Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation assay: negative

Mammalian cell gene mutation assay (thymidine kinase (TK) locus and structural chromosome aberrations): negative

Cytogenetic assay: negative (especially at non-cytotoxic concentrations)

UDS test: negative

The in various vitro tests (bacterial gene mutation test, mammalian cell gene mutation assay, cytogenetic tests, UDS test ) did not show genotoxic effects at non-cytotoxic concentrations. I

Therefore no further testing is required or initiated to assess the genotoxicity of n-butyl acrylate.

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Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-05-10 to 2016-04-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In vitro Mammalian Cell Gene Mutation Test
Specific details on test material used for the study:
n-butyl acrylate, purity 99.78 %, water content 0,04 %, batch taken from the cont. production 21 Jul 2015
GLP Characterisation report 16L00114 (20 Jun 2016)

Target gene:
ability to induce gene mutations at the thymidine kinase (TK) locus or structural chromosome aberrations at chromosome 11 in L5178Y TK+/- mouse lymphoma cells in vitro
Details on mammalian cell type (if applicable):
L5178Y TK+/- mouse lymphoma cells
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital and β-naphthoflavone induced rats (exogenous metabolic activation).
Test concentrations with justification for top dose:
SEE BELOW: ANY OTHER INFORMATION ON MATERIALS AND METHODS INCL. TABLES
Vehicle / solvent:
Due to the insolubility of the test substance in ultrapure water, DMSO was selected as vehicle, which had been demonstrated to be suitable in the mouse lymphoma assay and for
which historical control data are available. The final concentration of the vehicle DMSO in culture medium was 1% (v/v).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Preparation of test cultures:
Logarithmically growing cells in suspension culture (3 x 105 cells per 75 cm² flask in a total volume of 30 mL in exponential growth per treatment group required) were incubated 4 -
5 days prior to the start of the experiment.

Pretreatment of cells:
During the week prior to treatment, spontaneous TK deficient mutants (TK-/-) were eliminated from the stock cultures by incubating 3 x 105 cells per 75 cm² flask in a total volume of 30 mL
for 1 day in “THMG" medium (pretreatment medium A), and for the following 3 days in “THG" medium (pretreatment medium B).

Treatment of test cultures and expression period:
Following centrifugation and resuspension the cells were dispensed into 75 cm² flasks (4-hour exposure: 1.5 x 107 cells per culture; 24-hour exposure: 1 x 107 cells per culture).
Two cultures were treated in parallel for each test group. Subsequently the treatment medium was added (see table below). The cultures were incubated for the respective exposure period

Treatment of the cultures
Test groups RPMI-5 medium* [mL] Vehicle or test substance preparation in vehicle [mL] S9 mix [mL]
Without S9 mix
Vehicle control 19.8 0.2 -
Treatment groups 19.8 0.2 -
Positive control (MMS) 19.8 0.2 -
With S9 mix
Vehicle control 19.0 0.2 0.8
Treatment groups 19.0 0.2 0.8
Positive control (CPP) 19.0 0.2 0.8
Positive control (DMBA) 19.0 0.2 0.8
* For exposure conditions without S9 mix RPMI-10 medium was used. At the end of the exposure period, the cells were transferred in tubes, centrifuged for
5 minutes at 1000 rpm (173 g) and were resuspended in RPMI-5 medium. The washing of the cells was repeated at least once. Then the cells were centrifuged at 1000 rpm (173 g,
5 min) and were resuspended in RPMI-10 medium. From each culture a sample of treated cells (2 x 105 cells/mL or 6 x 106 cells/flask) was pipetted in 75 cm² flasks and was incubated
for a 2-day expression period.
To maintain exponential growth during this phase, each culture was counted daily and the cell numbers were adjusted at each day to 2 x 105 cells/mL in 30 mL RPMI-10 medium. The
cell numbers were determined using a cell counter (CASY, Roche Applied Science, Mannheim, Germany).

Selection period
For the selection of the mutants, the cells were centrifuged (173 g, 5 min) and 5 x 105 cells from each culture were resuspended in 50 mL selection medium (“TFT" medium;
1 x 104 cells/mL). Per culture 200 μL were dispensed in each well of two 96-well plates (2000 cells/well). After incubation for at least 9 days, both the number of negative wells and
the number of wells containing small or large colonies were scored for calculation of the mutant frequency (MF).

Cytotoxicity determination
Cloning efficiency 1 (survival)
At the end of the exposure period, the cells were centrifuged (173 g, 5 min) and 400 cells from each test group were resuspended in 50 mL RPMI-20 medium (8 cells/mL). Per culture
200 μL were dispensed in each well of two 96-well plates (1.6 cells/well). After incubation for 9 - 11 days the plates were scored for empty wells.
Cloning efficiency 2 (viability)
After the expression period, 2 days after end of exposure, the cells were centrifuged (173 g, 5 min) and 400 cells from each culture were resuspended in 50 mL RPMI-20 medium
(8 cells/mL). Per culture 200 μL were dispensed in each well of two 96-well plates (1.6 cells/well). After incubation for at least 9 days the plates were scored for empty wells.
Relative suspension growth and relative total growth
For calculation of the relative suspension growth (RSG) and the relative total growth (RTG) the cell counts determined within the expression period at 2nd and 3rd passage after
exposure in the case of 4-hour exposure and 1st, 2nd and 3rd passage after exposure in the case of 24-hour exposure were used.
In the pretest single cultures per test group were conducted only.

Treatment conditions
pH
The pH was measured at least for the top concentration and for the vehicle controls with and without S9 mix.
Osmolality
Osmolality was measured at least for the top concentration and for the vehicle controls with and without S9 mix.
Solubility
Test substance precipitation was checked immediately after treatment of the test cultures and at the end of treatment with the unaided eye.

