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EC number: 212-454-9
CAS number: 818-61-1
acrylate (HEA) did not induce gene mutations in the Salmonella
typhimurium / E. coli reverse mutation assay in the absence and the
presence of metabolic activation. HEA is not a mutagenic substance in
the HPRT locus assay using CHO cells (with and without metabolic
activation). HEA was positive at cytotoxic concentrations in the mouse
lymphoma assay (MLA) and induced an increase in chromosomal aberrations
and micronuclei in L5178Y mouse lymphoma cells. Since in the mouse
lymphoma assay preferentially small colonies were induced, the mutagenic
potential of HEA seems to be limited to clastogenicity.
result tables see attachment
Total no. AB
Cells with AB
AB: aberrations. 100
cells were scored for each data point.
calculated according to the method of Clive & Spector (1975).
The substance induced
a dose responsive increase in aberrations to a high frequency of 99
aberrations/100 cells at 20 µg/mL in the presence of increasing
cytotoxicity (survival at 20 µg/mL 15 %).
Per cent plating efficiency
Total mutant count
Mutant frequency [x10-6]
Per cent survival
Mutant frequency [S/L]
efficiency was based on plating 600 cells.
count is expressed as total mutants per 3 million cells plated in TFT
selection, and mutant frequency is expressed as per 1000000 surviving
cells. Percent survival is calculated according to the method of Clive
and Spector (1975).
small- and large-colony mutant frequencies (S/L) are also expressed as
per 1000000 surviving cells.
In vivo, HEA did not induce mutagenic effects in rat bone marrow cells in a chronic inhalation study. Furthermore, HEA did neither induce gene mutations nor chromosomal damage in a transgenic rodent mutation assay according to OECD TG 488. In addition, a micronucleus test with the structural analogue 2-hydroxypropyl acrylate (HPA) using the oral route in NMRI mice was negative. Based on the present results, HEA is not mutagenic in vivo. No carcinogenic effect was observed in the long-term inhalation carcinogenicity study with 2 -hydroxyethyl acrylate, supporting that 2 -hydroxyethyl acrylate is not mutagenic in vivo.
ratio of normochromatic to polychromatic erythrocytes was slightly
affected by the treatment with 2-hydroxypropyl acrylate at a dose of 600
mg/kg bw (at 24 and 48 hours in male mice and at 48 hours in female
mice). At this dose level, only slight toxic effects, as evidenced by
reduced spontaneous reactivity, were obtained up to 6 hours after
dosing. There was no increase in the frequency of micronuclei at any
dose level at either 24- or 48-hours after dosing compared to the
negative control group.
positive control compound, cyclophosphamide, produced significantly
increased frequencies of micronucleated polychromatic and normochromatic
are the results:
sacrificed at 24 hours:
Mean Micronuclei/2000 PCE
600 mg/kg bw
300 mg/kg bw
100 mg/kg bw
sacrificed at 24 hours:
and Females sacrificed at 48 hours:
600 mg/kg bw males
600 mg/kg bw females
There were no indications of alterations
related to exposure to either level of HEA for 1 year.
Examination of Bone Marrow Cells of Male and Female Rats exposed to
Vapours of 2-Hydroxyethyl Acrylate 5 Days/Week for 12 Months:
Exposure Level [ppm]
No. of Cells Scored
A gene mutation assay (gpt assay
and Spi- assay) with transgenic mice (gpt delta
mouse) was conducted to assess the potential of Hydroxyethyl acrylate to
induce gene point mutation and deletion mutations using the gpt
gene (gpt assay) and the red/gam genes (Spi-
assay) in the liver and stomach.
The test substance was administered to male
transgenic mice orally, once a day, for 28 consecutive days by gavage at
the dosage levels of 25.0, 100, and 275 mg/kg bw/day. After 3 days of
manifestation period, the mutant frequencies in the liver and stomach
were determined. One of seven mice in the 275 mg/kg bw/day group died
on Day 5. Furthermore,
the animals in the 275 mg/kg bw/day group showed a reduced bowy weight
gain which corresponded to approx. 80% of that of the vehicle control,
This observation was, however, not statistically significant. In the
histopathological examination, hyperkeratosis was observed in the
forestomach in the 275 mg/kg bw/day group. The average forestomach
weight in this group also showed a statistically significant increase as
compared to the vehicle control group.
