Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

2-Hydroxyethyl acrylate (HEA) did not induce gene mutations in the Salmonella typhimurium / E. coli reverse mutation assay in the absence and the presence of metabolic activation. HEA ist not a mutagenic substance in the HPRT locus assay using CHO cells (with and without metabolic activation). HEA was positive at cytotoxic concentrations in the mouse lymphoma assay (MLA) and induced an increase in chromosomal aberrations and micronuclei in L5178Y mouse lymphoma cells. Since in the mouse lymphoma assay preferentially small colonies were induced, the mutagenic potential of HEA seems to be limited to clastogenicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Sep 2016 to 14 Oct 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug 1998
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test substance: Hydroethylacrylate
- Test substance No.: 16/0173-1
- Analytical purity: 99.6 g/100g
- Physical state, appearance: liquid, colorless, clear
Target gene:
- S. typhimurium: his-locus
- E. coli: trp-locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and ß-napthoflavone induced male rat liver S9 mix
Test concentrations with justification for top dose:
1st Experiment: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (with and without S9 mix) Standard plate test

2nd Experiment: 0; 10; 33; 100; 333; 1000 and 2500 μg/plate (TA strains); 0; 33; 100; 333; 1000; 2500 and 5000 μg/Plate (E.coli) (with and without S9 mix) Preincubation test; No mutagenicity was observed in the standard plate test. Due to toxicity, the doses was adjusted in the preincubaton test.

3rd Experiment: 0; 1000; 2000; 2500; 3000; 4000 and 5000 μg/plate (E.coli) (with S9 mix) Preincubation test; Increased number of rebertants was observed in the preincubation test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility of the test substance in water
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene (2-AA); N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylenediamine (NOPD)
Details on test system and experimental conditions:
STANDARD PLATE TEST
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al. (1, 2).
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
The experimental procedure was based on the method described by Yahagi et al. (7) and Matsushima et al. (8). 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.

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a:
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.
Evaluation criteria:
ACCEPTANCE CRITERIA
Generally, the experiment was considered valid if the following criteria were met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain
- The sterility controls revealed no indication of bacterial contamination
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above
- Fresh bacterial culture containing approximately 10^9 cells per mL were used.

ASSESSMENT 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.

A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.
Key result
Species / strain:
S. typhimurium, other: TA1535, TA 100, TA 1537, TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed depending on the strain and the conditions from about 100 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed depending on the strain and the conditions from about 100 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 100 μg/plate.
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. However, using the tester strain E.coli WP2 uvrA with S9 mix in the preincubation test a single increase in the numbers of trp+ revertants was observed at a concentration of 2500 μg/plate (factor 2.8). In a repeat experiment (called 3rd Experiment) this finding was not reproduced and therefore these findings have to be regarded as biological irrelevant.

result tables see attachment

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Oct 2016 to 7 Mar 2017
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)
Version / remarks:
28 Jul 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
Aug 1988
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro gene mutation study in mammalian cells (HPRT)
Specific details on test material used for the study:
- Name of test substance: Hydroxyethylacrylate
- Test substance No.: 16/0173-1
- Analytical purity: 99.6 g/100g
- Physical state, appearance: liquid, colorless, clear
Target gene:
hypoxanthine-guanide phosphoribosyl transferase (HPRT)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
rat S9 mix (phenobarbital and ß-naphthoflavone induced) from male Wistar rat livers
Test concentrations with justification for top dose:
1st Exp: 2.5, 5, 10, 20, 30, 40 and 50 µg/mL (with and without S9 mix) (4hour)
2nd + 3rd Exp: 1.87, 3.75, 7.50, 15, 25, 35 and 50 µg/ml (without S9 mix) and 3.75, 7.5, 15, 25, 35, 45 and 60 µg/mL (with S9 mix) (4 hour)
4th Exp: 5, 10, 20, 30, 40, 50, 60 and 70 µg/mL (with S9 mix) (4 hour)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:culture medium (Ham´s F12)
- Justification for choice of solvent/vehicle: good solubility of the test substance in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 - 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): from day 16

SELECTION AGENT (mutation assays): 6-thioguanine (10µg/mL)

NUMBER OF REPLICATIONS: 2

Evaluation criteria:
ACCEPTANCE CRITERIA
The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
- The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
- The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range.

ASSESSMENT CRITERIA
A test substance is considered to be clearly positive if all following criteria are met:
- A statistically significant increase in mutant frequencies is obtained.
- A dose-related increase in mutant frequencies is observed.
- The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).

Isolated increases of mutant frequencies above our historical negative control range or isolated
statistically significant increases without a dose-response relationship may indicate a biological
effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
- The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R. If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY
After an exposure period of 4 hours without S9 mix, cytotoxic effects, as indicated by clearly reduced relative survival of about or below 20% of the respective negative control values, were observed from 40 μg/mL (RS: 3.6%) onward in the 1st Experiment and at 50 μg/mL (RS: 0.6%) in the 3rd Experiment. In addition, with S9 mix, there was a distinct decrease in relative survival in the 1st Experiment at 50 μg/mL (RS: 12.5%), in the 3rd Experiment from 45 μg/mL onward (RS: 20.9%) and in the 4th Experiment from 60 μg/mL onward (RS: 4.4%). In the 2nd Experiment which was considered invalid clearly reduced relative survival was obtained from 35 μg/mL onward (RS: 14.8%) in the absence of S9 mix and from 45 μg/mL onward (RS: 22.3%) in the presence of S9 mix.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study restrictions: no data on analytical purity, only concentrations tested at which considerable cytotoxicity, i.e. less than 50 % growth
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
The test was conducted according to the procedures described by Turner et al. (1984) and Doerr et al. (1989).

