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There are some studies with HPN, but also further studies with some metabolites and structural analogons:

IN VITRO

In the Ames Test for hydroxy pivalic acid neopentylglyco ester (HPN; 1115-20-4) (BASF, 1979, guideline 471 study with some restrictions) S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 were exposed with and without metabolic activation to dose levels of 0 (vehicle control), 4, 20, 100, 500, 2500 µg/plate. The positive and negative controls were valid. No cytotoxic effects were detected at these dose levels. No increase in revertants was found at any dose level in any strain studied. Conclusion: No mutagenic and no cytotoxic effects with and without metabolic activation at dose levels up to 2500 µg/plate.

In the GLP OECD476 study (BASF SE, 2010), hydroxypivalic acid neopentylglycol ester flakes (HPN) were tested in the in vitro mamalian cell gene mutation test. CHO cells were treated with up to 2100 µg/ml for 4 and 24 h with and without S9 mix. HPN did not lead to a relevant increase in the number of mutant colonies either with or without metabolic activation in 2 independent experiments. The mutant frequencies at any concentration were close to the range of the concurrent negative control values and clearly within the range of our historical negative control data. No cytotoxicity was observed. Vehicle and positive controls were valid. Therefore, HPN flakes is concluded to be not a mutagenic substances in the HPRT locus assay.

In a supporting in vitro chromosoem abberation study (Hatano Research Insitute, 1993) with the metabolite NPG (CAS 126 -30 -7), which was performed according to Japanese guideline and GLP, Chinese hamster cells were exposed +/- metabolic activation to dose levels of 0, 0.25, 0.5, 1.0 mg/ml. The high concentration corresponds to ca. 10 mM, the recommended limit dose according to OECD 471. Numerical and structural aberration were measured. Negative results were found at all dose levels in all trials with different experimental design. No cytotoxic effects were reported at concentrations up to 1 mg/ml. The relevant negative and positive controls were valid. Therefore, no chromosome mutagenic and no aneugenic activity were detected in Chinese hamster cells with and without metabolic activation at concentrations up to the recommended max. dose level of 1 mg/ml (ca. 10 mM).

The the GLP OECD473 study (BASF SE, 2010) hydroxypivalic acid neopentylglycol ester (HPN) was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in vitro +/- metabolic activation. V79 cells were exposed with up to 2050 µg/ml (about 10 mM) for 4 h +/- S9 mix and 18 h sampling time. Negative and positive controls were valid. A slight increase of aberrant metaphase cells observed in the 1st experiment +S9 mix This result was not confirmed in the 2nd experiment. No increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either. HPN, however, caused a statistically significant and biologically relevant increase in the number of structurally aberrant metaphases -S9 mix >= 1367 µg/ml.

 This positive result is unexpected, as chromosoal abberation test of NPG and pivalic acid and the in vivo micronucelus test of the structural analogon texanol were all negative. Beyond this, chemical structure of HPN dose not suggest any clastogenic potential.

One explanation on this in vitro result may be, that HPN was cleaved intracellularly to hydroxy pivalic acid and NPG. Intracellular cleavage resulting in an intracellular pH shift may have caused the effect. This mechanism is expected not to be relevant in the in vivo situation, as shown with the negative in vivo MNT test.

In a preGLP in vitro chromosmal abberation test (summarized in HPV ICCA 2006) a cultured rat liver cell line was incubated with the analogon pivalic acid (CAS 75 -98 -9) with concentrations of 0, 125, 250, 500 μg/ml for 24 h with and without metabolic activation. 100 cells were analyzed from each of 3 cultures/dose group. There was no increased incidence of chromosome aberrations in the treated cells. The top dose level resulted in 50% inhibition of cell growth in the presence of S9. Therfrore, pivalic acid is not genotoxic in rat liver cells in vitro under conditions of this assay.

IN VIVO

This study was performed to investigate the potential of HPN (1115 -20 -4)to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD 474 guideline and GLP (Harlan CCR, 2013).

Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated, based on results of a preexperiment:

24 h preparation interval: 500, 1000, and 2000 mg/kg b.w. and 48 h preparation interval: 2000 mg/kg b.w.

After treatment with the test item several animals showed transient clinical signs such as abdominal position, eyelid closure and/or ruffled fur.

After treatment with the test item the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that Hydroxypivalic acid neopentylglycolester did not exert a cytotoxic effect in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

In conclusion, it can be stated that under the experimental conditions reported, the test item Hydroxypivalic acid neopentylglycolester did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

In the in vivo Microncleus Test (Eastman KC, 1992, OECD/SIDS 1994), with the structural analogon Texanol (CAS 25265 -77 -4) mice were exposed to 200 - 2000 mg/kg bw according to OECD 474 and GLP. No significant increase in micronuclei in bone marrow polychromatic erythrocytes and no effect on Mitotic Index of PCE/NCE ratio were seen under the conditions of this assay in any dose group at any harvested time. The highest does produced transient acute toxicity in females, therefore the test substance was systemically available. Conclusion: The test article is negative in the in vivo mammalian bone marrow micronucleus assay.


Short description of key information:
- in vitro: negative (Ames test + HPRT locus assay), clastogenic (Chromomosome abberation test)
- in vivo: negative (Micronucleus test)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification and labelling concerning is required according to Annex VI of Directive 67/548/EWG or Annex I of Directive 1272/2008 (EU-GHS) as labelling criteria are not met.