The negative control group values obtained
for all organs were within the acceptable range of the historical
control data and thus considered as valid. In the gpt
assay and Spi- assay, the mutant frequencies in the liver and
stomach of the animals treated with Hydroxyethyl acrylate did not show
any increases as compared to the concurrent negative control value. All
group values were also within the historical control data. In some
cases the individual values surpassed the upper limit of the 95% control
limit of the historical data. However, these increases are considered
as not biologically relevant, since their distribution was sporadic and
the increase was also observed in the control group.
The mutant frequencies in the liver and
stomach in the positive control group, which was treated with benzo[a]pyrene,
were increased and these increases were statistically significant
compared with those of the negative control group. Therefore, the
present study was judged to be properly conducted.
these results, the in vitro genotoxicity profile of HEA is
identical to that of HPA and other short-chain alkyl acrylate. The
following table shows the comparison between these two substances and
the other analogues considered:
Bacterial reverse mutation
Gene mutations in mammalian cells
Negative(HPRT: BASF, 2017b)
Negative(HPRT: Evonik, 1995a)
Negative(Moore 1988 and others; MLA:BASF 2016 )
Clastogenicity in mammalian cells
Positive (cytotoxicity)(Dearfield 1989a,b)
Positive (cytotoxicity)(Evonik 2000)
Positive (cytotoxicity)(Ishidate 1981 and others)
In vivo clastogenicity
In a MLA
study included in the dossier, HEA showed a clastogenic effect at
cytotoxic concentrations; these observations are most probably due to
the high reactivity of the monomer and no real clastogenicity. In an in
vivo transgenic rodent assay, HEA was tested negative for
mutagenicity and clastogenicity as well. In addition, in the
carcinogenicity study performed on HEA the clastogenicity was tested in
parallel and came out negative in vivo. This is supported by
the negative in vivo chromosomal aberration studies / micronucleus assay
with the source chemicals HPA, MA and nBA. Therefore the overall picture
shows no genotoxicity on both HEA and the source substance HPA. Overall
also no carcinogenic effects was found in the long-term study.
common hydrolysis product (AA) of both HEA and HPA show a comparable
genetic toxicity profile in vitro, while the non-common
hydrolysis products (EG, PG) are overall negative. All hydrolysis
products have been tested for genetic toxicityin vivoand showed
AA (acrylic acid)
EG (ethylene glycol)
PG (propylene glycol)
Chromosomal aberrations in mammalian cells
Further details are
given under 13.2 Read-Across Justification
In vitro studies: bacterial systems
The potential to induce mutagenicity in bacteria was assessed in a study according to OECD 471 performed in compliance with GLP criteria (BASF, 2016). In this study the bacterial strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr were exposed to test substance concentrations between 33 µg - 5000 µg/plate (SPT) and 10 µg - 5000 µg/plate (PIT), in the presence and absence of a metabolic activation system (liver S9 mix from rats). A bacteriotoxic effect was observed depending on the strain and the conditions from about 100 µg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed.
In another supporting study, the test substance was not mutagenic in Salmonella typhimurium strain TA100 at 0.01-7.5 mg/plate (Rohm and Haas, 1982), nor in strains TA98, TA100, TA1535, TA1537 and TA1538 (no dose/plate given) with or without metabolic (S9) activation (Dow Chemical Co., 1976). The test substance was not mutagenic in Salmonella typhimurium strains TA102 and TA2638 (0.038 – 5 mg/plate) with or without metabolic (S9) activation, but gave inconclusive results (ca. 2-3 fold increase in revertants as compared to controls) at 1.25 mg/plate and above in E. coli strain WP2/pKM101 and at 2.5 mg/plate and above in strain WP2 uvrA/pKM101 (Watanabe et al., 1996).
