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Genetic toxicity in vitro

Description of key information

Valid experimental in vitro data are available to assess the genetic toxicity of trimethylolpropane triacrylate (TMPTA).

GENETIC TOXICITY IN VITRO

Gene mutation in bacteria in vitro:

Trimethylolpropane triacrylate (TMPTA) was tested for mutagenicity in the Ames standard plate test according to OECD 471 both in the presence and absence of rat liver S9 using the strains TA 1535, TA 100, TA 1537, and TA 98 (BASF, 1989). Without metabolic activation no increase in the number of revertant was observed up to 5000µg/plate, the highest dose tested. With S9-mix, there was a doubtful positive reaction for TA 1535 only. The detected increase varied between a factor of 1.6 and 4.8 with no dose dependency.

In a second Ames test similar to OECD471, no mutagenicity was observed in the above-mentioned strains TA 1535, TA 100, TA 1537, and TA 98 as well as in TA1538 and Saccharomyces cerevisiae strain D4 with or without rat liver S9 (Cytec 1976). The highest tested concentration was 10µl/plate and thus exceeded the maximum dose in the previously described assay.

In conclusion, because of lacking dose response and because the result was not confirmed in a second study report even tested at higher concentrations, the questionably positive result is not considered biologically relevant. Trimethylolpropane triacrylate is thus not mutagenic in bacteria.

Studies in mammalian cells in vitro:

The ability of TMPTA to induce point mutations in mammalian cells was investigated in an HPRT assay using CHO cells (Moore 1989). The protocol was similar to OECD 476, but no metabolic activation system was used. No increase in mutant frequency occurred, but consistent with results for other multifunctional acrylates, cytotoxicity was observed at low concentrations, ranging from 72% survival at 0.2µg/mL to 13% survival at 0.7µg/mL. In this study, chromosome aberrations were additionally examined. An increase in aberrations was observed at concentrations leading to approximately 50% or higher cytotoxicity. These results confirm the results of the mouse lymphoma assays described later on and experiences with other multifunctional acrylates in vitro.

An in vitro mammalian cell chromosomal aberration study was carried out with trimethylolpropane triacrylate in accordance with OECD TG 473 (Cytec, 2005). Primary human blood cells from two donors were incubated with the test substance at the following concentrations that were determined in a pre-test to achieve the maximum allowed cytotoxicity.

Cytotoxicity ranged from 45 to 100% in the first experiment and 26 to 83% in the second experiment at concentrations ≥ 12.5μg/mL. Statistically significant and concentration-related increases in the frequency of cells with structural chromosomal aberrations were noted with (≥ 9.38μg/mL) and without (> 28.13μg/mL) metabolic activation.

Two mouse lymphoma assays similar to OECD 476 were conducted to assess the mutagenic potential of the test substance on the TK +/-, locus of the L5178Y cells.

In the first assay, three independent experiments were carried out in the presence or absence of Aroclor induced rat liver S9 (Cytec 1979). The concentrations were selected based on a pre-study to achieve cytotoxicity in the highest dose between 10-20%. As discussed in detail in the corresponding study entry, the test substance consistently increased mutant frequencies only in the absence of S-9 mix and only at concentrations leading to a reduction in relative total growth to at least 33%. In the presence of S-9 activation, no increase in mutant frequency was observed at concentration leading to severe cytotoxicity (minimum total growth of 14.9%), but which were still within the acceptance criteria for this assay that require a total growth above 10% for the assay to be valid.

Similar results were also obtained in by Dearfield and Moore (1989), though no metabolic activation system was used in their studies. In two independent experiments a dose dependent increase in mutant frequency was only obtained at doses showing about 50% cytotoxicity or more. Additionally, performed colony sizing indicated, that trimethylolpropane triacrylate almost exclusively induced small colonies; the number of large colonies remained at control level. This indicates, that the test substance did not induce point mutations, but acts clastogenic in vitro.

