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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Hydroxypropyl acrylate was not mutagenic when tested under GLP in the bacterial reverse mutation assay test according to OECD TG 471 with Salmonella Typhimurium TA 1535, TA 100, TA1537, TA 98 and E. coli WP2 uvrA at concentrations between 33 µg - 5000 µg/plate (SPT) and 10 µg - 2500 µg/plate (PIT) with and without metabolic activation.

HPA was negative in the HPRT assay in Chinese hamster V79 cells with and without metabolic activation.

HPA induced chromosomal aberrations in V79 and CHO cells both in the presence and absence of metabolic activation at concentrations leading to cytotoxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial gene mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from male Wistar rats
Test concentrations with justification for top dose:
Standard plate test (with and without S9 mix): 0, 33, 100, 333, 1000, 2500 and 500 µg/plate
Preincubation test (with and without S9 mix): 0, 10, 33, 100, 333, 1000 and 2500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine; 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
Standard plate test:
Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants)
were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants)
were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Preincubation Test:
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
Species / strain:
other: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed depending on the strain and test conditions from about 333 μg/plate onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Solubility: No precipitation of the test substance was found with and without S9 mix.
Toxicity: A bacteriotoxic effect was observed depending on the strain and test conditions from about 333 μg/plate onward.
Mutagenicity: 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 the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.
Conclusions:
Under the experimental conditions of this study, the test substance Hydroxypropylacrylate is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced metabolic activation system.
Test concentrations with justification for top dose:
Experiment I:
- without S9 mix: 1, 3, 10*, 13, 17 and 20 µg/mL
- with S9 mix: 3, 10*, 30, 60*, 100 and 150 µg/mL
Confirmatory, Experiment II:
- without S9 mix: 1, 10*, 20, 30, 50, 70** and 100** µg/mL
- with S9 mix: 3, 30*, 60, 100 and 150 µg/mL respectively.
* = not evaluated, culture not continued
** = not able to evaluate, toxic effects.
Vehicle / solvent:
distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: ethylmethanesulfonate; with metabolic activation: 7,12- dimethylbenz(a)anthracene.
Details on test system and experimental conditions:
PRETEST
A preliminary toxicity experiment using the XTT-Assay at concentrations ranging from 3.0 to 1300 µg/mL with and without metabolic activation (S9
mix) was conducted to determine the appropriate concentration range for the mutagenicity assay. Based on the results of this study, the assay was
performed in two independent experiments with the described range of concentrations (µg/mL).


METHOD OF APPLICATION: in medium

The S9 liver microsomal fraction was obtained at the testing facility from the livers of 8-12 week old male Wistar rats which received a single i.p.
injection of 500 mg/kg b.w. Aroclor 1254 in olive oil five days previously. The protein concentration in the S9 preparation was 33.2 mg/mL. Before
the experiment, an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. Three-day old exponentially growing stock cultures were used.
Approximately 500 cells for toxicity or 1.5 E+06 cells for mutation rate were seeded. After 24 hours (Day 1), the medium was replaced with serum-free medium containing the test article with or without S9 mix. After 4 hours, the medium was replaced with untreated medium. The flasks were subcultured and incubated at 37°C (4.5% carbon dioxide) for 16 (toxicity) or 18 (mutation rate) days. At termination, the cells were fixed and stained with 10% methylene blue in 0.01% KOH. The stained colonies with more than 50 cells were counted.

DURATION
- Preincubation period: 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 18 d


NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
A test article was considered positive if it induced either a concentration related increase of the mutant frequency or a reproducible and positive response for one of the test points. A test article producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points was considered non-mutagenic in this system.
A significant response was described as follows:
A test article was considered mutagenic if it induced a reproducible mutation frequency that is at least three times higher than the spontaneous mutation frequency in the experiment at one or more of the concentrations. The test article is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency regardless of whether the 3-fold increase was observed. However, if there is a low spontaneous mutation rate in the normal range for the negative control, this is taken into account in determining whether the test substance is mutagenic in this system.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
30 µg/mL without activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

In the pre-test toxicity experiment, reduced cell viability was observed at concentrations greater than 10 µg/mL without metabolic activation and greater than 100 µg/mL with metabolic activation.

In the first experiment, the number of mutant colonies was similar across the solvent control (20.8 mutants/1E+06 cells) and treated (with or without metabolic activation) groups.

In the second experiment the values of the controls were exceeded at some concentrations both with and without activation. This "effect," however, was due to a low number of spontaneous mutant colonies in the solvent controls (4.3 mutants/1E+06 cells) and did not indicate a mutagenic effect of the test article. The absolute number of mutant colonies (range in both experiments of 3.1 to 24.9 mutants/1E+06 cells) was well within the historical control range for all concentrations tested.

The positive control agents produced the expected increase in mutations.

It was concluded that, under the conditions of this study, HPA was not mutagenic.

Results from Experiment 1:

Treatment

Conc.

S-9 mix

Cell density

Mutant colonies

 

[µg/mL]

 

[% of control]

[per E+06 cells]

Solvent control

0.00

-

100.0

16.8

Positive control

0.60

-

83.4

393.7

Test Substance

1.00

-

94.6

13.3

Test Substance

3.00

-

95.4

24.9

Test Substance

10.0

-

93.7

-

Test Substance

13.0

-

90.6

12.7

Test Substance

17.0

-

88.9

-

Test Substance

20.0

-

83.2

14.7

 

 

 

 

 

Solvent control

0.00

+

100.0

20.8

Solvent + DMSO

0.00

+

100.0

7.1

Positive control

3.85

+

83.4

284.8

Test Substance

3.00

+

99.7

11.7

Test Substance

10.0

+

100.8

-

Test Substance

30.0

+

100.8

15.3

Test Substance

60.0

+

88.8

-

Test Substance

100.0

+

43.2

18.8

Test Substance

150.0

+

15.7

21.7

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted in compliance with GLP regulations, with acceptable restrictions (no test substance purity given)
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced metabolic activation system.
Test concentrations with justification for top dose:
- Without S9 mix:
Experiments I, II (18 h): 1.0, 5.0, 10.0 µg/mL,
Experiment II (28 h): 10.0 µg/mL;
- With S9 mix:
Experiments I, II (18 h): 10.0, 50.0, 100.0 µg/mL,
Experiment II (28 h): 100.0 µg/mL.
Vehicle / solvent:
MEM (minimal essential medium, Seromed, Berlin)
- Justification for choice of solvent/vehicle: solubility of the substance, non-toxicity of the solvent
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
MEM (minimal essential medium)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
PRETEST
- The concentration range of the test article was determined in a pre-test (XTT-assay). After treatment with 30-1300 µg/mL (without S9 mix) and 300-1300 µg/mL (with S9 mix), toxic effects could be observed.


METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: 48 h (28 h preparation interval) and 55 h (18 h preparation interval), respectively
- Exposure duration: The treatment interval was 4 h with metabolic activation, and 18 and 28 h without. The three lowest concentrations were evaluated after 18 h, and the highest 28 h after initiation of the test.
- Time with spindle inhibitor: 2.5 h incubation with colcemid
- Fixation: After treatment with colcemid, the cells were treated in hypotonic solution on the slides for 20 min, then fixed and stained with Giemsa.


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS:
- 2 independent experiments were conducted.
- In each experimental group two parallel cultures were set up.


NUMBER OF CELLS EVALUATED:
Per culture 100 metaphases were scored for structural chromosomal aberrations.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
Analysis of metaphase cells:
Evaluation of the cultures was performed using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal desintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 +/- 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells was scored (% polyploid metaphases, in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).
A test article is classified as mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.
Statistics:
Chi-square test
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the highest concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

In the absence as well as in the presence of S-9 mix, in both experiments the mitotic indices were distinctly reduced after treatment with the highest evaluated concentrations at each fixation interval.

Without S-9 mix in experiment I the number of aberrant cells inclusive gaps was increased after treatment with 10 µg/mL of the test article (8.0%) as compared to the solvent control (1.5%). Exclusive gaps no relevant increase of the chromosome aberration frequencies after treatment with the test article was observed.

In experiment II, however, at fixation intervals 18 h and 28 h after treatment with 10 µg/mL hydroxypropyl acrylate the chromosome aberration frequency exclusive gaps was statistically significant increased whereas at lower concentrations no clastogenic effect occurred.

With S-9 mix in both independent experiments, there were statistically significant and biologically relevant increases in cells with structural aberrations after treatment with the test article at a concentration of 100 µg/mL. Again at lower concentrations (10 and 50 µg/mL) no clastogenic effect was observed.

In both experiments, no biologically relevant increase in the frequencies of polyploid metaphases was found after treatment with the substance compared to the frequencies of the controls.

Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosomal aberrations.

Summary of results:

Experiment

S9-mix

Conc.

Polyploid cells

Mitotic index

Aberrant cells in % of control

 

 

 

[µg/mL]

 

[%]

incl. gaps

excl. gaps*

exchanges

I

18 h

-

1.0

1.0

89.0

2.5

1.5

0.5

 

 

-

5.0

0.5

87.1

1.0

0.0

0.0

 

 

-

10.0

0.5

52.7

8.0

2.5

1.0

II

18 h

-

1.0

1.5

63.2

1.5

0.5

0.0

 

 

-

5.0

1.0

70.6

2.0

0.5

0.0

 

 

-

10.0

0.5

60.7

11.5

6.0**

1.0

II

28 h

-

10.0

2.0

58.2

15.5

14.5**

1.5

 

 

 

 

 

 

 

 

 

I

18 h

+

10.0

1.5

111.3

3.0

1.5

0.5

 

 

+

50.0

1.5

90.2

1.5

0.5

0.0

 

 

+

100.0

0.5

42.1

17.5

15.0**

7.5

II

18 h

+

10.0

2.5

114.4

1.5

0.0

0.0

 

 

+

50.0

2.5

80.2

3.5

2.5

0.0

 

 

+

100.0

2.0

52.1

31.0

28.0**

16.0

II

28 h

+

100.0

3.5

39.6

13.0

11.5**

3.5

* inclusive cells carrying exchanges

** statistically significant

Conclusions:
Interpretation of results: positive
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12 Oct 1999 - 18 Nov 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted in compliance with GLP regulations, with acceptable restrictions (no test substance purity given)
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
The S9 homogenate was prepared from the Iivers of live young male Sprague Dawley rats which had received prior treatment with phenobarbital and betanaphthoflavone to induce high levels of xenobiotic metabolizing enzymes.
Test concentrations with justification for top dose:
7.5, 15, 30, 45, 60 and 120 µg/mL in the absence of S9 metabolism,
100, 120, 140, 160, 180, 200, 220 and 240 µg/mL in the presence of S9 metabolism
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Culture medium (Ham's F10)
- Justification for choice of solvent/vehicle: The rest subsrance was found in a solubility pretest to be directly soluble in culture medium (Ham's F10)
at a concentration of 500 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin-C (0.30 µg/mL) in the absence of S9 metabolism and Cyclophosphamide (15.0 µg/mL) in the presence of S9.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 3 hr
- Harvest time: 20 hr (corresponding to 1.5 cell cycles)


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 100 metaphase spreads scored for chromosome aberrations


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, relative cell count

Evaluation criteria:
In this assay, the test substance was considered to have clastogenic properties if the following criteria were all fulfilled:
(i) Statistically significant increases in the incidence of cells bearing aberrations were observed at any dose-level over the concurrent control.
(ii) The increases must exceed the historical control values.
(iii) The increases were reproduced in both replicate cultures.
The evaluation was based on the set of results which excludes gaps.
Statistics:
Fisher's Exact Test
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9: severe toxicity > 45 µg/mL, with S9: severe toxicity > 160 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The test substance induced chromosomal aberrations in CHO cells under the present study conditions.

The observed aberrations consisted principally of chromatid deletions and exchanges and isolocus events.

 

In the absence of S9 metabolism significantly increases in the number of cells bearing aberrations (incl. and excl. gaps) were seen at higher dose levels (30 and 45 µg/mL). Moreover a dose-response relationship was observed. With S9 metabolism increases in aberrant cells (incl. gaps) were observed at the intermediate dose level of 140 µg/mL only.

 

Treatment

S-9

Dose level

Rel. cell count

Cells scored

Gaps

Chromatid

Chromosome

Isolocus

Other

Total abs.

Total abs.

Cells with abs.

Cells with abs.