Acceptance criteria :
The MLTK assay is considered valid if the following criteria are met considering the international guidelines and the current recommendations of the IWGT (7, 9, 10, 11, 12):
- The absolute cloning efficiency obtained at the time of mutant selection (CE2) of the negative/vehicle controls should fall in the range of 65 - 120%.
- The SG of the negative/vehicle controls referring to the expression period following treatment should fall in the range of 8 - 32 for 4-hour exposure and 32 - 180 for 24-hour
exposure.
- The mutant frequency of the negative/vehicle controls should fall within the range of 50 - 170 x 10-6 colonies.
- The positive controls should yield an absolute increase in total MF that is an increase above the spontaneous background MF (an induced MF [IMF]) of at least 300 x 10-6
colonies. The small colony MF should account for at least 40% of that IMF, means a small colony IMF of at least 120 x 10-6 colonies. Alternatively, the positive controls should
induce at least 150 x 10-6 small colonies above the spontaneous background MF. The upper limit of cytotoxicity observed in the positive controls should have a RTG that is
greater than 10%.
- The highest applied concentration of the test substance should be 2 mg/mL, 2 μL/mL or 10 mM, unless limited by cytotoxicity or solubility of the test substance. If toxicity occurs,
the highest concentration should lower the RTG to 10 to 20% of survival. If precipitation occurs, the highest evaluated concentration should be the lowest concentration where
precipitation is observed by the unaided eye.
Evaluation criteria:
Assessment criteria:
The test substance is considered mutagenic if all following criteria are met (11, 12):
- The mutation frequency exceeds a threshold of 126 mutant colonies per 106 cells (GEF: Global Evaluation Factor) above the concurrent negative/vehicle control value.
and
- Evidence of reproducibility of any increase in mutant frequencies, means the mutagenic response occurs at least in both parallel cultures of one experiment.
and
- A statistically significant dose-related increase in mutant frequencies using an appropriate statistical trend test.
The test substance is considered non-mutagenic if at least one of the following criteria is met (11, 12):
- The mutation frequency is below a threshold of 126 mutant colonies per 106 cells (GEF) above the concurrent negative/vehicle control value.
or
- No evidence of reproducibility of an increase in mutant frequencies is obtained.
or
- No statistically significant dose-related increase in mutant frequencies using an appropriate statistical trend test is observed.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and vehicle controls (95% control limit) and the mutation rates of all negative and
vehicle controls of this study were taken into consideration. Results of test groups were rejected if the RTG were less than 10% of the respective
negative/vehicle control.
Whenever a test substance is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from
clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose-related shift in the ratio of small versus large colonies clastogenic
effects are indicated.
Statistics:
STATISTICS
An appropriate statistical method to test for linear trend (MS EXCEL function RGP; 10) was performed to assess a possible linear dose-relation in mutant frequencies. The dependent variable was the corrected mutant frequency and the independent variable was the
concentration. A 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. However, both, biological and statistical significance has been considered together.
Species / strain:
mouse lymphoma L5178Y cells
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:
In this study, no biologically relevant increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolizing system. (see attached result table)

In this study, no biologically relevant increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolizing system. The corrected mutant. Unfortunately, in the 1st Experiment after adding a metabolizing system no clear cytotoxicity  was observed up to the highest applied concentration of 300 μg/mL. Thus, this experimental part failed the requirements of the current guidelines. Nevertheless, in this experimental part no relevant increase in corrected mutation frequencies was obtained.

In all experiments after 4 and 24 hours treatment in the absence and presence of metabolic activation the values for the corrected mutation frequencies (MFcorr.: 26.4 – 101.0 per 106 cells) were close to or within the respective vehicle control values (MFcorr.: 37.2 – 75.7 per 106 cells) and within the range of our historical negative control data (MFcorr.: 12.2 – 109.2 per 106 cells).

The statistical analyses by testing for linear trend led to clearly negative findings for the 1st Experiment in the presence of S9 mix and for the 2nd Experiment in the absence of S9 mix. Besides, in the 1st Experiment in the absence of metabolic activation and in the 2nd and 3rd Experiment in the presence of metabolic activation a statistically significant linear dose-relation in mutant frequencies was observed. However, all values were clearly within the range of our historical negative control data and below the respective mutant frequency threshold and, therefore, the statistical significance was considered as biologically irrelevant.

Both the positive control substances known to induce gene mutations, MMS (without S9 mix; 5 or 15 μg/mL) and DMBA (with S9 mix;1.0 μg/mL and 2.5 μg/mL), and the well-known clastogen CPP (with S9 mix; 2.5 μg/mL) led to clearly increased mutant frequencies as expected. Currently only rare data for the positive substance DMBA are available for comparison. The values of the corrected mutant frequencies (MFcorr.: 228.3 – 1074.7 per 106 cells) clearly exceeded the respective calculated thresholds for a mutagenic effect based on the GEF (126 plus the mutant frequency of the respective negative control). In addition, the corrected mutant frequencies were within our historical positive control data range (226.0 – 1496.6 per 106 cells). At least one positive control group per experimental part clearly fulfilled the criteria for positive controls as mentioned in the current OECD Guideline 490.

Conclusions:
Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in three experiments performed independently of each other.
Thus, under the experimental conditions described, n-Butyl Acrylate (n-BA) did not induce forward mutations or structural chromosome aberrations in vitro in the mouse lymphoma assay with L5178Y TK+/- cells in the absence and the presence of metabolic activation
Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented publication, acceptable with restrictions (no test substance purity).
Principles of method if other than guideline:
Measurement of unscheduled DNA synthesis (UDS) was carried out as described by Schiffermann D. et al. Canc. Lett. 23: 297-305, 1984 and Tsutsui T. et al. Cancer Res. 44: 184-189, 1984.
GLP compliance:
not specified
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Species / strain / cell type:
other: Syrian hamster embryo fibroblasts (SHE-cells)
Metabolic activation:
without
Test concentrations with justification for top dose:
1, 10, 50, 100, 200 and 400 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
Tertiary cultures (1.7 x 10E5 cells) were incubated with arginine-free medium (including 2.5 % fetal bovine serum) for 48 h. After change of medium 3H-thymidine and 10 mM hydroxyurea were added and the cultures were incubated with various concentrations (1 - 400 µg/ml) of the test compound (solvent: DMSO). After an incubation time of 5 h the cells were washed (phosphate-buffered saline) and solubilized (2 % SDS). Subsequently the cells were precipitated (20 % trichloroacetic acid) and the precipitate was washed with ethanol. Subsequently the precipitate was incubated with tissue solubilizer (6 h, 50°C) and measurement of radioactivity (liquid scintillation) was carried out.
Species / strain:
other: Syrian hamster embryo fibroblasts (SHE-cells)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Test concentration (µg/ml)