In summary, results from all in vitro studies showed that trimethylolpropane triacrylate induced chromosome aberrations in human lymphocytes and L5178Y cells in vitro. No indications for an increase in point mutations were observed in assays in bacteria or mammalian cells.

Supporting evidence from Literature (Johannson et al., 2008):

It is published that no evidence of point mutations was observed when more than 60 acrylates and methacrylates including acrylic acid were investigated in Salmonella bacterial tests or in hprt mammalian cell gene mutation tests. Also, these substances did not induce clastogenicity or aneuploidy in in vivo studies. Consistent with the in vivo testing results, acrylic acid exhibited no evidence of carcinogenicity in chronic rodent cancer bioassays. Nevertheless, positive results were often observed in vitro in the mouse lymphoma assay or other in vitro assays designed to detect clastogenicity. All substances for which colony sizing was performed in the mouse lymphoma assay, only small colonies were induced that indicate a clastogenic but not point-mutagenic potential. But the biological relevance of this in vitro response is questioned based on the non-concordance of in vitro and in vivo studies results.

Overview of available studies:

Gene mutation in bacteria:

S. typhimurium TA 1535, TA 100, TA 1537 and TA 98, with and without metabolic activation, OECD 471: positive with MA in TA 1535 all other negative (BASF, 1989)

S. typhimurium TA1535, TA 1537, TA 1538, TA 98 and TA 100 as well as Saccharomyces cerevisiae strain D4, with and without metabolic activation: negative (Cytec 1976).

Gene mutation and cytogenicity in mammalian cells:

HPRT without metabolic activation: negative (Moore et al. 1989)

Mouse Lymphoma, L5178Y, with and without metabolic activation OECD guideline 476: positive 1.0-2.5nL/mL without metabolic activation (Cytec 1979)

Mouse Lymphoma L5178Y without metabolic activation: exclusive induction of small colonies, clastogenic in vitro at cytotoxic concentrations (Moore et al., 1989, Dearfield et al., 1989)

Chromosome Aberration, human lymphocytes, with and without metabolic activation OECD 473: positive ≥ 9.38 μg/mL with metabolic activation and positive ≥ 28.13 μg/mL without metabolic activation (Cytec)

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

Genetic toxicity in vivo

Description of key information

Valid experimental in vivo data are available to assess the genetic toxicity of trimethylolpropane triacrylate (TMPTA).

GENETIC TOXICITY IN VIVO

In vivo cytogenicity and genotoxicity

In a micronucleus study according to GLP and OECD 474 four groups of 5 male and 5 female Swiss Ico: OF1 mice received a single oral dose of the test substance in corn oil of up to 1750 mg/kg for males and up to 2000 mg/kg for females (Haddouk H, 2006). Doses were selected in a preliminary toxicity test, in which 2 of 3 males died at 2000 mg/kg bw. Vehicle and cyclophosphamide (50mg/kg) treated mice served as negative and positive controls. The animals were killed 24 (positive and vehicle control, low, intermediate and high dose groups) or 48 h (vehicle control and high dose groups) after treatment. In males, no clinical signs and no mortality attributed to the treatment were observed in vehicle controls and low dose males. At 875 mg/kg bw, piloerection was noted and at 1750 mg/kg bw, two males were found dead after 24 h. Piloerection was noted in the surviving males. In females, no clinical signs or mortality were observed. Mean values of micronucleated cells (MPE) as well as the PE/NE ratios in the treated groups were equivalent to those of the control group for both harvest times. Cyclophosphamide induced a significant increase in the frequency of MPE, demonstrating the sensitivity of the test system under the experimental conditions of this study. In conclusion trimethylolpropane triacrylate (TMPTA) was considered to be non-mutagenic in the micronucleus test.