 

 

[µg/mL]

[%]

 

 

Deletion

Exchange

Deletion

Exchange

 

 

(+ gaps)

(- gaps)

(+ gaps)

(- gaps)

Untreated

-

0

100

100

0

1

0

0

0

2

0

3

3

3

3

-

100

1

2

0

0

0

0

0

3

2

3

2

TS

-

45.0

45

100

18

11

5

3

0

2

2H, 2PP

39

21

29***

18***

-

100

7

13

11

0

1

1

2H

33

26

21***

18***

TS

-

30.0

62

100

2

8

0

2

0

2

1H, 9PP

14

12

14**

12**

-

80

9

3

0

1

0

1

1PP

14

5

13**

5

TS

-

15.0

78

100

4

0

0

0

0

2

0

6

2

4

2

-

100

5

1

0

0

0

1

0

7

2

6

2

Mitomyc.

-

0.30

75

50

9

14

20

3

0

3

0

49

40

31***

27***

-

50

4

13

32

5

0

2

2H

56

52

36***

35***

Untreated

+

0

100

100

1

0

0

0

0

0

1PP

1

0

1

0

+

100

1

2

0

0

0

0

0

3

2

3

2

TS

+

160

52

100

2

0

0

0

0

0

2PP, 4ER

2

0

2

0

+

100

3

1

1

0

0

0

1PP

5

2

4

1

TS

+

140

58

100

9

2

5

1

0

1

2PP, 1ER

18

9

11**

6

+

100

9

3

6

1

0

1

1PP

20

11

12**

4

TS

+

100

73

100

2

1

0

0

0

0

1PP, 1ER

3

1

3

1

+

100

1

1

0

0

0

0

3PP, 2ER

2

1

2

1

Cycloph.

+

15.0

55

100

13

9

17

5

1

7

1PP

52

39

33***

27***

+

100

3

7

7

0

0

0

0

17

14

12***

11***

PP: Polyploid cells

H:Heavily damaged cells (more than 5 aberrations/cell)

ER:Endoreduplicated cells

Isolocus: - includes isochromatid and isolocus breaks

* statistically significant at P < 0.05

** statistically significant at P < 0.01

*** statistically significant at P < 0.001

In the second experiment, reduced cell counts were observed at all concentrations (3 to 75% of control) with relative cell counts for the 15, 30 and 45 µg/mL scored groups of 78, 62, and 45%, respectively for the nonactivated cultures and 73, 70 and 58% for the 100, 140 and 160 µg/mL cultures with metabolic activation. An approximate 5 -fold and 10 -fold

increase in chromosomal aberrations was observed at 30 and 45 µg/mL, respectively in the non-activated cultures. An approximate 10 -fold increase occurred at 140 µg/mL (but no change was observed at 100 or 160 µg/mL) with metabolic activation.

The positive control substances produced the expected increase in chromosomal aberrations.

It was concluded that HPA induced chromosomal aberrations under the conditions of this study.

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

Genetic toxicity in vivo

Description of key information

In vivo, a micronucleus test using the oral route in NMRI mice was negative. Furthermore, the structural analogue 2-hydroxyethyl acrylate did neither induce gene mutations nor chromosomal damage in a transgenic rodent mutation assay according to OECD TG 488 (Requested in the CCH Decision number: CCH-D-2114382275-45-01/F). In addition, no evidence of chromosomal damage was found in bone marrow cells from rats of the 12-months interim sacrifice as part of a chronic inhalation study with the structural analogue 2-hydroxyethyl acrylate.


Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2020 - March 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to IUCLID chapter 13.2 "Other assessment reports" for the read-across justification.

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.
 
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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)
Version / remarks:
2013
GLP compliance:
yes
Type of assay:
transgenic rodent mutagenicity assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: PAU 0118813

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Refrigerator (KS)
Species:
mouse
Strain:
C57BL
Remarks:
6JJmsSlc-Tg (gpt delta)
Details on species / strain selection:
C57BL/6JJmsSlc-Tg (gpt delta) mice are commonly used as transgenic animals, and animals of this strain are readily available in in vivo gene mutation assays.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Japan SLC, Inc.
- Age at study initiation: 9 weeks of age
- Weight at study initiation: ca.25 g
- Assigned to test groups randomly: yes
Animals were assigned to groups based on their body weights on Day 1 using LATOX-F/V5 computer system package. Unassigned animals were excluded from the study on Day 1 and will be treated as surplus animals.
- Housing: Animals were housed individually in a plastic cage (W 18.2 × D 26.0 × H 12.8 cm) with bedding (ALPHA-dri™; Shepherd Specialty Papers).
- Diet: pellet diet CRF-1 (Oriental Yeast) ad libitum
- Water: tap water from water bottles ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26
- Humidity (%): 35 to 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: 0.5 w/v% carboxymethyl cellulose sodium salt aqueous solution (0.5 w/v% CMC)

Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing volume (mL) was set at 0.1 mL per 10 g of body weight. The individual dosing volume (mL) was calculated on the basis of the most recent individual body weight measured.

Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Post exposure period:
none
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
275 mg/kg bw/day
No. of animals per sex per dose:
7
Control animals:
yes, concurrent vehicle
Positive control(s):
Benzo[a]pyrene (B[a]P)
The 37.5 mg of B[a]P was weighed, transferred into a graduated test tube and suspended to 3 mL with olive oil to make a 12.5 mg/mL solution. The positive control solution was prepared just before use. The dose was 125 mg/kg bw/day. The positive control substance was administered to animals orally once daily for 5 consecutive days by using a disposable syringe with a Teflon sonde.
Tissues and cell types examined:
Liver: Liver is a major site of xenobiotic metabolism.
Stomach: Stomach is the site of first contact, since administration is oral. The histopathological finding was observed in the stomach in dose range-finding study [BSRC’s Exp. No. I919 (652-023)].
Kidney, heart, bladder, lymph node (mesenteric), epididymis and testis were addtionally examined histopathologically by the Sponsor’s demand.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION
In a dose range-finding study [BSRC’s Exp. No. I919 (652-023)] performed in C57BL/6JJmsSlc mice treated with 0, 50.0, 100, 150, 200, 400 or 600 mg/kg bw/day of test substance in 0.5 w/v% CMC for a period of 14 days, 3/3 mice in the 600 mg/kg bw/day group died. No clinical signs of toxicity were observed in the 400 mg/kg bw/day or lower groups. In the histopathological examination, hyperkeratosis and squamous cell hyperplasia were observed in the forestomach in the 200 and 400 mg/kg bw/day groups, and inflammatory cell infiltration and erosion/ulcer with penetration were observed in the forestomach in the 400 mg/kg bw/day group.