GLP compliance:
not specified
Type of assay:
other: in vitro cytogenicity / chromosome aberration study in mammalian cells
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-Hydroxyethyl acrylate
- Source: Polysciences, Inc. (Warrington, PA)
- Analytical purity: no data
Target gene:
not applicable
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The TK+/- -3.7.2C heterozygote of L5178Y mouse lymphoma cells was used.
Metabolic activation:
without
Test concentrations with justification for top dose:
15, 18, 20 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
The positive control substance was tested in a trial with the structural analogue 2-ethylhexyl acrylate.
Details on test system and experimental conditions:
TEST PROCEDURE
L5178Y/TK+/- -3.7.2C cells were treated without exogenous activation for 4 h according to procedures described previously (Turner et al. 1984). For experiments including cytogenetic analysis, duplicate cultures were treated and one culture was used for mutation analysis and one for cytogenetics. No more than l00 uL of solvent (DMSO) was added to 10-mL culture. This concentration of solvent does not effect either the cytotoxicity or the mutagenicity. After 9-11 days incubation at 37°C, colonies were counted on an Artek model 880 automatic colony counter. Following cell treatment and wash, all cultures were divided and 10 uM BrdUrd was added to those cultures to be used for cytogenetic analysis. For aberration analysis cells were incubated for 14-15 h, with colcemid present for the last 2 h. Cells were treated with hypotonic KCl and fixed in acetic acid:methanol (1:3). Slides were made, stained using the fluorescence-plus-Giemsa method and blind coded. For each dose, 100 metaphase spreads that satisfied the criteria given below were analyzed for aberrations.

CONTROLS
- Negative control: DMSO
- Positive control: Methylmethane sulfonate (MMS: 15 ug/mL)
Evaluation criteria:
(i) well spread;
(ii) near normal centromere count (40 ± 3); and
(iii) first division cell (as indicated by the absence of BrdUrd-induced differential staining).
Aberrations were classified as chromatid breaks, deletions, and fragments (grouped as chromatid type breaks; triradials, quadraradials, and complex rearrangements (chromatid type rearrangements); chromosome breaks, deletions, fragments and minutes (chromosome type breaks); and dicentrics, rings and translocations (chromosome type rearrangements). A special category was used for metaphases with 10 or more aberrations. For these cells, aberrations were not characterized due to extreme amounts of damage. Chromatid and chromosome gaps were recorded but not included as aberrations.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
42 % survival at 15 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Aberration Analyses:

 

Dose [µg/mL]

Chromatid

Chromosome

Total no. AB

Cells with AB

Survival [%]

 

Breaks

Rearrangements

Breaks

Rearrangements

 

 

 

0

0

0

1

1

2

2

100

15

7

9

3

1

20

12

42

18

13

14

3

1

31

18

20

20

20

23

3

3

99

30

15

MMS: 15

4

11

11

3

39

23

9

AB: aberrations. 100 cells were scored for each data point.

Survival was calculated according to the method of Clive & Spector (1975).

 

 

 

The substance induced a dose responsive increase in aberrations to a high frequency of 99 aberrations/100 cells at 20 µg/mL in the presence of increasing cytotoxicity (survival at 20 µg/mL 15 %).

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Restrictions: no data on analytical purity
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The test was conducted according to the procedures described by Turner et al. (1984).

GLP compliance:
not specified
Type of assay:
other: in vitro gene mutation study in mammalian cells
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-Hydroxyethyl acrylate
- Source: Polysciences, Inc. (Warrington, PA)
- Analytical purity: no data
Target gene:
TK +/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The TK+/- -3.7.2C heterozygote of L5178Y mouse lymphoma cells was used.
Metabolic activation:
without
Test concentrations with justification for top dose:
1st experiment: 0, 15 µg/mL
2nd experiment: 0, 12, 16, 18 µg/mL
3rd experiment: 0, 10, 17, 18, 20, 23 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
TEST PROCEDURE
L5178Y/TK+/- -3.7.2C cells were treated without exogenous activation for 4 h according to procedures described previously (Turner et al. 1984). For experiments including cytogenetic analysis, duplicate cultures were treated and one culture was used for mutation analysis and one for cytogenetics. No more than l00 uL of solvent (DMSO) was added to 10-mL culture. This concentration of solvent does not effect either the cytotoxicity or the mutagenicity. After 9-11 days incubation at 37°C, colonies were counted on an Artek model 880 automatic colony counter using settings that optimize colony counts, compared to hand counts, without producing a spurious background. These counters have size discriminators that quantitate the colony size distribution. Colony sizing curves were generated by calculating the difference between adjacent counts of up to 16 uniformly spaced settings on the size discriminator. Each of these differences was converted to a mutant frequency by correcting for plating efficiency. The difference in mutant frequency between adjacent size settings was plotted as a histogram and small- and large-colony mutant frequency determined. Small colonies are generally < 0.6 mm and large colonies are > 0.6 mm. Survival was calculated according to the method of Clive and Spector (1975) and includes both a measure of growth in the suspension and cloning phases of the assay.

Clive D and Spector J (1975). Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. Mutat. Res. 31: 17-29

CONTROLS
- Negative control: DMSO
- Positive control: Methylmethane sulfonate (MMS: 15 µg/mL)
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 15 µg/mL 42-45 % survival
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Mutagenicity:

 

 

Concentration [µg/mL]

Per cent plating efficiency

Total mutant count

Mutant frequency [x10-6]

Per cent survival

Mutant frequency [S/L]

1stexperiment

 

 

 

 

 

 

 

0

97

229

79

100

-

 

0

80

187

78

100

57/21

 

10

92

388

141

72

-

 

15

66

923

466

24

375/91

 

20

35

1270

1210

4

-

 

25

18

1226

2270

1

-

 

EMS: 400

59

993

1116

42

-

2ndexperiment

 

 

 

 

 

 

 

0

76

203

89

100

67/22

 

0

69

211

102

100

-

 

6

74

175

79

86

-

 

10

92

500

181

68

-

 

11

99

473

159

68

-

 

12

91

622

228

60

177/51

 

13

82

642

261

52

-

 