UDS-Test (dpm/5 x 105 cells)

1

15400±869

10

13723±1006

50

10891±350

100

9545±269

200

5853±839

400

5084±262

Vehicle control

14002±215

Positive control

58098±802

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro cytogenicity / micronucleus study
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: Well documented publication, acceptable with restrictions. (no test substance purity)
Principles of method if other than guideline:
The test substance was investigated for its ability to induce micronuclei according to the method described by Schmuck et al. (Mutat Res. 203(6): 397-404, 1988).
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
other: Syrian hamster embryo fibroblasts (SHE-cells)
Metabolic activation:
without
Test concentrations with justification for top dose:
0.5 - 10 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Diethylstilbestrol
Details on test system and experimental conditions:
Tertiary cultures (1.5 x 10E5 cells) were incubated for 24 h at 37° C in a humified atmosphere in 12 % CO2 in air. The cultures were treated with various concentrations (0.5 - 10 µg/ml) of the test compound (solvent: DMSO). After incubation time of 5 h the compound was removed by medium change. After further incubation for 18 h cells were fixed, stained and scored for micronuclei. Only structures smaller than one third of the nucleus were counted in order to avoid confusion with dividing cells. For each concentration the number of cells containing single and multiple micronuclei was determined among a population of 2000 cells.
Species / strain:
other: Syrian hamster embryo fibroblasts (SHE-cells)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Test concentration (µg/ml)

Micronuclei per 2000 cells

0.5

13±4.7

1.0

10±4.8

5.0

15±6.3

10.0

10±3.1

Vehicle control

9±6.2

Positive control

72±3.0

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented publication, acceptable with restrictions. Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 were employed in this study for the detection of oxidising mutagens and cross-linking agents)
Principles of method if other than guideline:
According to Haworth et al. Environ. Mutagen. 5 (Suppl.1): 3-142, (1983).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidin auxotrophy
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced liver S-9 mix from the Sprague-Dawley rat and male Syrian hamster.
Test concentrations with justification for top dose:
up to 10000 µg/plate
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:
other: Without S9: Sodium azide (TA1535 and TA 100), 9-aminoacridine (TA 1537), and 4-nitro-o-phenylenediamine (TA98). With S9: 2-aminoanthracene for all strains.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
A chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In TA1535 and TA98 tested without S9, concentration of 333 µg/plate and higher were toxic; in TA1537 tested without S9, concentration of 1000 µg/plate and higher were toxic.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mean Number of Revertants

Dose (µg/plate)

TA100

TA1535

NA

10% HLI

10% RLI

NA

10% HLI

10% RLI

0

93

150

147

12

19

13

3.3

-

-

-

11

-

-

10

-

-

-

12

-

-

33

-

-

-

12

12

100

99

142

149

6

17

8

333

92

147

158

t

13

10

1000

97

155

138

-

7

5

3333

96

157

118

-

5

4

10000

94

104

102

-

0

4

POS

838

1718

1349

922

269

394

Dose (µg/plate)

TA1537

TA98

NA

10% HLI

10% RLI

NA

10% HLI

10% RLI

0

6

7

12

20

17

21

3.3

-

-

-

18

-

-

10

5

-

-

15

-

-

33

8

-

-

22

-

-

100

5

9

11

15

21

22

333

9

12

12

t

21

23

1000

0

7

9

-

16

18

3333

-

7

12

-

16

16

10000

-

5

9

-

10

15

POS

970

267

291

563

1095

587

RLI: Rat Liver S9
HLI: Hamster Liver S9
t: Toxic

- : not done

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceplate publication which meets basic scientific principles. Escherichia coli strain WP2 uvrA pKM101a nor S. typhimurium strain TA102 were employed in this study for the detection of oxidising mutagens and cross-linking agents)
Principles of method if other than guideline:
According to Ames et al. (Mutation Research 31: 347, 1975). In addition, n-Butyl acrylate was also tested in a modification of the liquid suspension test as described by Rannug et al. (Chem. Biol. Interact. 12: 251-263).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: TA1535, TA1537, TA1538, TA98, TA100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254- and phenobarbital- induced rat liver S9 fractions
Test concentrations with justification for top dose:
30 - 2000 µg/Plate
15, 150 and 1500 µg/ 2 mL incubation volume for liquid suspension test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
benzo(a)pyrene
other: Gylcidyl methacrylate, 2-aminoacridine, 2-aminoanthracene, 4-Nitro-o-phenylenediamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and liquid suspension tests (only strain TA100)

NUMBER OF REPLICATIONS: 3

Positive control substance: TA1535: Sodium azide, Gylcidyl methacrylate, 2-Aminoanthracene TA1537: 9-Aminoacridine, 2-Aminoanthracene TA1538 and TA98: 4-Nitro-o-phenylenediamine, 2-Aminoanthracene, Benzo(a)pyrene TA100: Sodium azide, Gylcidyl methacrylate, 2-Aminoanthracene, Benzo(a)pyrene.
Species / strain:
other: TA1535, TA1537, TA1538, TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the highest concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In addition, no genotoxicity effect was observed in the liquid suspension test.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Cytogenetic test in hamster via inhalation: negative

Cytogenetic test in rats via inhalation: negative

Long-term carcinogenicity tests: non-carcinogenic

supported by new data with the structural analog ethyl acrylate: gpttransgenic mouse (gptdelta mouse ) model according to OECD TG488 via gavage: negative

SEE ADDITIONAL INFORMATION BELOW

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
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:
other: Comparable to guideline study.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
no
Type of assay:
chromosome aberration assay
Species:
hamster, Chinese
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Zuchthygiene, Zuerich
- Weight at study initiation: males-34 (31-38) g and females-30 (25-34) g
- Housing: 2-3 hamsters per cage
- Diet (e.g. ad libitum): Herilan-Mrh supplied by H. Eggersmann KG
- Water (e.g. ad libitum): tap water
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:
A constant amount of n-Butyl acrylate (14.4ml/hr) was delivered using a continuous infusion piston pump to a heated (110° C) glass evaporator. The n-butyl acrylate vapors were diluted with dust-free conditioned air (3200 l/hr; temperature =22±2° C; humidity - 55±10 %) to deliver the desired concentration of test material to the test animals . The exposure chamber was 200 L. Control animals received fresh air only.