In 2005, the NTP dermally exposed groups of either 10 males and 10 female B6C3F or 15 male and 15 female Tg. AC hemizygous mice to 0.75 – 12 mg/kg trimethylolpropane triacrylate in acetone. Mice were treated 5 times per week for either 14 weeks (B6C3F) or 28 weeks (Tg. AC hemizygous). Doses were selected based on skin effects in a 2-weeks range finding study. No increase in the frequency of micronucleated NCEs was observed in peripheral blood samples from male or female mice. In the 3-month study, ratios of micronucleated polychromatic erythrocytes to NCEs in peripheral blood were unaltered by chemical treatment, indicating an absence of induced bone marrow toxicity. However, in the 6-month study, decreases in the percentages of circulating NCEs among total erythrocytes were noted in 12 mg/kg male and female mice, indicating a stimulation of erythropoiesis and the presence of increased numbers of immature erythrocytes in circulating blood. As a negative study this study support the lack of genotoxic potential of TMPTA. However, it should be acknowledged that use of transgenic mice is no longer an accepted model and also the use of acetone as vehicle is not considered appropriate for repeated dermal exposure due defatting and scaling of the skin resulting in artificial increase of dermal penetration of TMPTA. However, it may be noted that although both of these aspects increase the sensitivity of the test no genotoxic effect was found in the study.  

To further examine the potential for chromosome aberration as observed in vitro the toxicity of TMPTA with respect to DNA damage in bone marrow and liver cells was evaluated in vivo according to OECD TG 489 (In Vivo Mammalian Alkaline Comet Assay, July 2016). Female mice were - in order to ensure full bioavailability - dosed intravenously to TMPTA at the dose levels of 0, 5, 10, 20 mg/kg bw with PEG400 as vehicle (the administration divided on two injections with an interval of 23.5 hours).Ethyl methanesulfonate was used as positive controls. The treatment, did not induce increases in tail intensity (DNA damage) in the liver and bone marrow at any of the dose levels including the highest dose level of 20 mg/kg bw (the maximum tolerated dose) (Covance 2017).

Overview of available studies:

Micronucleus test in vivo, Swiss Ico: OF1 mice, OECD 474: negative, mean MPE as well as the PE/NE ratios in range of control, mortality in two high dose males (Cytec)

Micronucleus test in vivo in B6C3F mice and Tg. AC hemizygous mice, 3 and 6-month dermal application: no increase in micronucleated cells, but changes in the PCE/NCE ratio after 6 months (NTP, 2005).

In Vivo Mammalian Alkaline Comet Assay (OECD TG 489, July 2016). I.v. administration to Female mice. No increases in tail intensity (DNA damage) in liver and bone marrow cells at any of the dose levels including the highest dose level of 20 mg/kg bw (the maximum tolerated dose) (Covance 2017).

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

Additional information

Genotoxicity potential of TMPTA has been assessed in in vitro and in vivo assays.

Although negative results were obtained for point mutations in Ames tests and in an in vitro Mammalian cells mutation assay (HPRT assay in CHO cells), positive effects were found in mouse lymphoma assays (MLA) and in mammalian cell chromosomal aberration assays in CHO cells and in human lymphocytes.

Three MLA assays were available. In the first study (Litton Bionetics, 19793), the results were not consistent between the three experiments: increased mutant frequency was observed in all experiments without metabolic activation, but without any dose-relationship in the third experiment; in the presence of metabolic activation, increase in mutant frequency was only observed in a context of severe cytotoxicity. In a second study (Cameron, 19914), increased mutant frequency was reported only in the absence of metabolic activation at concentration leading to cytotoxicity (RTG comprised from 14.5% to 5%). In these two studies, the size of colonies was not reported to discriminate gene mutation and chromosomal aberration. In the last MLA study (Dearfield, 19895) performed without metabolic activation, a dose dependent increase in mutant frequency was obtained at doses showing about 50% cytotoxicity or more. Colony sizing indicated that TMPTA almost exclusively induced small colonies, suggesting a clastogenic mechanism.

In CHO cells exposed to TMPTA without metabolic activation, chromosome aberrations were increased at concentrations associated with 72% to 13% survival (Moore, 19896). In a more recent and well performed in vitro mammalian cell chromosomal aberration study in human lymphocytes (CIT, 20057), statistically significant and concentration-related increases in the frequency of cells with structural chromosomal aberrations were noted without and with metabolic activation. Therefore, results from in vitro studies showed that TMPTA may induce chromosome aberrations.

