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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several in vitro genotoxicity studies are available, including GLP guideline studies . Four Ames tests (using 4 -5 strains) are available, all showing negative results. Negative results were also obtained in an in vitro CAT and in an in vitro transformation assay. Two MLA studies are available: one key GLP study (1987) using piperazine phosphate (neutralized form) and one supporting study from 1980 (only an internal QA statement) using piperazine. The GLP study was negative up to concentrations of 400 ug/mL; in the 1980 study a positive result was seen at 400 ug/mL (with S9 -mix only) in the presence of cytotoxicity whereas the concentration of 500 ug/mL was too cytotoxic as to produce relevant results.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A well performed GLP guideline in vivo MN study is available.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In the EU RAR (2005) the following was stated:

In vitro genotoxicity

Using the strains TA 1535, TA 1537, TA 98, and TA 100, piperazine tested at the concentrations 33, 100, 333, 1,000, or 2,167 μg/plate was found to be negative in the Salmonella typhimurium reverse mutation test with and without metabolic activation (Haworth et al., 1983). In a study with piperazine phosphate conducted in accordance with OECD test guideline requirements these results could be confirmed (Marshall, 1986) using strains TA97 and TA98 (frameshift mutations) as well as with TA 100 and TA1535 (base-pair substitution) with concentrations ranging from 8 -5,000 μg/plate. Neither the citrate, adipate, mebendazole or thiabendazole salts of piperazine were found to induce reverse mutations, mitotic recombination, or gene conversion in Saccharomyces cervisiae (Hennig et al., 1987).

At concentrations ranging from 1.7 to 110 mg/ml, piperazine phosphate was also found to lack clastogenic properties in cultivated Chinese hamster ovary cells in presence and absence of metabolic activation in a GLP study (Allen et al., 1986). Conaway et al. (1982) reported, that piperazine induced mutations in the L5178 mouse lymphoma test upon metabolic activation in a poorly documented study.

However, in another mouse lymphoma test using test solutions containing 200, 250, 300, 350, and 400 μg/L of piperazine phosphate, negative results were reported both with and without metabolic activation (Cole and Arlett, 1976). A weak activity with respect to the induction of 6 -thioguanine resistance was subsequently found in the presence of rat-liver microsomes in an adequately reported guideline mouse lymphoma fluctuation assay conducted according to GLP and using piperazine phosphate at a concentration of 400 µg/L, but these increases were within the historical solvent control range, and lacked reproducibility (Kennelly, 1987).

Note: In Kennelly (1987) reference was made to the method used by Cole and Arlett (1976), so this is not a separate study.

In vivo genotoxicity

Upon dosing groups of CD-1 mice orally with 5,000 mg piperazine phosphate per kg, no significant increase in the level of micronuclei of polychromatic or normochromatic erythrocytes of the bone marrow could be detected in an adequately performed GLP study (Marshall, 1987). In an initial toxicity range-finder study, two male and 2 female mice each received the test article orally at a dose of 4,000, 4,500 and 5,000 mg/kg. No lethality was observed at 5,000 mg/kg, a dose that was subsequently utilised in this micronucleus test. Carboxymethyl cellulose in distilled water served as negative control. Cyclophosphamide (CPA), dissolved in water and administered orally at 80 mg/kg to one group of 5 male and 5 female mice which were killed after 48 hours provided the positive control. Groups of 5 male and 5 female mice treated at 5,000 mg/kg piperazine were sacrificed and sampled after 24, 48 and 72 hours. In general, positive control animals exhibited toxicity in the bone marrow as seen by an increased proportion of normochromatic erythrocytes (NCE), and increased numbers of micronucleated polychromatic erythrocytes (PCE) and NCE such that the micronucleus frequency in the positive control group was significantly greater than in controls (p < 0.001). Negative control mice exhibited normal ratios of PCE to NCE with group means for males and females ranging from 0.9 to 1.59, and normal frequencies of micronucleated PCE (mean 1.2 -2.8/1,000) and NCE (range 0.32 -1.8/1,000). Mice treated with piperazine phosphate exhibited ratios of PCE to NCE and frequencies of micronucleated PCE and NCE which were similar to controls. Group mean PCE/NCE ratios ranged from 1.16 to 2.04; mean frequencies of micronucleated PCE were 0.8 -2.8 per 1,000 and of micronucleated NCE, 0.9 -2.85. No statistically significant treatment-related increase in micronucleus frequency was found in any of the animals receiving piperazine phosphate at any sampling time.