TEST ATMOSPHERE
- Brief description of analytical method used: The n-butyl acrylate air mixture was measured continuously using a flame ionization detector (FID). Apparatus used was a calibrated total hydrocarbons analyzer (CARLO ERBA, mod. 370).
- Samples taken from breathing zone: yes.
Duration of treatment / exposure:
6 hours/day for 3 days and 5 hours/day for 1 day
Frequency of treatment:
6 hours/day and 5 hours/day
Post exposure period:
5 h
Remarks:
Doses / Concentrations:
817 ppm (corresponding to 4.28 mg/L) Calculation of concentrations (mg/L) based on Derelanko MJ (2000). Toxicologist's Pocket Handbook, CRC Press, conversion table, p. 57.
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed
Positive control(s):
see attached statement
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose was selected based on results of LC50 study. Dose of 65% of the LC50 was considered to be appropriate for this study. 65% of LC50 (1220 ppm) = ca. 800 ppm. Analytical concentration was found to be 817 ppm.


DETAILS OF SLIDE PREPARATION: Three hours before sacrifice, the animals were injected ip with 3.3 mg/kg of colcemid in order to arrest mitosis in the metaphase. Two hours after the colcemid injection the animals were sacrificed and bone marrow prepared according to the method of Schmid and Staiger (1969). Hundred metaphases were analyzed per animal.
Schmid and Staiger, Mut. Res. 7: 99-108, 1969.




Statistics:
Statistical evaluations was performed using the chi2- test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
The 4 days of exposure caused clinical signs of dyspnoea, disequilibrium, bloody discharge from eyes and nose, mortality of 4/10 animals and decrease in body weight (20 %).
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
There was no significant increase in the rate of chromosome aberrations in exposed hamsters with comparison to controls.
Gross pathological examination revealed bronchopneumonia of both lungs in the deceased animals. No gross lesions were observed in sacrificed animals after 4 days of exposure.

No. of animals male/femalea

Metaphases Analyzedb

Aberrant Metaphases

Mitotic index (%) male/female

Incl. Gaps

Excl. Gapsc

With Exchanges

n

%

n

%

n

%

Fresh air control

10/10 (9/9)

1800

8

0.44

3

0.17

0

0

3.17/2.97

n-butyl acrylate

10/10 (5/8)

1300

10

0.77d

4

0.31d

1

0.08

2.20/2.79

a-       The figures given in parentheses correspond to the number of animals analyzed

b-       100 metaphases/animal

c-       Breaks, fragments, exchanges. No multiple aberrant metaphases (cells with ≥ 5 aberrations) or pulverizations observed.

d-       P>0.05

Conclusions:
Interpretation of results (migrated information): negative
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
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:
other: Comparable to guideline study.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
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
- Source: SPF-Breeding, WIGA, Sulzfeld
- Weight at study initiation: males-242 g (215-259 g) and females-190 g (171-215 g)
- Housing: 2-3 rats per cage
- Diet (e.g. ad libitum): Herilan-Mrh supplied by H. Eggersmann KG
- Water (e.g. ad libitum): tap water
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:
A constant amount of n-Butyl acrylate (14.4ml/hr) was delivered using a continuous infusion piston pump to a heated (110° C) glass evaporator. The n-butyl acrylate vapors were diluted with dust-free conditioned air (3200 l/hr; temperature = 22±2° C; humidity - 55±10 %) to deliver the desired concentration of test material to the test animals. The exposure chamber was 200 L. Control animals received fresh air only.

TEST ATMOSPHERE
- Brief description of analytical method used: The n-butyl acrylate air mixture was measured continuously using a flame ionization detector (FID). Apparatus used was a calibrated total hydrocarbons analyzer (CARLO ERBA, mod. 370).
- Samples taken from breathing zone: yes.
Duration of treatment / exposure:
6 hours/day for 4 days
Frequency of treatment:
6 hours/day
Post exposure period:
5 h
Remarks:
Doses / Concentrations:
820 ppm (corresponding to approx. 4.3 mg/L) Calculation of concentrations (mg/L) based on Derelanko MJ (2000). Toxicologist's Pocket Handbook, CRC Press, conversion table, p. 57.
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed
Positive control(s):
see attached statement
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose was selected based on results of LC50 and subacute repeated dose studies. Dose of ca. 1/3 of LC50 was considered to be appropriate for this study. LC50/3 = 2145 ppm/3 = ca. 800 ppm. Analytical concentration was found to be 820 ppm.


DETAILS OF SLIDE PREPARATION: Three hours before sacrifice, the animals were injected ip with 3.3 mg/kg of colcemid in order to arrest mitosis in the metaphase. Two hours after the colcemid injection the animals were sacrificed and bone marrow prepared according to the method of Schmid and Staiger (1969). Fifty metaphases were analyzed per animal.

Schmid and Staiger, Mut. Res. 7 :99-108, 1969.



Statistics:
Statistical evaluations was performed using the chi2- test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
The 4 days of exposure caused clinical signs of dyspnoea, bloody discharge from eyes and nose and decrease in body weight (6-7 %).
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
There was no significant increase in the rate of chromosome aberrations in exposed rats with comparison to controls.
No mortality and pathological changes were observed in the exposed animals.

