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

425-430-1

EC Name:

-

Cas Number:

6746-94-7

Molecular formula:

C5 H6

IUPAC Name:

ethynylcyclopropane

Details on test material:

- Name of test material (as cited in study report): EthynyI cyclopropane (SD 957)
- Physical state: Colourless liquid
- Analytical purity: 97-100 %
- Lot/batch No.: 3590-129-D4
- Expiration date of the lot/batch: January 1999
- Storage condition of test material: -20°C, under nitrogen and in the dark
- Intended use: Pharmaceutical intermediate

Method

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 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix (Aroclor 1254-induced)

Test concentrations with justification for top dose:

First test: 0.625%, 1 .25%, 2.5%, 5% and 10% v/v (with and without S9 mix);
Second test: 1.25%, 2.5%, 5%, 7.5% and 10% v/v (with and without S9 mix);
Third test: 1.25%, 2.5%, 5%, 7.5% and 10% v/v (only without S9 mix);

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

TREATMENT
Formulation of the test substance
The test substance was tested in vapour phase. No carrier was employed. All concentrations cited in the report are expressed in terms of the SD957 sample as received.

Quality control of dosage form
Achieved concentration was estimated at the start and at the end of the exposure period by chemical analysis by gas chromatography of samples removed from the exposure vessels via ports specifically positioned for this purpose.

Positive controls
Appropriate positive control plates were included, in both the presence and absence of S9 mix, for each strain, as shown below . All positive control compounds were prepared as solutions in DMSO (Aldrich, A.C.S. spectrophotometric grade, 99.9% pure), except dichloromethane, which was used
in vapour phase.

Positive control / chemical / Concentration / Strain
2-Aminoanthracene 2 µg/plate TA1535 (Aldrich ; Lot No. 001 3406, 90% pure) . 10 µg/plate CM891
9-Aminoacridine 80 µg/plate TA1537 (Sigma; Lot No. 108C-0358, 99% pure).
2-Nitrofluorene 1 µg/plate TA98 (Aldrich ; Lot No. 012867, 98% pure).
Benzo[a]pyrene 5 µg/plate TA98, TA 100, TA 1537 (Sigma; Lot No . 51 F-0413, 98% pure).
N-Ethyl-N'-Nitro-N-nitrosoguanidine (ENNG) 2 µg/plate CM891 (Sigma; lot No . 2017-0235, 97% pure) . 3 µg/plate TA100; 5 µg/plate TA1535
Dichloromethane 7.5% v/v All strains (Fisher ; Lot No . 9627679 346, 99.99% pure).

Test organisms
Cultures of the histidine-dependent strains of Salmonella typhimurium were derived from cultures provided by Prof. Bruce Ames, University of California (received on 21 April 1995). The characteristics of the individual strains are as follows :
TA1535 - contains a histidine missense mutation (hisG46) but is also deficient in a DNA repair system (uvrB) and has a defective lipopolysaccharide coat on the cell wall (rfa mutation) . It is reverted by many agents causing base-pair substitutions, but is not sensitive to frameshift mutagens.

TA100 - is the same as TA1535 but contains a resistance transfer factor conferring ampicillin resistance and increasing sensitivity to some mutagens (plasmid pKM101). In addition to base-pair substitutions, it is also able to detect certain frameshift mutagens.

TA1537 - bears a histidine frameshift mutation (hisC3076). Like TA1535, it is defective in a DNA repair system and lipopolysaccharide coat. It is sensitive to agents causing frameshift mutations involving insertion or deletion of a single base-pair.

TA98 - contains another histidine frameshift mutation (hisD3052). Again it has a defective DNA repair system and lipopolysaccharide coat but also contains the pKM101 plasmid. It is reverted by a0gents causing deletion of two adjacent base-pairs (double frameshift mutations), but not by simple alkylating agents causing base-pair substitutions.

Cultures of the tryptophan-dependent strain CM891 of Escherichia coli were derived from cultures provided by the National Collections of Industrial and Marine Bacteria, Aberdeen (received on 16 May 1996). The characteristics of this strain are as follows:

CM891 - contains an ochre mutation. It is reverted by many agents causing A-T basepair substitutions at the irpE locus or by G-C base-pair substitutions in transfer RNA loci elsewhere in the chromosome. It is also deficient in a DNA repair system (uvrA), and is more readily reverted by certain mutagens
than its parent strain WP2 . It also contains the pKM 101 plasmid.

