Registration Dossier

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
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:
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. 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. The negative in vivo mutagenicity is supported by a very recent test according to OECD TG 488 with the structural analogue ethyl acrylate.

Link to relevant study records
Reference
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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals 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).

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. 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. The negative in vivo mutagenicity is supported by a very recent test according to OECD TG 488 with the structural analog ethyl 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.