In vitro studies: mammalian cell gene mutation test
The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro in a study according to OECD 476 and in compliance with GLP (BASF, 2017). Four experiments were carried out, both with and without metabolic activation (phenobarbital and ß-naphthoflavone induced) at concentrations up to 70 µg/mL. More specifically the following concentrations were tested:
1st Exp: 2.5, 5, 10, 20, 30, 40 and 50 µg/ml (with and without S9 mix) (4hour)
2nd + 3rd Exp: 1.87, 3.75, 7.50, 15, 25, 35 and 50 µg/ml (without S9 mix) and 3.75, 7.5, 15, 25, 35, 45 and 60 µg/ml (with S9 mix) (4 hour)
4th Exp: 5, 10, 20, 30, 40, 50, 60 and 70 µg/ml (with S9 mix) (4 hour)
Cytotoxicity was observed in this assay, at concentrations from 40 µg/ml onward (without S9 mix) and from 45 µg/mL onward (with S9 mix). From the data of this study it was concluded that in the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.
Dearfield et al. (1989) reported that the test substance produced a clear dose-response related increase in mutant frequency in the mouse lymphoma cell assay (L5178Y, TK+/-) (0, 10, 15, 20 and 25 µg/mL) up to 707 mutants per million surviving cells at a concentration of 18 µg/mL (survival 13 %) without metabolic (S9) activation.The majority of the mutant colonies were small colonies.
It has been demonstrated from several earlier reports that small colony mutant formation in cultured mouse lymphoma cells appears to represent chromosomal alterations to chromosome 11, which carries the tk locus, and that large colonies represent smaller scale, perhaps single gene mutations (Hozier et al. 1981, 1985; Moore et al. 1985). A previous study on the smaller molecular weight monofunctional acrylate/methacrylate esters (Moore et al.1988) demonstrated that this group of compounds induces primarily small-colony mutants, supported by clastogenic activity scored as aberrations in mouse lymphoma cells in vitro. The results with the current set of acrylate/methacrylate esters are consistent with this induced small-colony/clastogenicity mechanism. This supports the hypothesis that the in vitro genotoxicity of this group of compounds at cytotoxic concentrations including 2 -hydroxyethyl acrylate acts via a direct acting clastogenic mechanism (as no metabolic activation is required for the positive results) (Dearfield et al., 1989).
In vitro studies: chromosome aberration/clastogenicity
Dearfield et al. (1989) also reported a dose related increase (0, 15, 18 and 20 µg/mL) in chromosomal aberrations and micronuclei in L5178Y mouse lymphoma cells treated with test substance in the absence of metabolic (S9) activation.
The compound demonstrated significant cytotoxicity with a linear dose response. An increase in CA was observed only at cytotoxic concentrations at which the relative total growth was less than 50 % of the control value (e.g. greater than 50 % growth inhibition). More recent studies have indicated that there is an association between chromosomal aberrations and cytotoxicity at exposure concentrations which reduce cell growth to less than 50 % of the control value (Galloway, 2000 and references cited therein). These data suggest that the increase in CA reported in the assays with test substance may be an artifact of the experimental method. Effects on CA when cell growth was within the acceptable range (50 - 100 % of the control value) are not available in the present study by Dearfield et al. (1989). Though this statement was basically formulated for chromosomal aberration assays, it nevertheless also applies to micronucleus assays in vitro, as the same genotoxic mechanism, i.e. the formation of chromosomal aberrations, is investigated in both assays, albeit using different test procedures.
In vivo studies
In addition, a mouse micronucleus assay was carried out according to OECD TG 474 and GLP regulations with the structural analogue of the test substance using NMRI mice (5 males and 5 females per group) and administering single gavage doses of 0, 100, 300 and 600 mg/kg body weight, respectively (Evonik Roehm GmbH, 2000). The ratio of normochromatic to polychromatic erythrocytes was slightly affected by the treatment with 2-hydroxypropyl acrylate at a dose of 600 mg/kg bw (at 24 and 48 hours in male mice and at 48 hours in female mice). At this dose level, only slight toxic effects, as evidenced by reduced spontaneous reactivity, were obtained up to 6 hours after dosing. There was no increase in the frequency of micronuclei at any dose level at either 24- or 48-hours after dosing compared to the negative control group.