Cultures of all organisms were prepared using a gyratory incubator (New Brunswick Scientific, Model G25) by overnight incubation (10 hours) at 37°C of nutrient broth (Oxoid No. 2) freshly inoculated from a frozen culture stock.

Checking of tester strains
New batches of frozen culture stocks, stored at -80°C, are prepared at intervals from a central stock held in a liquid nitrogen refrigerator. Samples from each new batch are thawed at least 24 hours after freezing and checked for appropriate amino acid requirement and characteristic spontaneous reversion rate.

The Salmonella strains are checked by testing for growth inhibition in spot tests with:
crystal violet inhibition zone > 14 mm in diameter when 10 µl of a 1 mg/ml crystal violet solution is sported onto plate centre indicates possession of the
deep-rough (rfa) character.
mitomycin C inhibition zone > 20 mm in diameter when 10 µl of a 0.1 mg/ml
mitomycin C solution is spotted onto plate centre indicates a defective DNA repair system (uvrB) .

Further checks are conducted to confirm strain sensitivities to mutagens known to induce different types of mutational event, and to an antibiotic:

E. coli strain CM891 is checked for reversion in the presence of ENNG (2 lig/plate) and for growth inhibition on nutrient agar plates spread with Mitomycin C solutions at various concentrations up to 25 µg/plate: growth of strain CM891 is inhibited at lower Mitomycin C concentrations than its parent strain WP2. It is also tested for resistance to Ampicillin.

PROCEDURES
Preliminary (range-finding) test
Plates of Vogel-Bonner E minimal agar (25 ml) were seeded with two of the tester strains (TA98 and CM891) by applying an overlay consisting of 2 ml molten top-agar containing 0 .5mM histidine/0.5mM biotin or 0.5mM tryptophan, maintained at 45°C, 0.1 ml of a 10-hour culture and 0.5 ml rat liver
microsomal preparation (S9 mix) or 0.1 M sodium phosphate buffer (pH 7.4) as appropriate. The plates were placed in stainless steel vessels and were supported, with lids removed, on stainless steel racks inside these vessels, so as to permit circulation of test material. Plates containing S9 mix were placed in separate vessels from those not containing S9 mix. Three plates were prepared for each dose level. The vessels were sealed and partially evacuated.
Appropriate volumes of SD957 were injected via a PTFE-faced septum and allowed to vaporise at 37°C. In the absence of any bacterial toxicity data, the following nominal concentrations were used:
10%, 25%, 50%, 75% and 90% v/v.
The contents of the vessels were equilibrated to atmospheric pressure by admitting sterile air. Untreated control plates and plates treated with the vapour-phase, positive control dichloromethane were also prepared and were placed in stainless steel vessels under identical conditions.

The remaining positive control plates were prepared by placing aliquots (0.1 ml) of each control compound in sterile glass tubes. Where appropriate, 0.5 ml S9 mix or 0.1 M sodium phosphate buffer was added to each tube. Molten top-agar (2 ml) containing 0.5 mM histidine/ 0.5mM biotin or 0.5mM tryptophan, maintained at 45°C, and bacterial culture (0.1 ml) were then added, the tubes were inverted to mix thoroughly and the contents poured onto plates containing minimal agar (25 ml). The positive control plates were also placed in stainless steel vessels. Further plates were prepared without the inclusion of the test organisms to verify the sterility of the S9 mix and the test material.

Aliquots (0.1 ml) of a 10e-6 dilution of the overnight cultures were spread on the surface of plates of nutrient agar to measure the viability and cell-density of each culture. All plates were incubated at 37°C for 2 days. After this period, the plates were removed from the vessels and incubated for a further day to permit the growth of revertant colonies . After incubation, numbers of revertant colonies were counted with an automated colony counter. Total colonies on nutrient plates were counted in the same way. The minimal plates were also examined for the presence of a background lawn of non-revertant colonies ; toxicity of the test material is shown by a substantial reduction in revertant colony counts relative to untreated controls or by absence or
thinning of the background lawn. The concentration of test material chosen as the top concentration for the main mutation tests was the lowest concentration showing toxicity.
The control plates were checked for the absence of growth on sterility checks or normal counts on untreated controls in the presence and absence of S9 mix.
All plates and tubes were identified by the use of numbers indelibly marked on the plates and test tube racks.