No. of animals male/femalea

Metaphases Analyzedb

Aberrant Metaphases

Mitotic index (%) male/female

Incl. Gaps

Excl. Gapsc

With Exchanges

n

%

n

%

n

%

Fresh air control

10/10 (6/6)

1200

5

0.42

0

0

0

0

4.12/3.98

n-butyl acrylate

10/10 (8/8)

1600

2

0.13d

1

0.06d

0

0

3.16/3.43

a-       The figures given in parentheses correspond to the number of animals analyzed

b-       100 metaphases/animal

c-       Breaks, fragments, exchanges. No multiple aberrant metaphases (cells with ≥ 5 aberrations) or pulverizations observed.

d-       P>0.05

Conclusions:
Interpretation of results (migrated information): negative
Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study follows GLP and OECD TG 488
Guideline:
other: OECD Guidelines for the Testing of Chemicals 488 (26 July 2013: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays
GLP compliance:
yes (incl. certificate)
Type of assay:
transgenic rodent mutagenicity assay
Species:
mouse
Strain:
other: C57BL/6JJmsSlc-Tg (gpt delta) [SPF]
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan SLC, Inc.
- Age at study initiation:
At the time of purchase: 8 weeks of age
At the time of assignment to groups: 9 weeks of age
- Weight at study initiation: 24.2 to 26.8 g
- Assigned to test groups: Animals were assigned to groups based on their body weights on Day 1 using LATOX-F/V5 (FFC) computer system package. The weight range of the animals used was within ±20% of the overall mean weight. Unselected animals were excluded from the study on Day 1.
- Housing: Three animals were housed in a polycarbonate cage (W 18.2 × D 26.0 × H 12.8 cm) with bedding (ALPHA-driTM; lot No. 04114, Shepherd Specialty Papers). However, animals were housed individually from Day -6 because aggressive behavior was observed.
- Diet (e.g. ad libitum): Animals were allowed access to pellet diet CRF-1 sterilized by radiation (lot No. 140703, Oriental Yeast) ad libitum. BSRC obtained the certificate of analysis (report No. AR-14-JP-002550-01: July 22, 2014) on the contaminant levels for this lot from the manufacture and confirmed that such levels were within the acceptable limits proposed by the Japan Experimental Animal Feed Association. The food was also provided to animals at the time of exchanging the feeders.
- Water (e.g. ad libitum): Animals were provided access to tap water from water bottles ad libitum. The drinking water was examined for the quality at another inspection agency in April 2015 according to the specifications of the Water Works Law. In March and May 2015, BSRC examined the water for bacteria (common bacteria and Escherichia coli). The analytical results (report No. K15-0033, in-house data Nos. GT15-03 and GT15-05) of these analyses indicated that the levels of contaminants in the water were within the acceptable limits of the Tap Water Quality Standard and that no bacteria were detected in the water
- Acclimation period: The quarantine and acclimation period was from Day -7 to 1

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26°C (actual values: 22.8 to 23.1°C)
- Humidity (%): 35 to 70%RH (actual values: 42.2 to 58.3%RH)
- Air changes (per hr): 12 times or more/hour
- Photoperiod (hrs dark / hrs light): 12 hours (lights on: 7:00, lights off: 19:00)

IN-LIFE DATES: From: March 24,2015 To: June 5, 2015
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil (lot No. WEF2972), the vehicle to prepare the test substance formulations, was used
- Storage conditions: Room temperature
- Manufacturer: Wako Pure Chemical Industries, Ltd
- Lot/batch no. (if required): (lot No. WEF2972)
- Grade: For biochemistry
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The vehicle and the test substance were administered to mice orally once daily for 28 consecutive days at about 24-hour intervals using a disposable syringe with a Teflon sonde. The dosage volume (mL) was set at 0.1 mL per 10 g of body weight and was calculated on the basis of the most recent individual body weight measured in section 15.12.6.
The positive control substance was administered to mice orally once daily for 5 consecutive days at about 24-hour intervals using a disposable syringe with a Teflon sonde. The dosing volume was set at 0.1 mL per 10 g of body weight and was calculated on the basis of the most recent individual body weight measured.
Duration of treatment / exposure:
28 consecutive days at about 24-hour intervals
Frequency of treatment:
Once daily
Post exposure period:
3 days of manifestation period
Remarks:
Doses / Concentrations:
0, 8, 20, and 50 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
To ensure that the data would be available for 5 animals in each group, the number of the treated animals was 6 in each group.
Control animals:
yes
yes, historical
Positive control(s):
Benzo[a]pyrene (B[a]P)
- Justification for choice of positive control(s): Considering information in the academic documents, the following substance was selected as the positive control
- Route of administration: The positive control substance was administered to mice orally once daily for 5 consecutive days at about 24-hour intervals using a disposable syringe with a Teflon sonde.
- Doses / concentrations: The dose was set at 125 mg/kg in reference to a literature
Tissues and cell types examined:
The animals were necropsied after euthanasia by exsanguination under isoflurane anesthesia. The liver, stomach and testes were removed from the animals. The liver, stomach and testes were observed macroscopically. The organ weight of the liver was measured. The organ weight to body weight ratio (relative organ weight) was calculated from the body weight weighed on the day of necropsy and organ weight (absolute organ weight / body weight on the harvest day × 100). The liver was measured in grams (to 2 decimal places).
Details of tissue and slide preparation:
METHOD OF ANALYSIS: Pathological examinations consisted of macroscopic examination and histopathological examination. Because histopathological findings were observed in the forestomach in a carcinogenesis study[3], the stomach and liver were selected for histopathological examination. Fixed stomach and liver were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H.E.) routinely
Histopathological examination was conducted using all prepared specimens. All histopathological observations were graded according to severity and recorded.
Evaluation criteria:
VALIDITY OF STUDY
Since the following conditions were satisfied, the test will be considered successfully performed:
- The mutation frequency for the liver 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 calculated from our historical data.
Statistics:
The data on the mutant frequency from the negative control group and each test substance treated group were tested by Bartlett’s test for homogeneity of variance first. 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 was performed to analyze the differences.
The data on the mutant frequency from the negative control group and the positive control group were tested by F test for homogeneity of variance first. If homogeneity of variance was determined (not significant on F test), Student’s t test was performed to assess the statistical significance of differences between the negative control group and the positive control group. If there was no homogeneity (significant on F test), Aspin-Welch’s t test was performed to analyze the differences.
The significance level of 5% (two-sided) was selected for each test.
The results were evaluated as positive when the mutant frequency in the test substance treated group was significantly different from that in the negative control group. Final judgment was made in consideration of biological relevance under the test conditions.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
inflammatory cell infiltration (slight) in the forestomach
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
Liver
In the negative control group, the mean±SD of mutant frequency among the individuals was 3.14±1.35 (×10-6).
The means±SD of mutant frequencies in the ethyl acrylate treated groups, 8.00, 20.0 and 50.0 mg/kg/day, were 2.29±1.51 (×10-6), 1.67±0.98 (×10-6) and 1.68±1.16 (×10-6), respectively, indicating that ethyl acrylate was negative for liver genotoxicity in the Spi- assay . Furthermore, no statistically significant increases were observed as compared with the negative control group.
In the positive control group, the mean±SD of mutant frequency among the individuals was 12.17±7.03 (×10-6) and a statistically significant increase was observed compared with the negative control group.
Stomach
The means±SD of mutant frequencies in the ethyl acrylate treated groups, 8.00, 20.0 and 50.0 mg/kg/day, were 4.02±3.32 (×10-6), 3.36±3.38 (×10-6) and 2.00±0.31 (×10-6), respectively, indicating that ethyl acrylate was negative for stomach genotoxicity in the Spi-assay. Furthermore, no statistically significant increases were observed as compared with the negative control group.
In the positive control group, the mean±SD of mutant frequency among the individuals was 21.86±8.81 (×10-6) and a statistically significant increase was observed compared with the negative control group.

