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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Piperazine
EC Number:
203-808-3
EC Name:
Piperazine
Cas Number:
110-85-0
Molecular formula:
C4H10N2
IUPAC Name:
piperazine
Specific details on test material used for the study:
Piperazine phosphate CAS 1951-97-9 / EC 217-775-8 was used.
Piperazine phosphate (also referred to as ASCAREX P) was a white crystalline powder, batch WS 84/023.
The sample was 99.6% pure.
It was received on 25th April, 1986 and stored at room temperature in the dark.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
Liver S-9 mix
Test concentrations with justification for top dose:
For dose range-finder: 10, 2, 0.4, 0.08 and 0.016 mg/ml to give final concentrations of 5000, 1000, 200, 40 and 8 ug/plate
For mutation assay 1: 10, 2, 0.4, 0.08 and 0.016 mg/ml to give final concentrations of 5000, 1000, 200, 40 and 8 ug/plate
For mutation assay 2: 10, 5, 2, 0.4, 0.08 and 0.016 mg/ml to give final concentrations of 5000, 2500, 1000, 200, 40 and 8 ug/plate
Ttop dose is equivalent to maximum of 5 mg/plate.
Vehicle / solvent:
Distilled water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
Posiitive controls:
TA97: 9-Aminoacridine (AAC) (50 ug/plate - S-9)
TA97: 2-Aminoanthracene (AAN) ( 5 ug/plate + S-9)
TA98: 2-Nitrofluorene (2NF) (50 ug/plate - S-9)
TA98: 2-Aminoanthracene (AAN) ( 5 ug/plate + S-9)
TA100: Sodium Azide (NaN3) ( 2 ug/plate - S-9)
TA100: 2-Aminoanthracene (AAN) ( 5 ug/plate + S-9)
TA1535: Sodium Azide (NaNa) ( 2 ug/plate - S-9)
TA1535: 2-Aminoanthracene (AAN) ( 5 ug/plate + S-9)
Rationale for test conditions:
Toxicity range-finder
No more than 48 hours before test, a nutrient broth culture of strain TA100 was grown up overnight from a master plate which had been checked for strain characteristics (histidine dependence, rfa character and presence of pKM101l ampicillin resistance factor) according to Maron and Ames. Dilutions of piperazine phosphate were freshly prepared in DMSO as to give final concentrations of 5000, 1000, 200, 40 and 8 ug/plate together with a solvent control. Each dose was treated in triplicate with and without liver S-9; controls were treated in quintuplicate. Piperazine phospate treatments of TA100 at 5000 ug/plate, both in the absence and presence of S-9, were found to be non-toxic. A dose of 5000 ug/plate was therefore chosen as the top dose for all the tester strains in the mutation assays.

Mutation assays
Strains TA97, TA98, TA100 and TA1535 were checked and grown up as above on 2 separate occasions. As the range-finder revealed 5000 ug/plate to be a non-toxic dose, piperazine phosphate was dissolved in distilled water as to give final concentrations of 5000, 1000, 200, 40 and 8 ug/plate. For the second experiment strain TA100 was additionally treated with 2500 ug/plate piperazine phosphate in the absence of S-9.

The ingredients were rapidly mixed together and poured on to Minimal Davis agar plates. When the agar had set, plates were taped together in stacks, inverted and incubated at 37 degC in the dark for at least 2 days.
Colonies were counted using either a Biotran II electronic colony counter or manually and the background lawn of growth was inspected for signs of toxicity.

Each dose was tested in triplicate as described above, (solvent and
positive controls were tested in quintuplicate) and colony counts were
recorded on raw data forms in the study file.
Evaluation criteria:
Individual plate counts from both experiments were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined.
Spontaneous revertants on control plates are approximately normally distributed, such that 95% and 99% confidence limits can be established for historical control data to form a normal range and, for our laboratory, this appears in Table 5.
Statistics:
For evaluation of test agent and positive control data there are many statistical methods in use, of which no one method has been particularly recommended. For data of the kind produced in this type of study, analysis of variance (the F-test) is appropriate. This testdetermines whether different concentrations of test agent make a significantly greater contribution to the observed variation in revertant numbers than the variation due to replication of the treatments. In addition, this test shows whether the numbers of revertants resulting from treatment with positive control chemicals are significantly greater than those observed in solvent controls.
This method of data analysis has two advantages compared, for example, with Student's t—test. Firstly, the method enables each experiment to be analysed independently as there is no necessity to pool data from more than one experiment in order to generate sufficient statistical power and sensitivity. Secondly, the analysis incorporates all the data from one tester strain either with or without S-9 into one statistic, and thus it is less likely that small increases in revertant numbers of doubtful biological
significance will give rise to a statistically significant result.
If the observed variance ratio is statistically significant (p < 0.05) in any one experiment, sequential linear regression analyses, as suggested by Venitt, are performed on each set of three or more dose points in each experiment using the strain. Correlation coefficients are determined and assessed for statistical significance in order to determine whether a dose response has occurred.
A test compound would be considered positive in the assay if, in at least one strain and in more than one experiment, a significant F—test together with a significant dose response occurred.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The top dose of 5000 ug/plate chosen for the mutation assays proved to be non toxic except for strain TA1535 minus 8-9 in Experiment 2. For purposes of evaluation the means and standard deviations for each treatment in both experiments were calculated and are summarised in Tables 1 to 4 for strains TA97, TA98, TA100 and TA 1535, respectively (attached).

Applicant's summary and conclusion

Conclusions:
In a bacterial reverse mutation assay results were negative in Salmonella strains TA1535, TA97, TA98 and TA100, with and without metabolic activation.
Executive summary:

In a bacterial reverse mutation assay performed in principle according to OECD Guideline 471, piperazine phosphate was assayed for mutation in four histidine requiring strains (TA97, TA98, TAIOO and TA1535) of Salmonella typhimurium both in the absence and presence of metabolic activation by an Aroclor-1254 induced rat liver post-mitochondrial fraction (S-9).

An initial toxicity range-finder experiment was carried out in TA100, using final concentrations of piperazine phosphate at 5000, 1000, 200, 40, and 8 ug/plate plus a solvent control. No piperazine phosphate treatment proved to be

toxic, so the same concentration range was used for all strains in the main mutation assays.

The four tester strains of bacteria were treated in triplicate with piperazine phosphate as described above in the absence and presence of S -9 in two separate experiments. For the second experiment, an extra treatment of TA100 with 2500 ug/plate piperazine phosphate was included in the minus S-9 incubations. Both experiments included solvent controls and appropriate positive control chemicals all treated in quintuplicate. In each experiment and for each strain, the means of the numbers of revertants/plate in solvent control incubations were within the 99% confidence limits of pooled historical negative control data.

All chemicals used in positive control checks, in the absence or presence of liver S-9, induced marked increases in revertant numbers. These results validated the assay, confirming the mutability of the tester strains and the

metabolic capacity of the S -9 mix employed.

No treatment with piperazine phosphate gave rise to a statistically significant dose-related rise in mutation that could be reproduced in the second experiment. It is concluded that piperazine phosphate has no mutagenic activity in this assay system under these experimental conditions.