First test
Plates of Vogel-Bonner E minimal agar (25 ml) were seeded with the tester strains by applying an overlay consisting of 2 ml molten top-agar containing 0.5mM histidine/0.5mM biotin or 0.5mM tryptophan, maintained at 45° C, 0.1 ml of a 10-hour culture and 0.5 ml rat liver microsomal preparation (S9 mix) or 0.1 M sodium phosphate buffer (pH 7.4) as appropriate. The plates were placed in stainless steel vessels and were supported, with lids removed, on stainless steel racks inside these vessels, so as to permit circulation of test material. Plates containing S9 mix were placed in separate vessels from those not containing S9 mix. Three plates were prepared for each dose level. The vessels were sealed and partially evacuated. Appropriate volumes of SD957 were injected via a PTFE-faced septum and allowed to vaporise at 37° C. On the basis of toxicity data obtained in the preliminary test, the following nominal concentrations were used: 0.625%, 1.25%, 2.5%, 5% and 10% v/v. The contents of the vessels were equilibrated to atmospheric pressure by admitting sterile air. Untreated control plates and plates treated with the vapour-phase positive control dichloromethane were also prepared and were placed in stainless steel vessels under identical conditions.

The remaining positive control plates were prepared by placing aliquots (0.1 ml) of each control compound in sterile glass tubes . Where appropriate, 0.5 ml S9 mix or 0.1 M sodium phosphate buffer was added to each tube. Molten top-agar (2 ml) containing 0.5mM histidine/0.5mM biotin or 0 .5mM tryptophan, maintained at 45*C, and bacterial culture (0.1 ml) were then added, the tubes were inverted to mix thoroughly and the contents poured
onto plates containing minimal agar (25 ml).
Further plates were prepared without the inclusion of the test organisms to verify the sterility of the S9 mix and the test material. Aliquots (0.1 ml) of a 10e-6 dilution of the overnight cultures were spread on the surface of plates of nutrient agar to measure the viability and cell-density of each culture.
All plates were incubated at 37°C for 2 days. After this period, the plates were removed from the vessels and incubated for a further day to permit the growth of revertant colonies. After incubation, numbers of revertant colonies were counted with an automated colony counter. Total colonies on nutrient plates were counted in the same way. The minimal plates were also examined for the presence of a background lawn of non-revertant colonies. The control plates were checked for the absence of growth on sterility checks or normal counts on untreated controls in the presence and absence of S9 mix.
All plates and tubes were identified by the use of numbers indelibly marked on the plates and test tube racks.

Second test
The second test was conducted in exactly the same way as the first test, except that the following nominal concentrations were used: 1.25%, 2.5%, 5%, 7.5% and 10% v/v. The reason for the change was to include a concentration between 5% (where no toxicity was shown) and 10% (where toxicity was evident).

Additional test
A third test, in the absence of S9 mix only, but otherwise conducted in exactly the same way as the second test, was performed in an attempt to verify the actual test atmosphere concentrations vs. the nominal concentrations in the absence of S9 mix. This was considered necessary because no analytical data were obtained in the first main test in the absence of S9 mix (because of technical difficulties) and there was apparent loss of test material during the exposure period in the second main test in the absence of S9 mix with 5% and 7.5% atmospheres and in the presence of S9 mix with 5%, 7.5% and 10% atmospheres.
While this attempt was an apparent failure from an analytical viewpoint, because values obtained for achieved SD957 concentrations in the third test were generally low, the reproducibility of the biological response was clearly demonstrated.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY (RANGE-FINDING) TEST
The mean values quoted have been corrected to the nearest whole number.

Sterility checks, spontaneous reversion rate and viability checks
The absence of colonies on SD957 and S9 mix sterility check plates indicates that these preparations were free of microbial contamination.
The total colony counts on plates numbered 21 confirmed the viability and high cell density of the cultures of the individual organisms. The counts recorded on appropriate negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains and the absence of effects on these rates of inclusion.

Mutagenic activity of positive control chemicals
Appropriate positive control chemicals (with S9 mix where required) induced marked increases in revertant colony numbers in the appropriate strains, confirming sensitivity of the cultures and activity of the S9 mix.

Effect of SD957
No increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to SD957 at nominal concentrations from 10% to 90% in either the presence or absence of S9 mix. A marked reduction in revertant colony numbers was observed in all strains following exposure to SD957 at a nominal concentration of 10%, in both the presence and absence of S9 mix. At all higher concentrations, revertant colony numbers were reduced to zero. A top exposure concentration of 10% was therefore selected for use in the main mutation tests.