14

83

900

361

48

-

 

15

84

1108

440

45

-

 

16

73

1107

505

31

445/60

 

17

78

1110

474

30

-

 

18

56

1188

707

13

607/100

 

19

56

1209

720

9

-

 

20

51

1389

908

6

-

 

EMS: 400

63

1000

1053

52

-

3rdexperiment

 

 

 

 

 

 

 

0

73

191

87

100

57/30

 

0

83

203

82

100

-

 

4

94

196

70

100

-

 

8

95

217

76

96

-

 

10

85

324

127

70

92/35

 

15

71

739

345

42

-

 

16

77

783

339

38

-

 

17

74

878

395

35

321/74

 

18

67

907

455

20

362/93

 

19

61

834

456

17

-

 

20

59

985

560

15

479/81

 

21

49

1049

714

8

-

 

22

47

1130

801

6

-

 

23

43

1183

917

5

827/90

 

24

32

1144

1192

3

-

 

EMS: 400

54

884

1127

37

-

Plating efficiency was based on plating 600 cells.

Mutant count is expressed as total mutants per 3 million cells plated in TFT selection, and mutant frequency is expressed as per 1000000 surviving cells. Percent survival is calculated according to the method of Clive and Spector (1975).

The small- and large-colony mutant frequencies (S/L) are also expressed as per 1000000 surviving cells.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo, HEA did not induce mutagenic effects in rat bone marrow cells in a chronic inhalation study. In addition, a micronucleus test with the structural analogue 2-hydroxypropyl acrylate (HPA) using the oral route in NMRI mice was negative. Based on the present results, it is unlikely that HEA is mutagenic in vivo. No carcinogenic effect was observed in the long-term inhalation carcinogenicity study with 2 -hydroxyethyl acrylate, supporting that 2 -hydroxyethyl acrylate is not mutagenic in vivo.

A transgenic rodent mutation assay according to OECD TG 488 is ongoing with HEA to investigate the potential of gene mutations and chromosome damage in vivo.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please see for more information the read-across justification in Section 13.
Reason / purpose:
read-across source
Key result
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
Remarks on result:
other: Result read-across CAS No. 25584-83-2
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-Hydroxypropylacrylate
- Analytical purity: 97.8 %
- Batch No. 790201167; Lot No. 32114707
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/solvent used: CMC (carboxymethyl cellulose)
- Amount of vehicle: 33 mL/kg bw
Duration of treatment / exposure:
single doses
Frequency of treatment:
once
Post exposure period:
not applicable
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
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

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10 May 1974 to 07 May 1976
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Study restrictions: in negative control groups less than 50 cells scored
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Principles of method if other than guideline:
A chronic inhalation study in Sprague-Dawley rats included 12-month interim sacrifices for cytogenetic examinations. 4 male and 4 female rats per group were injected i.p. with colchicine (0.4 mg/kg bw), sacrificed 4 hours after injection and samples of bone marrow collected. Slides of the bone marrow were prepared for the microscopic examination of chromosomes. 50 cells per animal were scored for chromatid aberrations, chromosome aberrations and abnormal cells, with the exception of female controls where 35, 43, 19 and 25 cells were scored and one female in the 5 ppm group where only 2 cells were scored.
GLP compliance:
no
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Spartan substrain
- Source: Spartan Research Animals, Michigan
- Housing: 3-4 animals/cage
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): ambient
- Humidity (%): ambient
Route of administration:
inhalation: vapour
Vehicle:
- Vehicle(s)/solvent(s) used: none
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 3.7 cubic meters stainless steel chambers under dynamic airflov- conditions
- System of generating vapours: The exposure atmosphere in each chamber was generated by metering liquid HEA at a calculated rate into the top of
a 15 inch glass column ( 2" diameter) that was heated to a temperature (ca. 80°C) hot enough to vaporize the HEA. Dry compressed air was introduced at the bottom of the glass column to sweep the vapours into the chamber where they were diluted with room air at a rate calculated to provide the desired HEA Concentration.


TEST ATMOSPHERE
- The nominal concentration of HEA in the chamber was calculated from the ratio of the amount of liquid HEA used to the rate of total chamber airflow.
- Brief description of analytical method used:
The concentration of HEA in each chamber was determined three or more times daily.
Analytical concentrations for the first 6 1/2 months of exposure were obtained by drawing 10 liters of air from the chamber at a rate of 1 liter/min through a charcoal tube. The HEA absorbed on the charcoal was extracted into 2 mL of carbon disulfide. The quantity of HEA in a 2 µL sample of carbon disulfide extract was analysed by gas chromatography (detector: FID).
For the last 11 1/2 months of the study HEA samples were collected by bubbling chamber air through water instead of charcoal. Improved reproducibility of sample analysis and convenience were the primary reasons for using water instead of charcoal. Fifty liters of air from the chamber were drawn through 20 mL of distilled water in a fritted glass bubbler at a rate of 1 liter/min. The quantity of HEA in a 2 µL sample of the trapping solution was analyzed by gas chromatography using the same conditions as before.
- Samples taken from breathing zone: no
Duration of treatment / exposure:
12 months
Frequency of treatment:
6 hours per day, 5 days per week
Post exposure period:
Males: 5 months; females: 6 months
Dose / conc.:
0.024 mg/L air (nominal)
Remarks:
corresponding to 5 ppm
Dose / conc.:
0.002 mg/L air (nominal)
Remarks:
corresponding to 0.5 ppm
No. of animals per sex per dose:
4 animals/sex/dose group
Control animals:
yes, concurrent no treatment
Positive control(s):
none
Tissues and cell types examined:
Bone marrow cells
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable

There were no indications of alterations related to exposure to either level of HEA for 1 year.