Two in vivo micronucleus (MN) studies were available. The first study was performed by the NTP after dermal exposure of B6C3F or Tg.AC hemizygous mice for 14 weeks or 6 months, respectively (NTP, 20058). No increase in the frequency of micronucleated NCEs was observed. Although PCE/NCE ratio was unaltered after 3-month exposure, a decrease in the percentage of circulating NCEs among total erythrocytes was noted at the highest dose in both sexes. However, this study does not follow the appropriate guideline and no positive control was included to validate the protocol. The second study was performed according to OECD 474. Swiss Ico:OF1 mice received a single oral dose of TMPTA in corn oil of up to 1750 mg/kg for males and up to 2000 mg/kg for females (CIT, 20069). Mean values of micronucleated cells (MPE) as well as the PCE/NCE ratios in the treated groups were equivalent to those of the control group for both harvest times. In this study, PCE/NCE ratio was not altered and plasma levels of the test substance were not investigated.

Further, a Comet assay was performed in female mice by intravenous at the doses of 5, 10 and 20 mg/kg/day. Based on the dose-range finding study, the dose of 20 mg/kg/day is considered to be the MTD. Two target organs were evaluated: bone marrow and liver. There were no dose related increases in % hedgehogs in liver and bone marrow, thus demonstrating that treatment with TMPTA did not cause excessive DNA damage that could have interfered with comet analysis. TMPTA didn’t induce biologically relevant increases in tail intensity in the liver or bone barrow.

In summary, In vitro, TMPTA primarily induced clastogenicity, but such an effect was not evident in vivo in OECD test guideline compliant studies. In vivo, there are no studies that examined the induction of gene mutations in experimental animals treated with TMPTA. This endpoint was indirectly addressed using an in vivo comet assay in mice through evaluation of DNA single strand breaks in the bone marrow and liver cells. In this GLP and OECD test guideline compliant study, TMPTA did not induce DNA strand breaks when tested up to a maximum tolerated dose of 20 mg/kg/day (Keig‐Shevlin, 2017). The exposure of the bone marrow and the liver was assured in this study by the use of intravenous dose administration. There was also no evidence for TMPTA‐induced clastogenicity or aneugenicity in vivo. TMPTA did not induce micronuclei in mouse peripheral blood erythrocytes in B6C3F1/N or transgenic Tg.AC mice following dermal application for 14 or 28 weeks, respectively (NTP, 2005). Systemic exposure of TMPTA was assured in these studies based on prior data demonstrating absorption of the test material following dermal application (NTP, 2005). Similarly, TMPTA did not induce micronuclei in bone marrow polychromatic erythrocytes following oral gavage up to the limit dose of (2000 mg/kg bw for females, 1750 mg/kg bw for males) in a GLP and OECD test guideline compliant study (Haddouk, 2006). Although there are no experimental data to establish absorption of TMPTA following oral administration, it is reasonable to assume significant absorption based on the physical‐chemical properties and the dermal absorption data.

Justification for classification or non-classification

The mutagenic, clastogenic, and aneugenic properties of TMPTA were adequately investigated both in vitro and in vivo. In vitro, TMPTA primarily induced clastogenicity, but such an effect was not evident in vivo in OECD test guideline compliant studies. The in vivo comet assay is believed to be a reliable indicator assay for detecting gene mutagens as well as clastogens and this assay was negative with TMPTA. Thus, no data gaps were identified, and the database is sufficient to comprehensively assess the genotoxicity of TMPTA. Based on the available data, it is concluded that although TMPTA is an in vitro clastogen at cytotoxic concentrations, no such activity is likely to occur under normal in vivo conditions because of the cellular protective mechanisms operating in an intact animal. Based on available in vitro and in vivo data package, not classification is warranted.