Body weight and general conditions: Body weights in all the test substance treated groups were similar to those in the control group throughout the treatment periods. There was no change in the general condition in any of the test substance treated groups.

Liver weight and relative liver weight:There was no difference in the liver weight or relative liver weights of the test substance treated groups compared with those of the negative control group.

Macroscopic findings:There were no macroscopic findings related to test substance treatment in the liver, stomach or testis of the other animals.

Histopathological findings:Inflammatory cell infiltration (slight) in the forestomach was observed in 2 animals in the 8.00 mg/kg/day group and 1 animal in the 50.0 mg/kg/day group. Furthermore, inflammatory cell infiltration (slight) in the forestomach was observed in 4 animals in the positive control.There were no findings related to test substance treatment in the liver

Conclusions:
Interpretation of results (migrated information): negative
Ethyl acrylate did not induce gene mutation in transgenic mice under the conditions in this study.
Executive summary:

In 2014 IARC added Ethyl Acrylate as an agent for high priority reevaluation in 2015-2019.  Conduct of this study was required in preparation for IARC’s cancer classification reevaluation of Ethyl Acrylate. A gene mutation assay with transgenic mice (gpt delta mouse) was conducted to assess the potential of ethyl acrylate to induce gene point mutations and deletion mutations using the gpt gene and the red/gam genes (Spi⁻ selection), respectively, as mutation reporter genes in the liver and stomach.

In dose range-finding study in C57BL/6JJmsSlc mice treated for 28 days with 0, 25.0, 50.0, 100, or 200 mg/kg/day of ethyl acrylate in corn oil, gross observation revealed white nodules in the forestomach in all mice receiving 200 mg/kg/day and in one of the 3 mice receiving 100 mg/kg/day. Histopathological examination of animals in the 100 and 200 mg/kg/day groups revealed that there was an inflammatory response in the forestomach (consisting of squamous cell hyperplasia, fibrosis of mucosa, and infiltration of inflammatory cells). In addition, erosion was observed in 1 animal in the 200 mg/kg/day group. A local inflammatory response may interfere with the outcome of the mutagenicity data and should thus be avoided. Metabolism of ethyl acrylate occurs through carboxylesterases and through conjugation with glutathione. The latter metabolic reaction becomes saturated between 20 and 100 mg/kg, while dosages ≥ 100 mg/kg exhibit complete saturation. Therefore, a dosage of 50.0 mg/kg/day was selected as the high dose as this dose was without a potentially confounding inflammatory response and is anticipated to be at the threshold for saturation of GSH metabolism, based on previous rodent studies. Two additional dosage levels including 20.0 and 8.00 mg/kg/day were selected as lower doses in the present study.

The test substance was administered to male transgenic mice orally for 28 consecutive days by gavage and after 3 days of the manifestation period, the liver, stomach and testis were removed and mutant frequencies in the liver and stomach were determined.

The results showed that the mutant frequencies (6-thioguanine and Spi-selection) in the liver and stomach of all groups treated with ethyl acrylate were negative for genotoxicity under the conditions of this study . Furthermore, the mutant frequencies in the liver and stomach of all groups treated with ethyl acrylate did not show any statistically significant increases as compared with the negative control group.

The mutant frequencies in the liver and stomach of the positive control group, which was treated with benzo[a]pyrene (B[a]P, dosage level of 125 mg/kg/day), increased in both thegptand Spi-assays and these increases were statistically significant compared with that of the negative control group.

 

It is, therefore, concluded that ethyl acrylate did not induce gene mutation in transgenic mice (negative) under the conditions in this study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro studies

 

Bacterial systems

Three Ames tests with Butyl acrylate are available. Butyl acrylate was tested in the Ames test with Salmonella typhimurium TA98, TA100, TA1535, and TA1537 in concentrations from 3.15 up to 1000 nL/plate (2.84, 9.0, 28.4, 90.0, 283.5 and 900.0 µg per plate) with and without metabolic activation using the plate incorporation method. No mutagenic and cytotoxic effects were observed (BASF AG, 1977). In a study by Zeiger (1987), butyl acrylate was tested in Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 at concentrations up to 10000 µg/plate with and without metabolic activation using the preincubation method. No mutagenic effects were observed, and in TA1535 and TA98 tested without S9, concentration of 333 µg/plate and higher were toxic; in TA1537 tested without S9, concentration of 1000 µg/plate and higher were toxic. In a study by Waegemaekers (1984), butyl acrylate was tested in Salmonella typhimurium TA 1535, TA 1537, TA1538, TA 98 and TA 100 at concentrations from 30 to 2000 µg/plate with and without metabolic activation. No mutagenic effects were observed and cytotoxicity was observed in the highest dose tested.

 

Mammalian cell gene mutation tests

n-Butyl Acrylate (n-BA) was tested for its ability to induce gene mutations at the thymidine kinase (TK) locus or structural chromosome aberrations at chromosome 11 in L5178Y TK+/- mouse lymphoma cells in vitro with the microwell method.