Cytogenetic Examination of Bone Marrow Cells of Male and Female Rats exposed to Vapours of 2-Hydroxyethyl Acrylate 5 Days/Week for 12 Months: 

Exposure Level [ppm]

No. of Cells Scored

Chromatid Aberrations

Chromosome Aberrations

Abnormal Cells

Males

 

 

 

 

0

50

0

0

0

0

50

0

0

0

0

50

0

0

0

0

50

0

0

0

5.0

50

0

0

0

5.0

50

0

0

0

5.0

50

0

0

0

5.0

50

0

0

0

0.5

50

0

0

0

0.5

50

0

0

0

0.5

50

0

0

0

0.5

50

0

0

0

Females

 

 

 

 

0

35

0

0

0

0

43

0

0

0

0

19

0

0

0

0

25

1

0

1

5.0

2

0

0

0

5.0

50

0

0

0

5.0

50

0

0

0

5.0

50

0

0

0

0.5

50

0

0

0

0.5

50

0

0

0

0.5

20

0

0

0

0.5

50

0

0

0

Endpoint:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
ongoing
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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 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 will be assigned to groups based on their body weights on Day 1 using LATOX-F/V5 computer system package. Unassigned animals will be excluded from the study on Day 1 and will be treated as surplus animals.
- Housing: Animals will be 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) will be set at 0.1 mL per 10 g of body weight. The individual dosing volume (mL) will be 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 will be 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 will be prepared just before use. The dose will be 125 mg/kg bw/day. The positive control substance will be 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.
Kidney, heart, bladder, lymph node (mesenteric), epididymis and testis if requested by Sponsor
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 will be 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 will be 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 will be 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 will be measured on Day 1, 3, 8, 15, 22 and 24 (just before organ removal). Dead or moribund animals, if any, will be weighed when the condition is found.
The food weight (containing the feeder) of each animal in the negative control group and test substance-treated groups will be measured on Day 1, 3, 8, 15, 22 and 29, and the mean daily food consumptions (g/day) will be calculated.
Animals will be observed for clinical signs at least once daily until organ removal. In the administration period, animals will be observed for clinical signs at least twice daily. Moribund animals, if any, will be euthanized by CO2 inhalation when the condition is found.

Removal, macroscopic observation, organ weight and storage of organs (tissues):
The animals will be necropsied after euthanasia by exsanguination under isoflurane anesthesia. The liver, stomach, kidney, heart, bladder, lymph node (mesenteric), epididymis and testis will be removed from each animal. The organ weights of the liver and stomach will be measured in grams (to 2 decimal places). The organ weight to body weight ratio (relative organ weight) will be calculated from the body weight weighed on the day of necropsy and organ weight (absolute organ weight/final body weight × 100). The organs will be removed and stored according to the following methods.
Liver: Two samples will be prepared from the left lateral lobe using a biopsy trephine (BP-50F, Kai). Each sample will be separately put into microtubes and frozen in liquid nitrogen (LN2). The other lobes will be put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2. The remaining parts will be fixed in an adequate volume of 10 vol% neutral buffered formalin solution.
Stomach: The greater curvature of the stomach will be incised. The stomach contents will be removed by washing with physiological saline. The stomach piece (included forestomach and glandular stomach) will be cut to about 4 × 10 mm size and stuck on a thick paper to avoid the curl of the stomach tissue. This part will be fixed in an adequate volume of 10 vol% neutral buffered formalin solution.
Kidney: The capsule of left kidney will be removed and sliced in a thickness of approximately 1 to 2 mm (approximately 2 horizontal pieces in total). Each slice will be separately put into microtube and frozen in LN2. The capsule of right kidney will be removed and fixed in an adequate volume of 10 vol% neutral buffered formalin solution. The remaining parts will be put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2.
Heart: Heart will be put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2.
Bladder: The contents will be removed by washing with physiological saline. Bladder will be put into microtube and frozen in LN2.
Lymph node (mesenteric): About one-third of mesenteric lymph node will be fixed in an adequate volume of 10 vol% neutral buffered formalin solution. All remainder mesenteric lymph nodes will be put into a storage bag, and squashed and frozen with a flat-bottom metal container filled with LN2. The frozen tissues will be stored in an ultra-low temperature freezer.

Pathological examination
Pathological examinations will consist of macroscopic examination and histopathological examination.

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

Fixation
The liver, stomach, kidney and lymph node (mesenteric), epididymis and testis will be fixed in 10% neutral buffered formalin solution as described

Preparation of the histopathological specimens
Specimens for histopathological examination will be prepared for liver and stomach from all animals in negative control group and all test substance-treated groups. Fixed tissue samples will be embedded in paraffin, sectioned and stained with hematoxylin and eosin (H.E.) according to the routine method.

