Isobutyl acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro

Ames test (4 strains): negative with and without metabolic activation (NTP, 1982)

HPRT test (V79): negative with and without metabolic activation (BASF, 2014)

TK test (mouse lymphoma cells): negative with and without metabolic activation (BASF, 2016)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

February 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

- Name of test material: tert.-Butylacrylate
- Physical state: liquid, colourless, clear
- Lot/batch No.: 011631eda0

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and identity of media: MEM (minimal essential medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: MEM (minimal essential medium) containing Hank’s salts
supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %). The cell cultures were incubated at 37°C in a
1.5 % carbon dioxide atmosphere (98.5 % air).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/β-naphthoflavone induced rat liver
- method of preparation of S9 mix : An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-orthophosphate-buffer (100 mM, pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: final protein concentration in the culture: 0.75 mg/mL
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

pre test: 10 - 1280 µg/mL (with and without S9-mix)
The dose range of the main experiments was set according to the data generated in the preexperiment. The individual concentrations were generally spaced by a factor of 2.0. Narrower spacing was used at higher concentrations.

Experiment 1:
160, 320, 640, 960, 1280 µg/mL (with and without S9 mix)
Experiment 2:
320, 640, 800, 960, 1120 µg/mL (without S9 mix);
640, 800, 960, 1120, 1280 µg/mL (with S9 mix)

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The final concentration of DMSO in the culture medium was 0.5% (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

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: 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (incubation with a selection agent): 8 days

SELECTION AGENT: 6-thioguanine (11 μg/mL)

Rationale for test conditions:

- The pre-experiment was performed in the presence and absence (4 hours treatment) of metabolic activation. The highest concentration of the preexperiment was chosen with regard to the purity (99.83%) and the molecular weight (128.169 g/mol) of the test item.
- To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations. The cultures at the two lowest concentrations in experiment I with and without metabolic activation were not continued since a minimum of only four concentrations is required by the guidelines. In experiment II the cultures at the lowest concentration with metabolic activation were not continued for the same reason. The cultures at the highest concentration without metabolic activation were not continued to due exceedingly severe cytotoxic effects.

Evaluation criteria:

- A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
- The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in experiment I at 640 μg/mL and above without metabolic activation and at 960 μg/mL and above with metabolic activation. In experiment II relevant cytotoxic effects were noted at 640 μg/mL and above without metabolic activation. Cytotoxicity was more severe and the maximum concentration without metabolic activation was not analyzable. The relative cloning efficiency I was still acceptable with 16.3% and 17.0% but the cell density at the first subcultivation after treatment was too low to proceed. In the presence of metabolic activation cytotoxicity as described above was noted at 800 μg/mL and above. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation.

STUDY RESULTS
- No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls.
- The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of the first experiment at 960 μg/mL without metabolic activation. This effect however, was judged as biologically irrelevant, since it was based upon the rather low solvent control of 4.9 mutant colonies/10^6 cells and was not reproduced in the parallel culture under identical conditions.
- In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 4.9 up to 19.0 mutants per 10^6 cells; the range of the groups treated with the test item was from 4.5 up to 31.3 mutants per 10^6 cells.
- The viability (cloning efficiency II) of the solvent control of culture I of the first experiment with metabolic activation and of culture I of the second experiment with metabolic activation reached but did not exceed the lower limit of 50%. The data are valid however, as the solvent control of the parallel culture exceeded this limit.
- EMS (150 μg/mL) and DMBA (1.1 μg/mL in experiment I, 2.2 μg/mL in experiment II) were used as positive controls and showed a distinct increase in induced mutant colonies.
- Concurrent vehicle negative and positive control data: Please refer to the attached background material.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer to the attached background material.
- Negative (solvent/vehicle) historical control data: Please refer to the attached background material.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four strains tested; Salmonella TA102 or E.coli WP2 not tested. Preincubation according to Haworth et el. (1983).

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material: isobutyl acrylate

Target gene:

histidine operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

rat and hamster liver S9 fraction (Aroclor-induced)

Test concentrations with justification for top dose:

0; 100; 333; 1000; 3333; 10000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : one

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar; preincubation

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Key result

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:

3333 and 10000 µg/plate were slightly cytotoxic.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Please refer to the attachment.

Ames test:
- Mean number of revertant colonies per plate and standard deviation : Please refer to the attachment.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For justification for type of information please refer to the read across justification attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

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

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Reference 4

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

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. QA statement)

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 conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo

Micronucleus test: negative (BASF, 2001)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.07.2001-13.11.2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material: Isobutyl acrylate
- Lot/batch No.: S 122-01-GA

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd. Biotechnology and Animal Breeding Division; CH-4414 Fuellinsdorf
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males: mean 44.9 g; females: mean 33.6 g
- Housing: single
- Diet: pelleted standard diet, ad libitum
- Water: Tap water ad libitum



ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +/- 4
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle/solvent used: olive oil, O1500, Sigma-Aldrich Vertriebs-GmbH
- Amount of vehicle: 10 mL/kg

Duration of treatment / exposure:

single injection; harvesting of cells 24 and 48 hours after administration of test substance

Frequency of treatment:

single treatment

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

males/ 24 hours harvesting

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

males/ 24 hours harvesting

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

males/ 24 hours harvesting

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

males/ 48 hours harvesting

Dose / conc.:

312.5 mg/kg bw/day (nominal)

Remarks:

female/ 24 hours harvesting

Dose / conc.:

625 mg/kg bw/day (nominal)

Remarks:

female/ 24 hours harvesting

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Remarks:

female/ 24 hours harvesting

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Remarks:

female/ 48 hours harvesting

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

yes, historical

Positive control(s):

cyclophosphamide, 40 mg/kg bw, i.p.