TREATMENT AND SAMPLING TIMES

Administration period and manifestation time
[Negative control group and test substance treated groups]
Administration period: Day 1 to 28
Manifestation time: Day 29 to 31
The organs were removed 3 days after the last dosing (Day 31).
[Positive control group]
Administration period: Day 6 to 10
Manifestation time: Day 11 to 24
The organs were removed 14 days after the last dosing (Day 24).

DETAILS ON STUDY DESIGN
Individual body weights of the animals in the negative control group and test substance-treated groups were measured on Day 1 (day of assignment to groups), 3, 8, 15, 22, 29 and 31 (just before organ removal). In the positive control group, the individual body weights of the animals were measured on Day 1, 3, 8, 15, 22 and 24 (just before organ removal). Dead animal was weighed when the condition was found.
The food weight (containing the feeder) of each animal in the negative control group and test substance-treated groups were measured on Day 1, 3, 8, 15, 22 and 29, and the mean daily food consumptions (g/day) was calculated.
In the administration period, animals were observed for clinical signs twice daily. Then, animals were observed for clinical signs once daily until organ removal.

Removal, macroscopic observation, organ weight and storage of organs (tissues)

The animals were necropsied after euthanasia by exsanguination under isoflurane anesthesia. The liver, stomach, kidney, heart, bladder, lymph node (mesenteric), vas deferens/cauda epididymis and testis were removed from each animal. The organ weights of the liver and stomach were measured in grams (to 2 decimal places). The organ weight to body weight ratio (relative organ weight) was calculated from the body weight weighed on the day of necropsy and organ weight (absolute organ weight/final body weight × 100). The organs were removed and stored according to the following methods. The mouse preventer was set at the entrance of the dissecting-room.

Liver: Approximately 5-mm slice (1 horizontal piece) was cut from the left lateral lobe and fixed in an adequate volume of 10 vol% neutral buffered formalin solution. Two samples were prepared from the remaining parts of left lateral lobe using a biopsy trephine (BP-50F, Kai) and separately put into microtubes and frozen in liquid nitrogen (LN2). The other lobes were put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2.
Stomach: The greater curvature of the stomach was incised. The stomach contents were removed by washing with physiological saline. The stomach piece (included forestomach and glandular stomach) was cut to about 4 × 10 mm size and stuck on a thick paper to avoid the curl of the stomach tissue. This part was fixed in an adequate volume of 10 vol% neutral buffered formalin solution. The remaining part was separated into 3 piece (included forestomach and glandular stomach) and put into storage bag and frozen in LN2.
Kidney: The capsule of left kidney was removed and sliced in a thickness of approximately 1 to 2 mm (approximately 2 horizontal pieces in total). Each slice was separately put into microtube and frozen in LN2. The capsule of right kidney was removed and fixed in an adequate volume of 10 vol% neutral buffered formalin solution. The remaining parts were put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2.
Heart: Heart was put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2.
Bladder: The contents were removed by washing with physiological saline. Bladder was put into microtube and frozen in LN2.
Lymph node (mesenteric): About one-third of mesenteric lymph node was fixed in an adequate volume of 10 vol% neutral buffered formalin solution. All remainder mesenteric lymph nodes were put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2.
Germ cells: The right and left testis were put into a microtube separately and frozen in LN2.
The vas deferens/cauda epididymis was cut a little and be placed in a Petri dish containing 1.5 mL of cold Dulbecco’s phosphate-buffered saline (PBS). The germ cells suspended in this PBS were filtered using a cell strainer (pore size 40 µm). About 1 mL of cell suspension was put into microtube and frozen in LN2.

The frozen tissues were stored in an ultra-low temperature freezer.


Pathological examination
Pathological examinations consisted of macroscopic examination and histopathological examination.

Macroscopic examination (necropsy)
The external surface and orifices were observed, followed by observation of the organs and tissues in the abdominal, thoracic, pelvic and cranial cavities. All macroscopic abnormalities were recorded as to the location, size, color tone, etc.

Fixation
The liver, stomach, kidney lymph node (mesenteric) were fixed in an adequate volume of 10% neutral buffered formalin solution as described.

Preparation of the histopathological specimens
Specimens for histopathological examination were prepared for liver and stomach from all animals in negative control group and all test substance-treated groups (excpet animal No. 1302). Fixed tissue samples were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H.E.) according to the routine method.

Histopathological examination
The histopathological specimens were examined microscopically, and all histopathological findings including types and severity were recorded.

Extraction of genomic DNA (details on buffers and medium presented in "Any other information on materials and methods")
Extraction of genomic DNA in the liver and stomach were conducted.
Three milliliters of the buffer for tissue breakage (containing RNase) were poured into a Dounce-type homogenizer and cooled with ice. Each frozen tissue sample was put into the homogenizer and homogenized with a pestle. The homogenized tissue fragments were poured gently into an ice-cooled 15-mL centrifuge tube containing 3 mL of 0.5 mol/L sucrose solution, and centrifuged (centrifuge LC-122, Tomy Seiko) at 3000 rotations/min (1750 G) for 10 minutes. The supernatant was removed and 3 mL of cooled RNase-containing Dounce buffer were added to the tube and mixed well (nuclear/cell suspension).
Then, 3 mL of proteinase K solution were added to the nuclear/cell suspension and gently mixed by inversion. This suspension was incubated at 50°C for about 2 hours until it became clear. The same volume (about 6 mL) of Ph/Cl mixture was added to the solution and mixed by inversion a few times, mixed by using a rotator for 10 minutes, and finally centrifuged (centrifuge LC-122) at 2500 rotations/min (1220 G) for 10 minutes. Next, the upper layer (water layer) was gently collected and transferred into another 15-mL centrifuge tube by a transfer pipette. This procedure was repeated twice (volume of Ph/Cl mixture was same as the removed water layer). After removal of the water layer, the same volume of chloroform/isoamyl alcohol mixture was poured into the tube. The contents were mixed by inversion a few times, mixed by using a rotator for 10 minutes, and finally centrifuged at 2500 rotations/min for 10 minutes. Then, the water layer was transferred into another 50-mL centrifuge tube. Genomic DNA was extracted by gradually adding ethanol in the tube. Extracted genomic DNA was transferred into a microtube containing 70% ethanol and stood for about 10 minutes. The contents were centrifuged (centrifuge MX-160) at 13000 rotations/min (13230 G) for 10 minutes. After the supernatant was removed as much as possible using a micropipette, the tube was stood at room temperature to evaporate ethanol. An appropriate volume (100 µL) of TE buffer (lot No. 02548F, Nippon Gene) was added to the tube. The tube was stood overnight at room temperature to dissolve DNA residues. The DNA solution was stored in a refrigerator after preparation. The concentration of DNA in the genomic DNA solution was measured using a spectrophotometer (NanoDrop® ND-1000, AGC TECHNO GLASS) and adjusted to about 300 to 600 µg/mL with the TE buffer.