Histopathological examination
The histopathological specimens will be examined microscopically, and all histopathological findings including types and severity will be 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 glandular stomach will be conducted. Three milliliters of the buffer for tissue breakage (containing RNase) will be poured into a Dounce-type homogenizer and cooled with ice. Each frozen tissue sample will be put into the homogenizer and homogenized with a pestle.
The homogenized tissue fragments will be 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 will be removed and 3 mL of cooled RNase-containing Dounce buffer will be added to the tube and mixed well (nuclear/cell suspension).
Then, 3 mL of proteinase K solution will be added to the nuclear/cell suspension and gently mixed by inversion. This suspension will be incubated at 50°C for about 2 to 4 hours until it became clear. The same volume (about 6 mL) of Ph/Cl mixture will be 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) will be gently collected and transferred into another 15-mL centrifuge tube by a transfer pipette. This procedure will be repeated twice (volume of Ph/Cl mixture will be same as the removed water layer). After removal of the water layer, the same volume of chloroform/isoamyl alcohol at a volume ratio of 24:1 will be poured into the tube. The contents will be mixed by inversion a few times, mixed by using a rotator for 10 minutes, and finally centrifuged at 2500 rotations/min (1220 G) for 10 minutes. Then, the water layer will be transferred into another 50-mL centrifuge tube. Genomic DNA will be extracted by gradually adding ethanol in the tube. Extracted genomic DNA will be transferred into a microtube containing 70% ethanol and stood for about 10 minutes. The contents will be centrifuged (centrifuge MX-160) at 13000 rotations/min (13230 G) for 10 minutes. After the supernatant will be removed as much as possible using a micropipette, the tube will be stood at room temperature to evaporate ethanol. Appropriate volume of TE buffer (Nippon Gene) will be added to the tube. The tube will be stood overnight at room temperature to dissolve DNA residues. The DNA solution will be stored in a refrigerator after preparation. The concentration of DNA in the genomic DNA solution will be measured using a spectrophotometer (NanoDrop® ND-1000, AGC TECHNO GLASS) and will adjust to about 100 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) will be 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 will be inoculated into the flask. And it will be incubated overnight (14 to 18 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) will be poured into a 500-mL baffled Erlenmeyer flask. The pre-incubation culture (1.5 mL) will be inoculated into the flask and it will be incubated for about 2 to 6 hours (OD: approximately 0.6 or more) under the same conditions as the pre-incubation. Then, the bacterial suspension will be centrifuged at 2000 rotations/min for 10 minutes. The supernatant will be removed and the cells will be 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)
The preparation volume may be adjusted according to required volume.
Thirty milliliters of LB broth will be poured into a 200-mL baffled Erlenmeyer flask (total 2 bottles, confirmation of Spi- phenotypes: total 3 bottles). Each suspension (50 µL) of Escherichia coli [XL-1 Blue MRA, XL-1 Blue MRA (P2) or WL95 (P2)2)] that had been thawed after being frozen at -80°C will be inoculated into the flask. And they will be incubated overnight (14 to 18 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) will be poured into a 500-mL baffled Erlenmeyer flask (total 2 or 3 bottles). Each pre-incubation culture (1.5 mL each) will be inoculated into the flask and they will be incubated for about 2 to 6 hours (OD: approximately 0.6 or more) under the same conditions as the pre-incubation. Then, the bacterial suspensions will be centrifuged at 2000 rotations/min for 10 minutes. The supernatant will be removed and the cells will be suspended in LB broth (the volume half of the bacterial suspension collected) containing 10 mmol/L magnesium sulfate (E. coli suspension). The E. coli suspension [XL-1 Blue MRA or XL-1 Blue MRA (P2)] will be stored on ice or in a refrigerator and used within 3 days. The other E. coli suspension [WL95 (P2)] will be stored on ice or in a refrigerator and used within that day.

Packaging of genomic DNA (common to the gpt and Spi- assays)
Packaging will be conducted according to the instruction manual attached to Transpack packaging extract (Agilent Technologies).
Three red tubes per animal (in gpt assay) or one red tube per animal (in Spi- assay) of Transpack packaging extract will be thawed. Using a pipette, 10 µL of genomic DNA solution will be transferred to each red tube. The packaging reaction will be mixed by pipetting and the tube will be incubated at 30°C for 90 minutes. Next, a blue tube of Transpack packaging extract will be thawed, and 10 µL of it will be transferred to the red tube containing a packaging reaction, and mixed in the same manner. It will be 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 will be dispensed into each tube [2 tubes for calculating total number of colonies (for tittering), 5 tubes for calculating mutant frequency (for selection)]. LB broth containing 10 mmol/L magnesium sulfate 495 µL will be dispensed into a tube for dilution. Then, 5 µL of the packaged DNA sample will be added to the tube for dilution and mixed (diluted suspension). The diluted suspension 5 µL will be added to the 2 tubes for tittering and mixed. About 60 µL of the packaged DNA will be added to the 5 tubes for selection and mixed. The tubes for tittering and selection will be 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 will be added to the tube for tittering and mixed. The contents will be poured on an M9+Cm agar plate. To the tube for selection, 2.5 mL of 6TG top agar will be added and the contents will be poured on an M9+Cm+6TG agar plate. The agar plate for tittering will be incubated in an incubator at 37°C for 3 days. The agar plate for selection will be incubated in an incubator at 37°C for 5 days.
The above packaging procedure or genomic extraction-to-packaging procedures will be repeated until the total number of colonies per animal reached 300000.

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

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

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

Confirmation of mutant phenotypes (gpt assay)
All gpt mutant candidate colonies on the agar plate for the selection will be picked up by sterile toothpicks. After the tip of toothpick will be rinsed well with 50 µL of 1/15 mol/L Na-K buffer, suspended cells will be streaked on the M9+Cm agar plate first and on the M9+Cm+6TG agar plate next. The streaked parts on the agar plates will be identified by attaching a cross-section paper and writing a serial number. The plates will be incubated in an incubator at 37°C for 2 to 3 days. Only gpt mutant candidate colonies growing on both agar plates will be 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 will be dispensed into each tube. Then, 2.5 mL of λ-trypticase top agar will be added to the tube and mixed. The contents will be poured on a λ-trypticase agar plate. The agar plates will be stand for about 1 hour at room temperature to vaporize the surface.
All Spi- candidates on the agar plate for the selection will be punched out with sterilized Pasteur pipette or bore-wide tip. The agar plug will be suspended with 50 µL of SM buffer (confirmation solution). One to two microliters of the confirmation solution will be spotted on the λ-trypticase agar plates where each of XL-1Blue MRA, XL-1Blue MRA (P2) and WL95 (P2) strains have been spread with λ-trypticase top agar. The spotted parts on the agar plates will be identified by attaching a cross-section paper and writing a serial number. The plates will be incubated overnight (14 to 18 hours) in an incubator at 37°C. Only Spi- candidates made plaque on the three agar plates will be called a mutant plaque.

