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EC number: 239-701-3
CAS number: 15625-89-5
experimental in vitro data are available to assess the genetic toxicity
of trimethylolpropane triacrylate (TMPTA).
TOXICITY IN VITRO
mutation in bacteria in vitro:
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
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.
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.
in mammalian cells in vitro:
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.
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.
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.
mouse lymphoma assays similar to OECD 476 were conducted to assess the
mutagenic potential of the test substance on the TK +/-, locus of the
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.
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.
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.
evidence from Literature (Johannson et al., 2008):
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
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
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
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).
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.
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.
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