Wistar rats were partially hepatectomized and the liver labeled during regeneration using tritiated tymidine. After 2 weeks a single dose of 50 mg piperazine, 10 -50 mg/kg N,N-dinitrosopiperazine were administered by i.p. injection. Liver DNA was isolated and single and double strand breaks deteermined by the alkaline elution technique. Whereas the dinitrosopiperazine gave positive results, there was no indication of any DNA damage induced by piperazine as such (Stewart and Farber, 1973). Likewise, piperazine alone was without effect in the host-mediated S. typhimurium (TA 1950) mouse assay (Braun et al., 1977).

N-mononitrosopiperazine (NPZ) as well as N,N'-dinitrosopiperazine (DNPZ) have been found to induce mutations in vivo in the host-mediated Salmonella typhimurium mouse assay (Zeiger et al., 1972). Further, using this assay a positive response was also obtained upon co-administration of piperazine dihydrochloride and nitrite (Braun et al., 1977).

Conclusion in RAR (2005):

Studies conducted in vitro, as well as in vivo indicate that piperazine does not induce point mutations or chromosome aberrations.

Comments to the currently available studies:

The additional strains mentioned in the current OECD TG 471, viz. E. coli WP2 uvrA, E. coli WP2 uvrA (pKM101) or S. typhimurium TA102, have been added to OECD TG 471 to increase the sensitivity of the Ames assay in order to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, the structure of Piperazine does not give rise to such concerns. Piperazine is not a hydrazine. In addition, mechanistic profiling (QSAR Toolbox version 4.4.1) does not show alerts for DNA reactivity pointing at possible oxidizing mutagenic or crosslinking effects:

QSAR Toolbox v. 4.4.1:

Piperazine, CAS 110-85 -0

General mechanistic:

DNA binding by OECD:Shiff base formers - Chemicals Activated by P450 to Glyoxal

Structural alert: Ethylenediamines (including piperazine)

Mechanism

Ethylenediamine and derivatives have been suggested to be metabolised to Glyoxal by cytochrome P450 (Combourieu B et al 2000, Enoch SJ et al 2009, Marqui C 2001). Glyoxal can undergo multiple Schiff base reactions resulting in DNA cross-linking.

Structural alert mitigating factors

·        No mitigating factors have been reported for the chemicals in this mechanistic alert 

References

Combourieu B et al (2000) Applied and Environmental Microbiology, 66, p3187-3193

Enoch SJ et al (2009) Chemical Research in Toxicology, 22, p1447-1453

Marqui C (2001) Journal of Agricultural and Food Chemistry, 49, p4676-4681

Thus, only one profiler is triggered, viz. DNA binding by OECD: Shiff base formers - Chemicals Activated by P450 to Glyoxal.The relevance of this alert is limited because ADME data on Piperazine are indicating that most of the substance is excreted unchanged. (Study in pigs: “the major part of the resorbed compound is excreted as unchanged piperazine during the first 48 h”). Noteworthy is that specifically ‘DNA binding by OASIS’ and also other genotoxicity alerts are not triggered.

Secondly, the possible metabolite of concern is Glyoxal. In the Ames test, Glyoxal has indeed shown a weak positive response with and without S9 in strain TA102, as well as a slight increase in mutants with S9 in E. coli WP2 uvr A but at cytotoxic concentrations.

Thus, Glyoxal induced a stronger response in all studies in strain TA100, which strain has been used in the tests with Piperazine, and which did not result in a positive response.

There is an additional alert for H-acceptor-path3-H-acceptor for in vivo mutagenicity (Micronucleus) by ISS (see also at item B1). This micronucleus alert is not predictive for bacterial mutagenicity, and, in addition, the predictive value is very low considering that an overwhelming majority of all substances in Toolbox firing this alert are negative in in vitro genotoxicity testing.

Also, DEREK (Derek Nexus version 6.1.0) classifies Piperazine as inactive for ‘Mutagenicity in vitro in bacterium’, with “no misclassified or unclassified features”.