Calculation of total number of colonies (gpt assay)
The number of colonies (N) in the plates for tittering will be counted, and then the total number of colonies will be calculated using the equations presented in "Any other information on materials and methods".
Evaluation criteria:
If the following conditions will be satisfied, the test will be considered successfully performed:
- the mutant frequency in the positive control group markedly increases with a statistically significant difference from the negative control group.
- the mutant frequency in the negative control group should be within the acceptable range (mean±3SD) calculated from the historical data at BSRC.
Statistics:
Data on mutant frequency from negative control group and each test substance-treated group will be tested by Bartlett’s test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity will be determined (not significant on Bartlett’s test), then Dunnett’s multiple comparison test will be performed to assess the statistical significance of differences between negative control group and each test substance-treated group (two-sided, familywise significance level of 0.05). If there will be no homogeneity (significant on Bartlett’s test), Steel’s test (two-sided, significance level of 0.05) will be performed to analyze differences. Data on mutant frequency from negative control group and positive control group will be tested by F test for homogeneity of variance (two-sided, significance level of 0.05) first. If homogeneity of variance will be determined (not significant on F test), Student’s t test (two-sided, significance level of 0.05) will be performed to assess statistical significance of differences between negative control group and positive control group. If there will be no homogeneity (significant on F test), Aspin-Welch’s t test (two-sided, significance level of 0.05) will be performed to analyze differences.
Data of body weight, body weight gain, organ weight, absolute organ weight / body weight ratio and food consumption from the negative control group and each test substance-treated group will be tested by Bartlett’s test for homogeneity of variance first. If homogeneity will be determined (not significant on Bartlett’s test), then Dunnett’s multiple comparison test will be 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 will be no homogeneity (significant on Bartlett’s test), Steel’s test (two-sided, significance level of 0.05) will be performed to analyze differences.
Key result
Sex:
male
Remarks on result:
other: study ongoing, results will be presented in an update
Endpoint:
in vivo mammalian somatic cell study: gene mutation
Remarks:
Dose-Range Finder
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 February 2020 - 20 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)
Version / remarks:
2013
Deviations:
yes
Remarks:
14 days treatment/Dose-Range Finder for OECD TG 488/ No extration of genomic DNA
GLP compliance:
no
Type of assay:
other: Dose Range Finder for OECD TG 488
Specific details on test material used for the study:
SOUSOURCE 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 mice are used as the non-transgenic animals of same rodent strain of in vivo gene mutation assay. It is assumed that the observed toxicity profile in the wild-type animals reflect that observed in the transgenic animals.
Sex:
male
Details on test animals 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.
- Housing: individually in a plastic cage (W 18.2 × D 26.0 × H 12.8 cm) with bedding (ALPHA-dri™; lot Nos. 03119 and 04119, 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:
1) The test substance was weighed and put into a graduated cylinder
2) An appropriate volume of vehicle was added to the same cylinder
3) The test substance was suspended by vortexing.
4) It was diluted with vehicle to prepare formulation, and stirred using a vortex mixer.

The stability of the test substance formulation in vehicle (0.5 w/v% CMC) is confirmed after storage for 7 days at room temperature. The dosing volume (mL) was set at 0.1 mL per 10 g of body weight.
Duration of treatment / exposure:
14 days
Frequency of treatment:
daily
Post exposure period:
none
Dose / conc.:
50 mg/kg bw/day
Remarks:
Experiment I
Dose / conc.:
100 mg/kg bw/day
Remarks:
Experiment I
Dose / conc.:
150 mg/kg bw/day
Remarks:
Experiment I
Dose / conc.:
200 mg/kg bw/day
Remarks:
Experiment II
Dose / conc.:
400 mg/kg bw/day
Remarks:
Experiment II
Dose / conc.:
600 mg/kg bw/day
Remarks:
Experiment II
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Positive control(s):
None
Details of tissue and slide preparation:
1. Measurement of body weight and observation of clinical signs
Observation of clinical signs was observed daily. Individual body weights of the animals in the negative control group and the test substance-treated groups were measured on Day 1 (day of assignment to groups), 3, 7 and 15 (just before necropsy).
2. Food consumption
The food weight (containing the feeder) of each animal was measured on Day 1, 3, 7 and 15, and the mean daily food consumptions (g/day) was calculated.
3. Pathological examination
Pathological examinations consisted of organ weight measurement, macroscopic examination (necropsy) and histopathological examination. The animals for scheduled necropsy were necropsied after euthanasia by exsanguination under isoflurane anesthesia.
4. Organ weight measurement
The liver and stomach of animals of scheduled necropsy were weighed. Before the weighing, the grater curvature of the stomach was incised and the stomach contents were removed by washing with physiological saline. The organ weight ratio to body weight (relative organ weight) was calculated from the body weight measured on the day of necropsy and organ weight (absolute organ weight / final body weight × 100). The organ was measured in grams (to 2 decimal places).
5.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.
6. Fixation
The liver, stomach and all organs with macroscopic abnormalities were fixed in an adequate volume of 10 vol% neutral buffered formalin solution. Before fixation, the stomach was stuck on a thick paper to avoid the curl of the stomach tissue.
7. Preparation of the histopathological specimens
Specimens for histopathological examination were prepared for liver and stomach from all animals survived on the day of necropsy (Day 15). Fixed tissue samples were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H.E.) according to the routine method.
Statistics:
The data of body weight, organ weight and absolute organ weight / body weight ratio were tested by Bartlett’s test for homogeneity of variance (two-sided, significance level of 0.05). If homogeneity was determined (not significant on Bartlett’s test), 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. 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.
Key result
Sex:
male
Genotoxicity:
not determined
Remarks on result:
other: Dose range finder for OECD TG 488
Additional information on results:
RESULTS OF RANGE-FINDING STUDY I
- Dose range: 50, 100, 150 mg/kg bw/day

- Clinical signs of toxicity in test animals:
All animals survived the entire observation period. No suppression of body weight gain was observed in all groups. No clinical signs of toxicity was observed in all groups. No apparent suppression of food consumption was observed in all groups. The relative stomach weight was significantly increased in the 100 mg/kg bw/day group when compared to the negative control group. However, no dose relationship was observed. There were no differences from the negative control group in the absolute liver weight, relative liver weight or absolute stomach weight. Black patch in the spleen was observed in one mouse in the 100 mg/kg bw/day group. However, this finding was considered to be a spontaneous lesion because it was also found in the negative control group.
There were no findings related to test substance treatment in the liver and stomach (forestomach and glandular stomach).
Since no toxicity signs were observed in this range finder a second experiment was conducted.