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Dose selection: on basis of the results from a pre-experiment.

TREATMENT:
10 animals per group were evaluated for micronuclei. At least 2000 polychromatic erythrocytes (PCE) per animal were scored. Cytotoxicity was evaluated by the ratio of polychromatic to normochromatic erythrocytes (determined in the same samples as used for micronuclei scoring).

Evaluation criteria:

A test substance is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes, which clearly exceeds the negative control range or a relevant positive response for at least one of the test points.
Statistical methods (nonparametric Mann-Whitney test) can be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test substance producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

A non-parametric Mann-Whitney-Test was performed.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical signs in the high dose animals

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: male: 0, 1250 mg/kg bw; female: 0, 1500 mg/kg bw
- Clinical signs of toxicity in test animals: In the dose range-finder pre-experiment, 1250 mg/kg bw i.p. were lethal to both tested male animals. In females, 1500 mg/kg bw i.p. were lethal for 1 of 2 animals.

RESULTS OF DEFINITIVE STUDY
The test item did not induce micronuclei at any dose level or any harvesting time used in this study. It was considered non-clastogenic and non-aneugenic in this assay.
The mean number of normochromatic erythrocytes was not increased after treatment with the test substance as compared to the controls, indicating that the test item had no cytotoxic effect on the bone marrow. The positive control substance induced a distinct increase of micronuclei.
- Clinical signs of toxicity in test animals: males at 1000 mg/kg bw: reduced spontaneous activity, eyelid closure, apathy, prostrate position. females at 1250 mg/kg bw: reduced spontaneous activity, eyelid closure, apathy, prostrate position. 1000 mg/kg bw induced clear signs of toxicity in 7 male animals (reduced activity, eyelid closure, apathy, prostrate position). In females, at 1250 mg/kg bw clear signs of toxicity were observed in 7 female animals (reduced activity, eyelid closure, apathy, prostrate position).

Summary of Micronucleus Test Results

Males

Test group	dose (mg/kg bw)	sampling time (h)	PCEs with micronuclei (‰)	range	PCE / NCE
vehicle	0	24	1.20	2 - 3	2000 / 1965
test substance	250	24	0.60	0 - 3	2000 / 1888
test substance	500	24	0.60	0 - 3	2000 / 2141
test substance	1000	24	1.20	1 - 4	2000 / 2371
positive control	40	24	14.90	17 - 36	2000 / 1771
vehicle	0	48	0.00	0 - 0	2000 / 2022
test substance	1000	48	0.40	0 - 2	2000 / 2642

Females:

Test group	dose (mg/kg bw)	sampling time (h)	PCEs with micronuclei (‰)	range	PCE / NCE
vehicle	0	24	0.10	0 - 1	2000 / 1965
test substance	312.5	24	0.50	0 - 3	2000 / 1888
test substance	625	24	0.40	0 - 3	2000 / 2141
test substance	1250	24	0.20	0 - 2	2000 / 2371
positive control	40	24	12.10	11 - 43	2000 / 1771
vehicle	0	48	0.00	0 - 0	2000 / 2022
test substance	1250	48	0.10	0 - 21	2000 / 2642

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies: Bacterial systems

Isobutyl acrylate was tested in the assay with Salmonella typhimurium TA98, TA100, TA1535, and TA1537 in concentrations from 100 up to 10000 µg/plate with and without metabolic activation. No mutagenic effects were observed. At 3333 and 10000 µg/plate slight cytotoxicity was observed (NTP 1982, Zeiger 1987).

 

Mammalian cell gene mutation tests

There are no mammalian cell gene mutation assays available for isobutyl acrylate. But the structurally-related acrylate esters n-butyl acrylate and tert-butyl acrylate were tested for gene mutations in mammalian cells.

 

HPRT

A study was performed to investigate the potential of tert.-butylacrylate to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The main experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. The maximum concentration of 1280 μg/mL was equal to approximately 10 mM. The test item was dissolved in DMSO. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, tert.-butylacrylate is considered to be non-mutagenic in this HPRT assay (BASF, 2014).

 

MLA

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 (BASF, 2016).

 

There are no in vitro micronucleus assays or chromosome aberration tests available for isobutyl acrylate. In accordance with column 2 of REACH Annex VIII, no study has to be conducted since adequate data from an in vivo cytogenicity test are available.

 

In vivo studies

 

In a micronucleus test performed with isobutyl acrylate in accordance with OECD guideline 474 under GLP conditions, mice were dosed intraperitoneally with up to 1000 mg/kg bw (males) and 1250 mg/kg bw (females). Isobutyl acrylate did not induce micronuclei in bone marrow at any dose level or any harvesting time used in this study (24 and 48 hours after the end of exposure). Isobutyl acrylate was considered non-clastogenic and non-aneugenic in this assay. The mean number of normochromatic erythrocytes was not increased after treatment with the test substance as compared to the controls, indicating that isobutyl acrylate had no cytotoxic effect on the bone marrow, but there were clear clinical signs of toxicity at the highest dose-levels tested (reduced activity, eyelid closure, apathy, prostrate position). The positive control substance, cyclophosphamide, induced a distinct increase of micronuclei (BASF, 2001).

 

Conclusion

Isobutyl acrylate was not mutagenic in the Ames test and not clastogenic in vivo in the mouse micronucleus test. In addition, the source substances for read-across n-butyl and tert-butyl acrylate were not mutagenic in mammalian cell gene mutations assays in the presence and absence of a metabolic activation system (S9).

Justification for classification or non-classification

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

The available data on genetic toxicity are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available data, the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) 2020/1182.