Preparation of test strains (for gpt assay)
Thirty milliliters of LB broth, 300 µL of maltose solution (200 mg/mL) and 30 µL of kanamycin solution (20 mg/mL) were poured into a 200-mL baffled Erlenmeyer flask. A suspension (50 µL) of Escherichia coli strain YG6020 that had been thawed after being frozen at -80°C was inoculated into the flask. And it was incubated overnight (about 15 to 16 hours) at 37°C with a shaker at 120 strokes/min as the pre-incubation culture.
One hundred milliliters of LB broth, 1 mL of maltose solution (200 mg/mL) and 100 µL of kanamycin solution (20 mg/mL) were poured into a 500-mL baffled Erlenmeyer flask. The pre-incubation culture (1.5 mL) was inoculated into the flask and it was incubated for about 2 to 3 hours (OD: about 0.9) under the same conditions as the pre-incubation. Then, the bacterial suspension was centrifuged at 2000 rotations/min for 10 minutes. The supernatant was removed and the cells were suspended in LB broth (the volume half of the bacterial suspension collected) containing 10 mmol/L magnesium sulfate (E. coli suspension).

Preparation of test strains (for Spi- assay)
Thirty milliliters of LB broth was poured into a 200-mL baffled Erlenmeyer flask. Each suspension (50 µL) of Escherichia coli [XL-1 Blue MRA, XL-1 Blue MRA (P2) or WL95 (P2))] that had been thawed after being frozen at -80°C was inoculated into the flask. And they were incubated overnight (15 to 16 hours) at 37°C with a shaker at 120 strokes/min as the pre-incubation culture.
One hundred milliliters of LB broth and 1 mL of maltose solution (200 mg/mL) were poured into a 500-mL baffled Erlenmeyer flask. Each pre-incubation culture (1.5 mL each) was inoculated into the flask and they were incubated for about 2 to 3 hours (OD: about 0.9 to 1.0) under the same conditions as the pre-incubation. Then, the bacterial suspensions were centrifuged at 2000 rotations/min for 10 minutes. The supernatant was removed and the cells were suspended in LB broth (the volume half of the bacterial suspension collected) containing 10 mmol/L magnesium sulfate (E. coli suspension). The E. coli suspensions [XL-1 Blue MRA and XL-1 Blue MRA (P2)] were stored on ice or in a refrigerator and used within 3 days. The other E. coli suspension [WL95 (P2)] was stored on ice or in a refrigerator and used within that day.

Packaging of genomic DNA (common to the gpt and Spi- assays)
Packaging was conducted according to the instruction manual attached to Transpack packaging extract (lot No. 0006517934 or 0006543965, Agilent Technologies).
Three red tubes per animal (in gpt assay) or one red tube per animal (in Spi- assay) of Transpack packaging extract were thawed. Using a pipette, 10 µL of genomic DNA solution was transferred to each red tube. The packaging reaction was mixed by pipetting and the tube was incubated at 30°C for 90 minutes. Next, a blue tube of Transpack packaging extract was thawed, and 10 µL of it was transferred to the red tube containing a packaging reaction, and mixed in the same manner. It was incubated at 30°C for another 90 minutes, and diluted up to 100 µL (total 300 µL in gpt assay) or 300 µL (Spi- assay) of SM buffer, and mixed (packaged DNA sample).

Plating of packaged DNA sample (gpt assay)
E. coli (YG6020 strain) suspension 200 µL was dispensed into each tube [2 tubes for calculating total number of colonies (for titering), 5 tubes for calculating mutant frequency (for selection)]. LB broth containing 10 mmol/L magnesium sulfate 495 µL was dispensed into a tube for dilution. Then, 5 µL of the packaged DNA sample was added to the tube for dilution and mixed (diluted suspension). The diluted suspension 5 µL was added to the 2 tubes for titering and mixed. About 60 µL of the packaged DNA was added to the 5 tubes for selection and mixed. The tubes for titering and selection were incubated at 37°C for approximately 20 minutes without shaking and subsequently at 37°C for 30 minutes with a shaker at 120 strokes/min. Then, 2.5 mL of top agar was added to the tube for titering and mixed. The contents were poured on an M9+Cm agar plate. To the tube for selection, 2.5 mL of 6TG top agar was added and the contents were poured on an M9+Cm+6TG agar plate. The agar plates for titering were incubated in an incubator (ILL-60 or SSV-R11DA, Ikeda scientific) at 37°C for 3 days. The agar plates for selection were incubated in the incubator at 37°C for 5 days.
In this single packaging procedure, the total number of colonies per animal reached 300000. Therefore, no further packaging procedure was required.

Plating of packaged DNA sample (Spi- assay)
E. coli (XL-1 Blue MRA) suspension 200 µL was dispensed into each tube [2 tubes for calculating total number of plaques (for titering)]. E. coli [XL-1 Blue MRA (P2)] suspension 200 µL was dispensed into each tube [2 tubes for calculating mutant frequency (for selection)]. LB broth containing 10 mmol/L magnesium sulfate 495 µL was dispensed into a tube for dilution. Then, 5 µL of the packaged DNA sample was added to the tube for dilution and mixed (diluted suspension). The diluted suspension 5 µL was added to the 2 tubes for titering and mixed. About 150 µL of the packaged DNA sample was added to the 2 tubes for selection and mixed. The tubes for titering and selection were incubated at 37°C for approximately 20 minutes without shaking. Then, 2.5 mL of λ-trypticase top agar was added to the tube for titering and selection, and mixed. The contents were poured on a λ-trypticase agar plate. The agar plates were incubated overnight an incubator (SSV-R11DA, Ikeda scientific) at 37°C.
The above packaging procedure was repeated until the total number of plaques per animal reached 300000.