MAIN MUTATION TEST 1
The mean values quoted have been corrected to the nearest whole number.

Sterility checks, spontaneous reversion rate and viability checks
The absence of colonies on SD957 and S9 mix sterility check plates indicates that these preparations were free of microbial contamination.
The total colony counts on plates numbered 21 confirmed the viability and high cell density of the cultures of the individual organisms. The counts recorded on appropriate negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains and the absence of effects on these rates of inclusion.

Mutagenic activity of positive control chemicals
Appropriate positive control chemicals (with S9 mix where required) induced marked increases in revertant colony numbers in the appropriate strains, confirming sensitivity of the cultures and activity of the S9 mix.

Effect of SD957
No increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to SD957 at nominal concentrations of 0.625%, 1 .25%, 2.5%, 5% and 10% in either the presence or absence of S9 mix.
A marked reduction in revertant colony numbers was observed in all strains following exposure to SD957 at a nominal concentration of 10%, in both the presence and absence of S9 mix.

Results of chemical analysis of test atmospheres
Because of technical difficulties, no results are available for analysis of the test atmospheres in the first main test conducted in the absence of S9 mix.
The results of the analysis of test atmospheres in the first main test conducted in the presence of S9 mix indicate that, in general, the average achieved concentrations were 113-137% of those intended.

MAIN MUTATION TEST 2
The mean values quoted have been corrected to the nearest whole number.

Sterility checks, spontaneous reversion rate and viability checks
The absence of colonies on SD957 and S9 mix sterility check plates indicates that these preparations were free of microbial contamination.
The total colony counts on plates numbered 21 confirmed the viability and high cell density of the cultures of the individual organisms. The counts recorded on appropriate negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains and the absence of effects on these rates of inclusion.

Mutagenic activity of positive control chemicals
Appropriate positive control chemicals (with S9 mix where required) induced marked increases in revertant colony numbers in the appropriate strains, confirming sensitivity of the cultures and activity of the S9 mix.

Effect of SD957
No increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to SD957 at nominal concentrations of 1 .25%, 2.5%, 5%, 7 .5% and 10% in either the presence or absence of S9 mix.
A marked reduction in revertant colony numbers was observed in all strains following exposure to SD957 at a nominal concentration of 10%, in both the presence and absence of S9 mix.

Results of chemical analysis of test atmospheres
The results of the analysis of test atmospheres in the second main test conducted in the presence and absence of S9 mix indicate that, in general, the average achieved concentrations were 90-117% of those intended.

ADDITIONAL MUTATION TEST
The mean values quoted have been corrected to the nearest whole number.

Sterility checks, spontaneous reversion rate and viability checks
The absence of colonies on SD957 sterility check plates indicates that these preparations were free of microbial contamination.
The total colony counts on plates numbered 21 (see Tables) confirmed the viability and high cell density of the cultures of the individual organisms. The counts recorded on appropriate negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains and the absence of effects on these rates of inclusion.

Mutagenic activity of positive control chemicals
Appropriate positive control chemicals induced marked increases in revertant colony numbers in the appropriate strains, confirming sensitivity of the cultures.

Effect of SD957
No increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to SD957 at nominal concentrations of 1 .25%, 2 .5%, 5%, 5 .5% and 10% in the absence of S9 mix.
A marked reduction in revertant colony numbers was observed in all strains following exposure to SD957 at a nominal concentration of 10%.

Results of chemical analysis of test atmospheres
Values obtained for the achieved concentration of SD957 in this third main test conducted in the absence of S9 mix were 56-63% of those intended. The reason for the unusually low values is unknown, but it is suspected that the characteristics of the GC column used for this analysis were different from those of the column used for the rest of the study which resulted in artificially low analytical values. This is supported by the cytotoxicity profile exhibited by the bacterial strains. In addition, it strongly suggests that the exposures were approximately two times higher than the analytical values.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Although some apparently low values for achieved concentrations were obtained in the chemical analyses, the results obtained in the mutation tests were highly reproducible. In this regard, the highest exposure conditions produced desired levels of cytotoxicity and demonstrated acceptable

levels of exposure in the test system. It is therefore concluded that the test material, SD957, did not exhibit any mutagenic activity under the conditions of the test.