As part of a chronic inhalation study (exposure of test substance to 0.5 and 5 ppm; 6 h/day, 5 days/week) some of the rats (i.e. 4 rats/sex/dose group) were sacrificed after 12-months exposure and the bone marrow cells examined for chromosomal damage. No evidence of chromosomal damage was seen at either dose level (Rampy et al., 1978; Dow Chemical Co., 1979).
In their decision on compliance check ECHA requested a further in vivo genotoxicity study to follow up the concern on gene mutation and chromosomal aberrations. ECHA requested a study according OECD TG 489 (Comet Assay) in rats on the following tissues: liver, glandular stomach and duodenum.
Assessment of the biological relevance of in vitro positive mutation studies is usually achieved by performing an in vivo follow up study. The in vivo follow up studies which can be used are the in vivo micronucleus assay for the determination of clastogenic and aneugenic potential of a compound. For the in vivo assessment of gene mutations and chromosome damage (clastogenicity) two guideline conform assays can be used, namely the in vivo comet assay as well as the in vivo transgenic rodent assay (TGR).
The in vivo comet assay is a genotoxicity test detecting and quantifying single and double strand breaks. This assay is not a true mutation assay and is regarded as an indicator test, since the fate of the cell with the DNA damage is not considered. The TGR is a true mutation assay, since the detected mutants represent survivors of a mutagen exposure. The TGR assay using the GPT model with the read outs using the GPT as well as SPI-modules is able to detect both mutations on gene level (GPT module) as well as deletion process representing chromosome breakage (SPI-module). Hence both assays are able to detect DNA alterations on the gene and chromosome level. However, the TGR assay is a true mutation assay and less prone to confounding factors (e.g. cytotoxicity). Thus, the preferred in vivo follow up assay for the detection of gene and chromosome mutations is the TGR assay.
Therefore, the registrant conducted a gene mutation assay (gpt assay and Spi- assay) with transgenic mice (gpt delta mouse) according to OECD TG 488 to assess the potential of Hydroxyethyl acrylate to induce gene point mutation and deletion mutations using the gpt gene (gpt assay) and the red/gam genes (Spi- assay) in the liver and stomach.
The test substance was administered to male transgenic mice orally, once a day, for 28 consecutive days by gavage at the dosage levels of 25.0, 100, and 275 mg/kg bw/day. After 3 days of manifestation period, the mutant frequencies in the liver and stomach were determined. One of seven mice in the 275 mg/kg bw/day group was died on Day 5. The negative control group values obtained for all organs were within the acceptable range of the historical control data and thus considered as valid. In the gpt assay and Spi- assay, the mutant frequencies in the liver and stomach of the animals treated with Hydroxyethyl acrylate did not show any increases as compared to the concurrent negative control value. All group values were also within the historical control data. In some cases the individual values surpassed the upper limit of the 95% control limit of the historical data. However, these increases are considered as not biologically relevant, since their distribution was sporadic and the increase was also observed in the control group. In the histopathological examination, hyperkeratosis was observed in the forestomach in the 275 mg/kg bw/day group.
The mutant frequencies in the liver and stomach in the positive control group, which was treated with benzo[a]pyrene, were increased and these increases were statistically significant compared with those of the negative control group. Therefore, the present study was judged to be properly conducted.
Considering all information available, including statistical analysis, it was concluded that Hydroxyethyl acrylate did not induce gene mutation in the liver or stomach of transgenic mice (negative) under the conditions in this study.
Labelling, and Packaging Regulation (EC) No 1272/2008
available data are reliable and suitable for classification purposes
under Regulation 1272/2008. As a result the substance is not considered
to be classified for mutagenicity under Regulation (EC) No 1272/2008, as
amended for fourteenth time in Regulation (EU) No 2020/217.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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