Colony counting (gpt assay)
The number of colonies was counted manually after the incubation (5th days after the incubation). However, gpt mutant candidate colonies on the agar plate for the selection were marked during incubation (on the 3rd and 4th days), because the colonies were not easily distinguished from the precipitation of 6TG on 5th days after the incubation.

Plaque counting (Spi- assay)
The number of plaques was counted manually after the incubation.

Confirmation of mutant phenotypes (gpt assay)
All gpt mutant candidate colonies on the agar plate for the selection were picked up by sterile toothpicks. After the tip of toothpick was rinsed well with 50 µL of 1/15 mol/L Na-K buffer, suspended cells were streaked on the M9+Cm agar plate first and on the M9+Cm+6TG agar plate next. The streaked parts on the agar plates were identified by attaching a cross-section paper and writing a serial number. The plates were incubated in an incubator (ILL-60, Ikeda scientific) at 37°C for 2 days. Only gpt mutant candidate colonies growing on both agar plates were called mutant colonies.

Confirmation of Spi- phenotypes (Spi- assay)
E. coli [XL-1 Blue MRA, XL-1 Blue MRA (P2) and WL95 (P2)] suspension 200 µL was dispensed into each tube. Then, 2.5 mL of λ-trypticase top agar was added to the tube and mixed. The contents were poured on a λ-trypticase agar plate. The agar plates were stood for about 1 hour at room temperature to vaporize the surface.
All Spi- candidates on the agar plate for the selection were punched out with sterilized Pasteur pipette or bore-wide tip. The agar plug was suspended with 50 µL of SM buffer (confirmation solution). One to two microliters of the confirmation solution was spotted on the λ-trypticase agar plates where each of XL-1Blue MRA, XL-1Blue MRA (P2) and WL95 (P2) strains had been spread with λ-trypticase top agar. The spotted parts on the agar plates were identified by attaching a cross-section paper and writing a serial number. The plates were incubated overnight (about 17 to 18 hours) in an incubator (ILL-60, Ikeda scientific) at 37°C. Only Spi- candidates made plaque on the all three agar plates of XL-1Blue MRA, XL-1Blue MRA (P2) and WL95 (P2) strains were called a mutant plaque.

Calculation of total number of colonies (gpt assay)
The number of colonies (N) in the plates for tittering was counted, and then the total number of colonies was calculated using the equations presented in "Any other information on materials and methods".
Evaluation criteria:
-The test substance-treated groups exhibits a statistically significant increase of the mutant frequency compared with the negative control.
-The mutant frequency (mean of group value) in the test substance-treated group is outside the 95% control limit of the laboratory historical negative control data.

If all of the above criteria are met, the test result was considered to be positive. In addition, the biological relevance of the results was taken into consideration for the final judgment.

VALIDITY OF STUDY
Since the following conditions were satisfied, the test was considered successfully performed:
-The mutant frequency in the positive control group markedly increased with a statistically significant difference from the negative control group.
-The mutant frequency in the negative control group was within the acceptable range (95% confidence interval) calculated from the historical data at BSRC.
Statistics:
The data on the mutant frequency from the negative control group and each test substance-treated group were tested by Bartlett’s test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity was determined (not significant on Bartlett’s test), then Dunnett’s multiple comparison test was performed to assess the statistical significance of differences between the negative control group and each test substance-treated group (two-sided, familywise significance level of 0.05). If there was no homogeneity (significant on Bartlett’s test), Steel’s test (two-sided, significance level of 0.05) was performed to analyze the differences.
The data on the mutant frequency from the negative control group and the positive control group were tested by F test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity of variance was determined (not significant on F test), Student’s t test (two-sided, significance level of 0.05) was performed to assess the statistical significance of differences between the negative control group and the positive control group. If there was no homogeneity (significant on F test), Aspin-Welch’s t test (two-sided, significance level of 0.05) was performed to analyze the differences.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
In the forestomach, hyperkeratosis was observed in all 6 mice in the 275 mg/kg bw/day group.
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mutant frequency (gpt assay)

Liver
In the negative control group, the group mean±SD of mutant frequency among the individuals was 2.46±1.00 (×10-6). The individual values ranged between 1.43-4.05 (×10-6).
The group mean±SD of mutant frequencies in the test substance-treated groups, 25.0, 100 and 275 mg/kg bw/day, were 2.12±0.69 (×10-6), 2.45±0.78 (×10-6) and 2.00±1.39 (×10-6), respectively, and no statistically significant increase was observed compared with the negative control group. The individual values ranged between 0.00-3.55 (×10-6).
All mean values of the negative control and treatment groups were within the 95% control limit range of the historical negative control group data [0.6-4.2 (×10-6)]. The obtained values from the individual animals were also within the 95% control limit of the historical individual control data [0.0-5.6 (×10-6)].
When compared to the negative control group, a statistically significant increase of the positive control group mutant frequency was observed [13.96±3.08 (×10-6)].

Stomach
In the negative control group, the group mean±SD of mutant frequency among the individuals was 3.61±1.53 (×10-6). The individual values ranged between 1.26-5.77 (×10-6).
The group mean±SD of mutant frequencies in the test substance-treated groups, 25.0, 100 and 275 mg/kg bw/day, were 2.86±1.94 (×10-6), 3.06±1.03 (×10-6) and 2.84±1.80 (×10-6), respectively, and no statistically significant increase was observed compared with the negative control group. The individual values ranged between 0.94-6.32 (×10-6).
All mean values of the negative control and treatment groups were within the 95% control limit range of the historical negative control group data [0.0-4.4 (×10-6)]. The obtained values from the individual animals were also within the 95% control limit of the historical individual control data [0.0-5.3 (×10-6)], excpet animal No. 1001 (negative control animal), animal No. 1101 (dose group 25.0 mg/kg bw/day) and animal No. 1305 (dose group 275 mg/kg bw/day).
When compared to the negative control group, a statistically significant increase of the positive control group mutant frequency was observed [21.68±5.61 (×10-6)].