RESULTS OF RANGE-FINDING STUDY II
- Dose range: 200, 400, 600 mg/kg bw/day

- Clinical signs of toxicity in test animals: In the 600 mg/kg bw/day group, 3 animals died on Day 1, and mortality was 3/3. In the 200 and 400 mg/kg bw/day groups, all animals survived the entire observation period, and mortality was 0/3. No suppression of body weight gain was observed in the 200 and 400 mg/kg bw/day groups. No clinical signs of toxicity was observed in the 200 and 400 mg/kg bw/day groups. No apparent suppression of food consumption was observed in the 200 and 400 mg/kg bw/day groups.
The relative liver weight and absolute stomach weight were statistically significantly increased in the 400 mg/kg bw/day group when compared to the negative control group. The relative stomach weight was obviously increased in the 400 mg/kg bw/day group, but no statistically significant difference was observed when compared to the negative control group. Since the Bartlett’s test showed no homogeneity, the Steel’s test was performed, but the Steel’s test was inappropriate for this small sample size (N=3). Thickening of the forestomach was observed in all animals in the 400 mg/kg bw/day group. In addition, adhesion of forestomach and diaphragm was observed in one animal in this group. 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. No remarkable changes were observed in the liver and glandular stomach.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

With these results, the in vitro genotoxicity profile of HEA is identical to that of HPA and other short-chain alkyl acrylate. The following table shows the comparison between these two substances and the other analogues considered:

Endpoint

HEA

HPA

MA

nBA

Bacterial reverse mutation

Negative
(BASF, 2016)

Negative
(BASF, 2017)

Negative
(BASF 1977)

Negative
(BASF 1977)

Gene mutations in mammalian cells

Negative
(HPRT: BASF, 2017b)

Negative
(HPRT: Evonik, 1995a)

Negative
(Moore 1991)

Negative
(Moore 1988 and others;
MLA:BASF 2016 )

Clastogenicity in mammalian cells

Positive (cytotoxicity)
(Dearfield 1989a,b)

Positive (cytotoxicity)
(Evonik 2000)

Positive (cytotoxicity)
(Ishidate 1981 and others)

Negative
(Wiegand 1989)

In vivo clastogenicity

Negative

Negative

Negative

Negative

In a MLA study included in the dossier, HEA showed a clastogenic effect at cytotoxic concentrations; these observations are most probably due to the high reactivity of the monomer and no real clastogenicity. In addition, in the carcinogenicity study performed on HEA the clastogenicity was tested in parallel and came out negative in vivo. This is supported by the negative in vivo chromosomal aberration studies / micronucleus assay with the source chemicals HPA, MA and nBA. Therefore the overall picture shows no genotoxicity on both HEA and the source substance HPA. Overall also no carcinogenic effects was found in the long-term study.

The common hydrolysis product (AA) of both HEA and HPA show a comparable genetic toxicity profile in vitro, while the non-common hydrolysis products (EG, PG) are overall negative. All hydrolysis products have been tested for genetic toxicityin vivoand showed no effects:

Endpoint

AA (acrylic acid)

EG (ethylene glycol)

PG (propylene glycol)

Bacterial reverse mutation

Negative

Negative

Negative

Gene mutations in mammalian cells

Negative (HPRT)

Negative (HPRT)

N/A

Chromosomal aberrations in mammalian cells

Positive

Negative

Negative

In vivo clastogenicity

Negative

Negative

Negative

Further details are given under 13.2 Read-Across Justification

Additional information

In vitro studies: bacterial systems

The potential to induce mutagenicity in bacteria was assessed in a study according to OECD 471 performed in compliance with GLP criteria (BASF, 2016). In this study the bacterial strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr were exposed to test substance concentrations between 33 µg - 5000 µg/plate (SPT) and 10 µg - 5000 µg/plate (PIT), in the presence and absence of a metabolic activation system (liver S9 mix from rats). A bacteriotoxic effect was observed depending on the strain and the conditions from about 100 µg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed.

In another supporting study, the test substance was not mutagenic in Salmonella typhimurium strain TA100 at 0.01-7.5 mg/plate (Rohm and Haas, 1982), nor in strains TA98, TA100, TA1535, TA1537 and TA1538 (no dose/plate given) with or without metabolic (S9) activation (Dow Chemical Co., 1976). The test substance was not mutagenic in Salmonella typhimurium strains TA102 and TA2638 (0.038 – 5 mg/plate) with or without metabolic (S9) activation, but gave inconclusive results (ca. 2-3 fold increase in revertants as compared to controls) at 1.25 mg/plate and above in E. coli strain WP2/pKM101 and at 2.5 mg/plate and above in strain WP2 uvrA/pKM101 (Watanabe et al., 1996).

In vitro studies: mammalian cell gene mutation test

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro in a study according to OECD 476 and in compliance with GLP (BASF, 2017). Four experiments were carried out, both with and without metabolic activation (phenobarbital and ß-naphthoflavone induced) at concentrations up to 70 µg/mL. More specifically the following concentrations were tested:

1st Exp: 2.5, 5, 10, 20, 30, 40 and 50 µg/ml (with and without S9 mix) (4hour)

2nd + 3rd Exp: 1.87, 3.75, 7.50, 15, 25, 35 and 50 µg/ml (without S9 mix) and 3.75, 7.5, 15, 25, 35, 45 and 60 µg/ml (with S9 mix) (4 hour)

4th Exp: 5, 10, 20, 30, 40, 50, 60 and 70 µg/ml (with S9 mix) (4 hour)

Cytotoxicity was observed in this assay, at concentrations from 40 µg/ml onward (without S9 mix) and from 45 µg/mL onward (with S9 mix). From the data of this study it was concluded that in the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Dearfield et al. (1989) reported that the test substance produced a clear dose-response related increase in mutant frequency in the mouse lymphoma cell assay (L5178Y, TK+/-) (0, 10, 15, 20 and 25 µg/mL) up to 707 mutants per million surviving cells at a concentration of 18 µg/mL (survival 13 %) without metabolic (S9) activation.The majority of the mutant colonies were small colonies.