Three independent experiments were carried out with and/or without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats (exogenous metabolic activation). Cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for an expression period of about 48 hours and then cultured in selection medium for another approx. 10 days. Finally, the number of large and small colonies was determined.

The negative controls gave mutant frequencies within the range expected for the L5178Y TK+/- mouse lymphoma cell line. All positive controls either for the induction of gene mutations or clastogenicity – methyl methanesulfonate (MMS), cyclophosphamide (CPP) and 7,12-dimethylbenz[a]anthracene (DMBA) - led to the expected increase in the frequencies of forward mutations. Cytotoxicity indicated by reduced relative total growth (RTG) of below 20% of control was observed in all experiments in the absence and presence of metabolic activation, except in the 1st Experiment with metabolic activation.

In this study, the corrected mutant frequencies in the exposed test groups did never exceed the corresponding Global Evaluation Factor (GEF) under any experimental condition. The test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in three experiments performed independently of each other.

Thus, under the experimental conditions described, n-butyl acrylate did not induce forward mutations or structural chromosome aberrations in vitro in the mouse lymphoma assay with L5178Y TK+/- cells in the absence and the presence of metabolic activation.

 

In an in vitro UDS-Test with Syrian hamster embryo fibroblasts, butyl acrylate was tested in concentrations from 1- 400 μg/mL. No induction of unscheduled DNA synthesis in SHE-cells was observed (Wiegand et al., 1989). No cytotoxicity was reported by the authors. No exogenous metabolic activation system was used in the cytogenetic study (described below) and the UDS test, since intrinsic metabolic capacity had been shown in SHE cells previously (Poiley et al. 1980). However, a negative result in an UDS assay alone is not a proof that a substance does not induce gene mutation as this assay is sensitive to some but not all DNA repair mechanisms.

 

Mammalian cell cytogenetic tests

In a cytogenetic study with Syrian hamster embryo (SHE) fibroblasts, no induction of micronuclei was observed at concentrations of 0.5 - 10 μg/mL without metabolic activation (Wiegand et al., 1989). At the tested concentrations no cytotoxicity of the test substance was observed.

The negative result of the above cited cytogenetic study was confirmed in the new in vitro gene mutation test in L5178Y mouse lymphoma cells, in which n-butyl acrylate did not induce structural chromosome aberrations in the absence and the presence of metabolic activation. This test was performed according to the current OECD TG 490 up to cytotoxic concentrations under GLP conditions.

In 1991 NTP published result tables of clastogenetic assays performed by Litton Bionetics (1985). The information within these tables is not comprehensive; cytotoxicity is provided as reduced number of cells examined. It is distinguished between complex and simple aberrations without providing any definition, i.e. whether simple aberrations are gaps, which generally are not included in the total aberration frequency. The overall result of the test was interpreted as negative. Only in trials without S9 addition, and in concentrations where high cytotoxicity (only 5 - 50% of 200 cells could be evaluated) occurred, a significant increase of aberrant cells was found. According to Galloway (2000) cytotoxicity is a confounding variability in chromosome aberration assay. In a survey, looking at 253 compounds tested in 27 different laboratories, it was shown that chromosome aberrations can occur secondary to toxicity and these data are not relevant for human risk. This occurs when toxicity exceed 40 – 50 %. Therefore, the observed increase of aberrations is probably due to the cytotoxicity. In one trial with metabolic activation, a slight, but statistically significant increase of simple chromosomal aberrations (gaps ?) was observed in the highest concentration without cytotoxic effect.

 

Conclusion :

Within the ECHA final decision, there was a certain concern about the reliability of the older genotoxicity tests. Within the new performed mouse lymphoma assay in L5178Ytk+/-3.7.2C cells the negative results from the older tests in regard to cell gene mutations as well as chromosomal aberrations were confirmed. Genetic events detected using the tklocus include both gene mutations and chromosomal events. The cell lines used in these tests measure forward mutations in reporter genes, specifically the endogeneous thymidine kinase gene (TK for human cells and Tk for rodent cells, collectively referred to as TK in this Guideline). The OECD TG 490 is intended for use with two cell lines: the L5178Y TK+/--3.7.2C mouse lymphoma cell line (generally called L5178Y) and the TK6 human lymphoblastoid cell line (generally called TK6). Although the two cell lines vary because of their origin, cell growth, p53-status, etc., the TK gene mutation tests can be conducted in a similar way in both cell types as described in this guideline.The autosomal and heterozygous nature of the thymidine kinase gene enables the detection of viable colonies whose cells are deficient in the enzyme thymidine kinase following mutation from TK+/- to TK-/-. This deficiency can result from genetic events affecting the TK gene including both gene mutations (point mutations, frame-shift mutations, small deletions, etc.) and chromosomal events (large deletions, chromosome rearrangements and mitotic recombination). The latter events are expressed as loss of heterozygosity, which is a common genetic change of tumor suppressor genes in human tumorigenesis. (OECD, 2016)

 

Overall, the available in vitro tests give no indication for a genotoxic potential (gene mutations and chromosomal events) of n-butyl acrylate.

 

In vivo studies

Butyl acrylate was tested in two cytogenetic assays in vivo. In those studies, Sprague-Dawley rats and Chinese hamsters were exposed by inhalation 6 hours per day for 4 consecutive days to vapour concentrations of 820 ppm and 817 ppm (corresponding to approx. 4.3 and 4.28 mg/L), respectively. Clear signs of toxicity (dyspnoea, bloody discharge from eyes and nose, decrease in body weight, lethality) were observed in both species. The chromosome analysis carried out in the bone marrow of the animals after the 4-day inhalation did not indicate any chromosome-damaging effect of butyl acrylate in either species or sex (BASF AG 1978, Engelhardt & Klimisch 1983).

 

The study design of exposing rats and hamsters on 4 consecutive days to 817 / 820 ppm for 6 h /day (last exposure period 5 h) and killing the animals 5 hours after the last exposure (i.p. Colchemid treatment 3 h before sacrifice) allows the analysis of numerous post-exposure duration periods at this single harvest time point (i.e. 5h, 29h, 53h, etc.). Hence, the sampling period provides opportunities to investigate the immediate and delayed effects in the same animals.