In conclusion, Piperazine does not contain structural elements that are linked to oxidizing mutagens, cross-linking agents or hydrazines. Even when the possible mutagenic metabolite Glyoxal could have been formed in the test, this would have been picked up by strain TA100, which means that a test with the additional strain TA102 or E. coli WP2 uvrA would be of no added value.

In the MLA (1980) study report it was mentioned that a dark pink coloration (due to the phenol red component) was noted which indicated an alkaline pH at concentrations exceeding approximately 125 to 313 ug/ml.

A solution of phenol red is often used as a pH indicator in cell cultures. Its color exhibits a gradual transition from yellow (labda max = 443 nm) to red (labda max = 570 nm) over the pH range 6.8. to 8.2. Above pH 8.2, phenol red turns a bright pink (fuchsia color) (From <https://en.wikipedia.org/wiki/Phenol_red>). It can therefore be concluded that exposures above 300 ug/mL have a pH > 8.2

With regard to the possible impact of pH on the mutation frequency in the MLA study the following is noted:

Current OECD TG 490:

8. Care should be taken to avoid conditions that could lead to artifactual positive results (i.e. possible interaction with the test system) not caused by interaction between the test chemical and the genetic material of the cell; such conditions include changes in pH or osmolality, interaction with the medium components (20) (21), or excessive levels of cytotoxicity (22) (23) (24). Cytotoxicity exceeding the recommended top cytotoxicity levels as defined in paragraph 28 is considered excessive for the MLA and TK6.

Measuring cytotoxicity and choosing treatment concentrations

22. When determining the highest test chemical concentration, concentrations that have the capability of producing artifactual positive responses, such as those producing excessive cytotoxicity (see paragraph 28), precipitation (see paragraph 29) in the culture medium, or marked changes in pH or osmolality (see paragraph 8), should be avoided. If the test chemical causes a marked change in the pH of the medium at the time of addition, the pH might be adjusted by buffering the final treatment medium so as to avoid artifactual positive results and to maintain appropriate culture conditions.

Effect of pH shifts on the mutant frequency at the thymidine kinase locus in mouse lymphoma L5178Y TK+/− cells MA Cifone et al., 1987, Mutation Research/Genetic Toxicology, Volume 189, Issue 1, September 1987, Pages 39-46 https://doi.org/10.1016/0165-1218(87)90031-0 In this study, in the absence of S9 mix, no changes in mutant frequency were observed over a pH range of 6.4 -9.2; a small, 1.9 -fold increase was observed for a moderately toxic treatment (24% relative growth) at pH 6.3. However, in the presence of S9 mix, the mutant frequency increased sharply for pH values below 6.8. At pH 6.4, a 4 -fold increase was induced, and pH 6.0 resulted in a 10 -fold increase in mutant frequency. Basic pH shifts in the presence of S9 mix caused no changes in mutant frequency up to pH 8.0; treatment with pH 8.8 was highly toxic (5.3% relative growth) and caused a 3-fold increase in mutant frequency. Thus from the results of this study it can be concluded that at pH 6.4 and lower can result in an increase in mutant frequency. Also at high (alkaline) pH an increase has been seen, viz. no effect up to pH 8.0 and a 3 -fold increae at pH 8.8. However, at that pH there is a high increase in toxicity (at pH 8.8 with 5.3% relative growth, toxicity too high for normal evaluation.

This pH effect may have caused the positive result in case of testing piperazine (Myhr, 1980), such effect was not present when using the neutralized piperazine phosphate (1987 study) using similar test concentrations.

Based on the available genotoxicity data in vitro and in vivo it can be conluded that piperazine is not a genotoxic compound and as such should not be classified.

Again, in the EU RAR (2005) referring to the same studies, it was concluded that the studies conducted in vitro, as well as in vivo, indicate that Piperazine does not induce point mutations or chromosome aberrations. MAK (1998) also referring to the same studies, did not conclude a concern for Piperazine with regard to genotoxicity. And finally, the European Agency for the Evaluation of Medicinal products (EMEA, 2002) – Committee for Veterinary Medicinal Products - concluded that (based on the same studies) a series of GLP compliant mutagenicity studies in prokaryotic and eukaryotic cells, both in vitro and in vivo has been completed and showed no evidence of mutagenic effect [EMEA/MRL/807/01-FINAL].

 

Justification for classification or non-classification

Based on the available genotoxicity data in vitro and in vivo it can be conluded that piperazine is not a genotoxic compound and as such should not be classified.