It has been demonstrated from several earlier reports that small colony mutant formation in cultured mouse lymphoma cells appears to represent chromosomal alterations to chromosome 11, which carries the tk locus, and that large colonies represent smaller scale, perhaps single gene mutations (Hozier et al. 1981, 1985; Moore et al. 1985). A previous study on the smaller molecular weight monofunctional acrylate/methacrylate esters (Moore et al.1988) demonstrated that this group of compounds induces primarily small-colony mutants, supported by clastogenic activity scored as aberrations in mouse lymphoma cells in vitro. The results with the current set of acrylate/methacrylate esters are consistent with this induced small-colony/clastogenicity mechanism. This supports the hypothesis that the in vitro genotoxicity of this group of compounds at cytotoxic concentrations including 2 -hydroxyethyl acrylate acts via a direct acting clastogenic mechanism (as no metabolic activation is required for the positive results) (Dearfield et al., 1989).

In vitro studies: chromosome aberration/clastogenicity

Dearfield et al. (1989) also reported a dose related increase (0, 15, 18 and 20 µg/mL) in chromosomal aberrations and micronuclei in L5178Y mouse lymphoma cells treated with test substance in the absence of metabolic (S9) activation.

The compound demonstrated significant cytotoxicity with a linear dose response. An increase in CA was observed only at cytotoxic concentrations at which the relative total growth was less than 50 % of the control value (e.g. greater than 50 % growth inhibition). More recent studies have indicated that there is an association between chromosomal aberrations and cytotoxicity at exposure concentrations which reduce cell growth to less than 50 % of the control value (Galloway, 2000 and references cited therein). These data suggest that the increase in CA reported in the assays with test substance may be an artifact of the experimental method. Effects on CA when cell growth was within the acceptable range (50 - 100 % of the control value) are not available in the present study by Dearfield et al. (1989). Though this statement was basically formulated for chromosomal aberration assays, it nevertheless also applies to micronucleus assays in vitro, as the same genotoxic mechanism, i.e. the formation of chromosomal aberrations, is investigated in both assays, albeit using different test procedures.

In vivo studies

In addition, a mouse micronucleus assay was carried out according to OECD TG 474 and GLP regulations with the structural analogue of the test substance using NMRI mice (5 males and 5 females per group) and administering single gavage doses of 0, 100, 300 and 600 mg/kg body weight, respectively (Evonik Roehm GmbH, 2000). The ratio of normochromatic to polychromatic erythrocytes was slightly affected by the treatment with 2-hydroxypropyl acrylate at a dose of 600 mg/kg bw (at 24 and 48 hours in male mice and at 48 hours in female mice). At this dose level, only slight toxic effects, as evidenced by reduced spontaneous reactivity, were obtained up to 6 hours after dosing. There was no increase in the frequency of micronuclei at any dose level at either 24- or 48-hours after dosing compared to the negative control group.

As part of a chronic inhalation study (exposure of test substance to 0.5 and 5 ppm; 6 h/day, 5 days/week) some of the rats (i.e. 4 rats/sex/dose group) were sacrificed after 12-months exposure and the bone marrow cells examined for chromosomal damage. No evidence of chromosomal damage was seen at either dose level (Rampy et al., 1978; Dow Chemical Co., 1979).

In their decision on compliance check ECHA requested a further in vivo genotoxicity study to follow up the concern on gene mutation and chromosomal aberrations. ECHA requested a study according OECD TG 489 (Comet Assay) in rats on the following tissues: liver, glandular stomach and duodenum.

 

Assessment of the biological relevance of in vitro positive mutation studies is usually achieved by performing an in vivo follow up study. The in vivo follow up studies which can be used are the in vivo micronucleus assay for the determination of clastogenic and aneugenic potential of a compound. For the in vivo assessment of gene mutations and chromosome damage (clastogenicity) two guideline conform assays can be used, namely the in vivo comet assay as well as the in vivo transgenic rodent assay (TGR).

 

The in vivo comet assay is a genotoxicity test detecting and quantifying single and double strand breaks. This assay is not a true mutation assay and is regarded as an indicator test, since the fate of the cell with the DNA damage is not considered. The TGR is a true mutation assay, since the detected mutants represent survivors of a mutagen exposure. The TGR assay using the GPT model with the read outs using the GPT as well as SPI-modules is able to detect both mutations on gene level (GPT module) as well as deletion process representing chromosome breakage (SPI-module). Hence both assays are able to detect DNA alterations on the gene and chromosome level. However, the TGR assay is a true mutation assay and less prone to confounding factors (e.g. cytotoxicity). Thus, the preferred in vivo follow up assay for the detection of gene and chromosome mutations is the TGR assay.

 

Therefore, the registrant concluded to perform a study according OECD TG 488 (TRG-Assay) in C57BL/6JJmsSlc mice on the following tissues: liver, glandular stomach and duodenum. A dose range-finding study for a OECD TG 488 study in C57BL/6JJmsSlc mice treated with 0, 50, 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 was performed (Hydroxy REACH Task Force, 2020). All 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. In conclusion, 275 mg/kg bw/day is considered to be the maximum tolerated dose and will used as the highest dosage level in the main gene mutation assay with gpt delta mice. For the lower dose levels 100 and 25 mg/kg bw/day will be used. The study is ongoing

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No 1272/2008, as amended for fourteenth time in Regulation (EU) No 2020/217.