 

The inhalation route was chosen as appropriate route of exposure, because it is the only relevant route of exposure for humans and in parallel the 2 inhalation tests was performed. The dose was selected based on results of LC50 and subacute repeated dose studies, representing ca. 1/3 of LC50 for rats and and 65 % of the LC50 for hamsters. Within the study severe toxicity was observed in rats and hamsters, dyspnoea, disequilibrium, bloody discharge from the eyes and noses and death of some animals demonstrating systemic availability of the substance and its metabolites. These clinical observations (including mortality) clearly indicate that an MTD was achieved in these genotoxicity studies. Demonstration of target tissue exposure is further evidenced by the slight to moderate reduction of the mitotic index of 23 % / 14 % in male and female rats and 31 % / 6 % in male and female hamsters. In addition within various toxicokinetic studies it was shown that after oral uptake the fast hydrolysis into butanol and acrylic acid (first path) is the major metabolic pathway (Both butanol and acrylic acid give no suspicion concerning mutagenicity in vivo). Whereas after parenteral uptake the conjugation with GSH seems to be an important metabolic pathway and mercapturic acid metabolites were determined in faeces and urine – showing the systemic availability of the substance and its metabolites.

 

In both studies with n-butyl acrylate no positive control groups were run in parallel. However, in 1977 these standards were not set, yet, but many chromosome aberration studies in different species were part of the laboratory’s routine testing program at that time. Unfortunately, data or records of the in-house validation are not available from the pre-computer era. Therefore (raw)data of studies performed in this time were compiled. A set of 7 studies was performed from Feb to Oct 1975 in Chinese hamsters which elucidated clastogenic effects in test substance exposed animals (see attached statement 7.6.2). The observation of increased chromosomal aberration rates in these studies clearly demonstrates the proficiency of the laboratory of BASF AG, Ludwigshafen, Germany, in performing this test system. Besides, in these studies several routes of administration were used (e.g. oral [gavage], intraperitoneal, inhalation). From 1980 onwards chromosome aberration studies in Chinese hamsters included positive control groups (cyclophosphamide). In 1974 a cytogenetic study in Sprague-Dawley rats, both sexes, with single and multiple intraperitoneal administration of the test substance was performed in the BASF test facility. The positive control substance Trenimon® led to clearly increased numbers of aberrant meta-phases 6, 24 and 48 hours after the last administration. Thus, also the proficiency of the laboratory to perform the chromosome aberration test in bone marrow of rats was demonstrated (see attached statement 7.6.2).

.

Finally, it has to be considered that one of the reasons for the absence of positive control groups in the published studies was that it was unusual to use them at that time. Additionally, both studies used the inhalation route and for this administration route no positive control substance was known. Thus, the scientists decided to run the studies without them.

 

Although these tests do not exactly meet current OECD TG, the data clearly indicate a lack of genotoxic potential for n-butyl acrylate. The studies are reliable and very valuable for assessing the genotoxic potential of n-butyl acrylate as the studies included robust numbers of animals, demonstrated systemic n-butyl acrylate exposure at a clear MTD, aBend are consistent with the lack of carcinogenic response in long-term rodent studies.

According to the ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a: Endpoint specific guidance , Version 3.0 August 2014, the weight-of-evidence (WoE) approach shouldinclude an evaluation of the available data as a whole,i.e.bothover and acrosstoxicological endpoints, e.g. for mutagenicity the existing carcinogenicity data. ECHA’s endpoint specific guidance indicates that “there is considerable positive correlation between the mutagenicity of substances in vivo and their carcinogenicity in long-term studies with animals.

Taking the weight- of- evidence approach (WoE) to assess the mutagenic potential of n-butyl acrylate, this includes the in vitro and in vivo mutagenicity data supplemented with the results of two carcinogenicity studies.

Based on the available in vitro tests there is no specific indication for a genotoxic potential (gene mutations and chromosomal events) of n-butyl acrylate. In support of the two in vivo cytogenetic assays in rats and hamster with negative result,it is scientifically justified that n-butyl acrylate’s lack of carcinogenicity also demonstrates a lack of in vivo genotoxicity (chromosomal aberrations and/or mutagenicity)of this substance. Therefore the carcinogenicity studies data indicates that the substance does not have the potential for genotoxic effects in vivo and should be considered to be equivalent to data generated by the in vivo chromosomal aberration and mutagenicity test methods referred to in Article 13. 

 

In addition the negative findings in the in vivo studies is in line with the data of the other category members. In a very recent study ethyl acrylate was also tested as not mutagen in the gpt transgenic mouse (gpt delta mouse ) model according to OECD TG488. After repeated oral gavage no increase in mutant frequency in the target organs stomach and liver were found. (BAMM 2015)

 

Overall conclusion

In various vitro tests (bacterial gene mutation test, mammalian cell gene mutation assay, cytogenetic tests, UDS test ) n-butyl acrylate did not show genotoxic effects at non-cytotoxic concentrations. In addition, two older but generally reliable in vivo chromosome aberration tests, using the most appropriate route of exposure, tested up to severe toxic (lethal) concentrations and clear indications of systemic availability of the product / metabolites did not show any cytogenetic effect. Furthermore, the chemical class and the metabolic degradation products give no suspicion that n-butyl acrylate has a genotoxic potential in vivo. Finally, two reliable long-term carcinogenicity tests did not show any indication of a possible carcinogenic effect. n-Butyl acrylate gives no indication for a genotoxic potential.

Therefore no further testing should be required to assess the genotoxicity of n-butyl acrylate.

 

References

- Galloway SM., Cytotoxicity and chromosome aberrations in vitro: experience in industry and the case for an upper limit on toxicity in the aberration assay., Environ Mol Mutagen. 2000;35(3):191-201.

- Poiley JA et al., Metabolic activation by hamster and rat hepatocytes in the Salmonella mutagenicity assay., J Natl Cancer Inst. 1980 Dec;65(6):1293-8.

- OECD TG 490, In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene, Adopted July 29, 2016

Justification for classification or non-classification

CLP classification (Regulation (EC) No 1272/2008): no classification required