Mutant frequency (Spi- assay)

Liver
In the negative control group, the group mean±SD of mutant frequency among the individuals was 1.84±1.12 (×10-6). The individual values ranged between 0.00-3.28 (×10-6).
The group mean±SD of mutant frequencies in the test substance-treated groups, 25.0, 100 and 275 mg/kg bw/day, were 2.31±2.32 (×10-6), 1.76±1.07 (×10-6) and 2.22±1.60 (×10-6), respectively, and no statistically significant increase was observed compared with the negative control group. The individual values ranged between 0.00-5.65 (×10-6).
All mean values of the negative control and treatment groups were within the 95% control limit range of the historical negative control group data [0.0-5.7 (×10-6)]. The obtained values from the individual animals were also within the 95% control limit of the historical individual control data [0.0-7.6 (×10-6)].
When compared to the negative control group, a statistically significant increase of the positive control group mutant frequency was observed [13.99±2.59 (×10-6)].

Stomach
In the negative control group, the group mean±SD of mutant frequency among the individuals was 2.81±1.93 (×10-6). The individual values ranged between 0.00-5.42 (×10-6).
The group mean±SD of mutant frequencies in the test substance-treated groups, 25.0, 100 and 275 mg/kg bw/day, were 2.84±1.84 (×10-6), 2.11±1.31 (×10-6) and 2.38±1.53 (×10-6), respectively, and no statistically significant increase was observed compared with the negative control group. The individual values ranged between 0.00-5.70 (×10-6).
All mean values of the negative control and treatment groups were within the 95% control limit range of the historical negative control group data [0.0-4.4 (×10-6)]. The obtained values from the individual animals were also within the 95% control limit of the historical individual control data [0.0-5.7 (×10-6)].
When compared to the negative control group, a statistically significant increase of the positive control group mutant frequency was observed [14.16±4.62 (×10-6)].

Body weight and general conditions
There were no statistically significant differences in animal body weights between the negative control group and each of the test substance-treated groups during the administration period.
In the 275 mg/kg bw/day group, one animal (animal No. 1302) was dead on Day 5. In other animals, no clinical signs of toxicity was observed in any of the test substance-treated groups.

Food consumption
There were no statistically significant differences in food consumption between the negative control group and each of the test substance-treated groups.

Organ weight and relative organ weight
The absolute stomach weight and relative stomach weight were statistically significantly increased in the 275 mg/kg bw/day group when compared to the negative control group.
There were no statistically significant differences in the absolute liver weight and relative liver weight between the negative control group and each of the test substance-treated groups.

Gross findings (necropsy)
There were no macroscopic findings related to test substance-treatment in any of the test substance-treated groups.
Black in the spleen was observed in one animal in the 100 mg/kg bw/day group and one animal in the 275 mg/kg bw/day group. This finding was considered to be a spontaneous lesion because it was also found in the negative control group.

Histopathological findings
In the forestomach, hyperkeratosis was observed in all 6 mice in the 275 mg/kg bw/day group.
There were no findings related to test substance treatment in the liver and glandular stomach.
Conclusions:
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.
Executive summary:

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.

 

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelman, D-33178 Borchen
- Assigned to test groups randomly: yes
- Housing: 5 animals of identical sex/cage
- Diet (ad libitum): pelleted standard diet (Altromin, D-32791 Lage/Lippe)
- Water (ad libitum): tap water
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 55±10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent used: CMC (carboxymethyl cellulose)
- Amount of vehicle (if gavage): 33 mL/kg bw
Duration of treatment / exposure:
single doses
Frequency of treatment:
once
Post exposure period:
not applicable
Remarks:
Doses / Concentrations:
100, 300 and 600 mg/kg body weight
Basis:
actual ingested
No. of animals per sex per dose:
5 animals/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide

- Route of administration: single i.p. injection of 10 mL/kg bw cyclophosphamide in 0.9 % NaCl.
- Doses / concentrations: 30 mg/kg b.w
Tissues and cell types examined:
One bone marrow smear was prepared per animal from the tissue cleared from each femur.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
An initial experiment to determine the toxicity of the test substance was conducted. Three male and three female mice were administered the test
substance orally at 1000 mg/kg b.w. This dose resulted in only slight toxicity and was therefore chosen as the top dose. In the main experiment,
two animals died within the first 6 hours of dosing at 1000 mg/kg b.w. so a dose of 600 mg/kg b.w. was chosen as the highest dose that could be used for analysis of micronuclei. All 10 mice at 1000 mg/kg b.w. died within 24 hours of dosing.


TREATMENT AND SAMPLING TIMES:
Five males and five females from each group were sacrificed 24 hours after dosing. Forty eight hours after dosing five animals per sex from the 600 mg/kg dose level were killed.

DETAILS OF SLIDE PREPARATION:
Stained smears were examined by light microscopy for incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. To describe a cytotoxic effect, the ratio of polychromatic to normochromatic erythrocytes was assessed by the examination of at least 1000 erythrocytes.

Evaluation criteria:
Evaluation of Results:
Cells were evaluated for large (aneugenic effects) and small (clastogenic effects) micronuclei. The test substance was classified as mutagenic if it induced either a statistically significant (Mann-Whitney test), dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible, statistically significant positive response for at least one of the test points.
Statistics:
Mann-Whitney test
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
600 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

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.

The positive control compound, cyclophosphamide, produced significantly increased frequencies of micronucleated polychromatic and normochromatic erythrocytes.

 

Following are the results:

 

Males sacrificed at 24 hours:

 

 

Mean Micronuclei/2000 PCE

 

Dose group

All (%)

Small (%)

Mean PCE/NCE

Negative control

3.2 (0.16)

2.8 (0.14)

1000/873.6

600 mg/kg bw

4.4 (0.22)

3.8 (0.19)

1000/1056.8

300 mg/kg bw

5.4 (0.27)

5.4 (0.27)

1000/1177.6

100 mg/kg bw

4.8 (0.24)

3.8 (0.19)

1000/974.6

Positive control

20.2 (1.01)

18.8 (0.94)

1000/739.6

 

 

 

Females sacrificed at 24 hours:

 

 

Mean Micronuclei/2000 PCE

 

Dose group

All (%)

Small (%)

Mean PCE/NCE

Negative control

3.2 (0.16)

2.8 (0.14)

1000/737.4

600 mg/kg bw

2.8 (0.14)

2.0 (0.10)

1000/854.6

300 mg/kg bw

5.2 (0.26)

4.8 (0.24)

1000/773.8

100 mg/kg bw

3.2 (0.16)

2.8 (0.14)

1000/918.8

Positive control

19.6 (0.98)

18.4 (0.92)

1000/688.6

 

 

 

Males and Females sacrificed at 48 hours:

 

 

Mean Micronuclei/2000 PCE

 

Dose group

All (%)

Small (%)

Mean PCE/NCE

600 mg/kg bw males

2.2 (0.11)

2.0 (0.10)

1000/986.2

600 mg/kg bw females

2.2 (0.11)

1.8 (0.09)

1000/1065.4

 

 

 

Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro Studies: Bacterial systems

Hydroxypropyl acrylate was tested under GLP in the bacterial reverse mutation assay test according to OECD TG 471 with Salmonella Typhimurium TA 1535, TA 100, TA1537, TA 98 and E. coli WP2 uvrA at concentrations between 33 µg - 5000 µg/plate (SPT) and 10 µg - 2500 µg/plate (PIT) with and without metabolic activation. Hydroxypropyl acrylate was not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay (BASF SE, 2017).

Hydroxypropyl acrylate was tested in the bacterial reverse mutation assay test according to OECD TG 471 with Salmonella typhimurium TA98, TA100, TA1535, and TA1537 at concentrations up to 5000 µg/plate with and without metabolic activation. Hydroxypropyl acrylate was considered not mutagenic under the conditions of the assay (Evonik Roehm GmbH, 1995). HPA was not mutagenic in Salmonella typhimuriumstrain TA100 at 0.079 – 7.9 µg/plate (Rohm and Haas, 1982). In a publication, in which the results are only give in a table and no information about the purity of the test material is given hydroxypropyl acrylate was described positive in two E. coli strains (WP2uvrA/pKM101 and WP2/pKM101) and negative in two Salmonella typhimurium strains (TA 102 and TA 2638) when tested with metabolic activation (Watanabe et al., 1996). Based on the poor description of the study details, the missing information about the purity of the material tested and that the results could not be confirmed in reliable OECD guideline studies, the study resutls could not be used for the assessment of the substance. .

 

In-vitro studies: Mammalian cell gene mutation tests and Genotoxicity tests

A mammalian cell (CHO V79) mutation assay according to OECD TG 476 was negative with and without metabolic activation at concentrations up to 150 µg/mL. Cytotoxicity was observed in this assay, however, at concentrations as low as 30 µg/mL (Evonik Roehm GmbH, 1995).

Hydroxypropyl acrylate was tested in a cytogenetic assay with Chinese hamster V79 cells according to OECD TG 473 at concentrations up to 100 µg/mL and was positive with and without metabolic activation in this assay (Evonik Roehm GmbH, 1995). In this assay cytotoxicity was observed only at the highest test concentration. In a chromosomal aberration assay with CHO cells, hydroxypropyl acrylate tested at concentrations up to 240 µg/mL was positive for chromosomal damage. Reduced cell counts were observed at all concentrations (3 to 75 % of control) with relative cell counts for the 15, 30 and 45 µg/mL scored groups of 78, 62, and 45 %, respectively for the nonactivated cultures and 73, 70 and 58 % for the 100, 140 and 160 µg/mL cultures with metabolic activation. An approximate 5 -fold and 10 -fold increase in chromosomal aberrations was observed at 30 and 45 µg/mL, respectively in the non-activated cultures. An approximate 10 -fold increase occurred at 140 µg/mL (but no change was observed at 100 or 160 µg/mL) with metabolic activation (Evonik Roehm GmbH, 2000).

 

Other (meth)acrylates, including acrylic acid, methyl (meth)acrylate, ethyl (meth)acrylate, 2- ethylhexyl acrylate, 2-hydroxyethyl acrylate, and several multifunctional (meth)acrylates (Moore and Doerr, 1990), have been evaluated in in vitro mutagenicity assays with mammalian cells. In these studies, positive results were reported at concentrations that led to a clearly reduced cell survival rate. 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 mutagenicity reported in some of the chromosomal aberration assays with hydroxypropyl acrylate may be an artifact of the experimental method.

 

In vivo studies

A mouse micronucleus assay was carried out according to OECD TG 474 and GLP regulations with hydroxypropyl acrylate (HPA) 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. The ratio of normochromatic to polychromatic erythrocytes was slightly affected by the treatment with 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 (Evonik Roehm GmbH, 2000).

A gene mutation assay (gpt assay and Spi- assay) with transgenic mice (gpt delta mouse) according to OECD TG 488 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 (major site of xenobiotic metabolism) and stomach (stomach is the site of first contact, since administration is oral) (HARTF, 2021).

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.

Considering all information available including statistical analysis, it was concluded that Hydroxyethyl acrylate was clear negative at any assay points and both endpoints. Hydroxethyl acrylate did not induce gene mutation in the liver or stomach of transgenic mice (negative) under the conditions in this study.

In addition, as part of the chronic inhalation study conducted with the structural analogue 2-hydroxyethyl acrylate (exposure 0.5 and 5 ppm HEA; 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).

 

Conclusion

 

Hydroxypropyl acrylate was not mutagenic when tested under GLP in the bacterial reverse mutation assay test according to OECD TG 471 with Salmonella Typhimurium TA 1535, TA 100, TA1537, TA 98 and E. coli WP2 uvrA at concentrations between 33 µg - 5000 µg/plate (SPT) and 10 µg - 2500 µg/plate (PIT) with and without metabolic activation.

HPA was negative in the HPRT assay in Chinese hamster V79 cells with and without metabolic activation.

HPA induced chromosomal aberrations in V79 and CHO cells both in the presence and absence of metabolic activation at concentrations leading to cytotoxicity.

In vivo, a micronucleus test using the oral route in NMRI mice was negative. Furthermore, the structural analogue 2-hydroxyethyl acrylate did neither induce gene mutations nor chromosomal damage in a transgenic rodent mutation assay according to OECD TG 488. In addition, no evidence of chromosomal damage was found in bone marrow cells from rats of the 12-months interim sacrifice as part of a chronic inhalation study with the structural analogue 2-hydroxyethyl acrylate.

 

Justification for classification or non-classification

Based on the available data, classification as a genotoxic substance is not triggered according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.