Docosyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The target substance Docosyl methacrylate is considered to be non-mutagenic in a Salmonella typhimurium reverse mutation assay. In addition, its metabolite Docosan-1-ol (Behenyl alcohol) was shown to be non-mutagenic in the Ames test as well as in a Chromosome Aberration and HPRT test. For the structural analogue substance Isodecyl methacrylate no genotoxicity was observed in the Ames test and the HPRT test and no chromosomal aberrations were reported. For the structural analogue monomer mix (methacrylic acid ester of an alcohol mixture with an average C-number of 14.8) two additional in vitro studies received negative results as well.  

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline available

Principles of method if other than guideline:

The potential for Docosan-1-ol (Behenyl alcohol) to induce gene mutations, in vitro, at the HGPRT locus was evaluated in Chinese hamster V79 cells, with and without metabolic activation.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell transformation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Liver microsomal fractions from 8- to 12-week-old male Wistar rats

Test concentrations with justification for top dose:

2.0, 7.5, 15.0, and 20.0 μg/mL
The selection of doses was based on the results of a previously conducted rangefinding experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
The final concentration of ethanol in the culture medium did not exceed 1% v/v.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours

SELECTION: Mutant frequency was determined by seeding known numbers of cells in medium containing thioguanine (11 μg/mL to detect mutant cells, and in medium without thioguanine to determine the total number of surviving cells.

STAIN: Methylene blue (10%) was used for staining in 0.01% KOH solution.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Stained colonies with more than 50 cells were counted with a preparation microscope.

Evaluation criteria:

For the test substance to be considered positive, a significant dose-related increase in the mutant frequency or a reproducible and significant positive response for at least one of the test points was required. The test substance also was considered to be mutagenic if there was a reproducible concentration-related increase in the mutation frequency.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline available

Principles of method if other than guideline:

The potential for Docosan-1-ol (Behenyl alcohol) to induce structural chromosome aberrations in Chinese hamster V79 cells was evaluated in vitro, with and without metabolic activation.

GLP compliance:

not specified

Type of assay:

other: chromosome aberration assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Cytokinesis block (if used):

Colcemia

Metabolic activation:

with and without

Metabolic activation system:

Liver microsomal fractions from 8- to 12-week-old male Wistar rats

Test concentrations with justification for top dose:

Cultures prepared at 7 and 24 h were treated with 20 μg/mL behenyl alcohol, while cultures prepared at 18 h were treated with 0.6, 10.0, and 20.0 μg/mL behenyl alcohol. The concentrations used in this study were based on the results from a preexperiment, which used the plating efficiency assay as an indicator for toxicity response.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

Assays were initiated by seeding approximately 5×10E4 to 1×10E5 cells per dish in minimal essential medium. After 48 h (for cells harvested at 7 and 24 h) and 55 h (for cells harvested at 18 h), the medium was replaced with serum-free medium containing behenyl alcohol at the appropriate dose.

DURATION
- Exposure duration: 4 hours

SPINDLE INHIBITOR: Colcemia (0.2 μg/mL) was added to the cultures for the last 2 h (for cells harvested at 7 h) or for the last 2.5 h (for cells harvested at 18 and 28 h)
STAIN: Giemsa

NUMBER OF CELLS EVALUATED: One hundred metaphases were scored for cytogenic damage per slide.

DETERMINATION OF CYTOTOXICITY
- Method: platting efficiency

Evaluation criteria:

For the test substance to be considered mutagenic, either a significant dose-related increase in the number of structural chromosomal aberrations or a significant positive response at one of the test points was required.

Statistics:

Statistical analysis included use of the chi2 test, which was only performed for cells carrying aberrations exclusive gaps. However, both biological and statistical
significance was considered.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from male Wistar rats livers

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (with and without S9 mix) (SPT and PIT)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, acetone was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine; 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al. (1, 2).
Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. (7) and Matsushima et al. (8).
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with
metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis
System.

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

Species / strain:

other: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 1000 μg/plate onward

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

SOLUBILITY: Precipitation of the test substance was found in the SPT from about 1000 μg/plate onward and from 333 μg/plate in the PIT both with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 1000 μg/plate onward.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

Conclusions:

Under the experimental conditions of this study, the test substance Docosyl methacrylate is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay 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 mouse micronucleus tests with the structural analogue substances Dodecyl methacrylate and Octadecyl methacrylate supports the absence of a mutagenic potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09-July-1989 - 31-Aug.- 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Pre-Experiment for Toxicity
A preliminary study on acute toxicity was performed with 2 animals per sex under identical conditions as in the mutagenicity study concerning:
starvation period, animal strain; vehicle; route, frequency, and volume of administration.
The animals were treated orally with a single dose of 5000 mg/kg bw in 1% CMC with the test item and examined for acute toxic symptoms.

GLP compliance:

yes

Type of assay:

other: Micronucleus Assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm, Füllinsdorf, Switzerland
- Number of animals. 84 (42 males/42 females)
- Age at study initiation: minimum 11 weeks
- Weight at study initiation: approximately 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: 18 hours before treatment
- Housing: single
- Diet: pelleted standard diet,ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±3°C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs artifical light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: vehicle was chosen to its relative non-toxicity for animals. All animals received a single standard volume
of 10 mL/kg body weight orally.
- Concentration of test material in vehicle:
- Supplier: Fluka; SIGMA-Aldrich Vertiebs-GmbH, 82041 Deisenhofen, Germany
- Lot/batch no.: no data
- Catalogue no.: 21902
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test substance was formulated in 1% CMC. All animals received a single standard volume of 10 ml/kg body weight
orally.

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data

Duration of treatment / exposure:

24 h, 48 h and 72 hours

Frequency of treatment:

single dose

Dose / conc.:

5 000 other: mg/kg

Remarks:

nominal conc., suspended in Carboxymethylcellulose (1 %)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA; cyclophosphamide
- Supplier: SERVA, Heidelberg, Germany
- Purity: commercial grade
- Dissolved in: physiological saline
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg bw
Volume administered: 10 mL/kg bw

The stability of CPA at room temperature is sufficient. At 20 °C only 1% of CPA is hydrolysed per day in aqueous solution.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or
the highest dose that can be formulated and administered reproducibly.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 72
hours.
The volume to be administered should be compatible with physiological space available.

TREATMENT AND SAMPLING TIMES:
Approximately 18 hours before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including
the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time.
Sampling of bone marrow was done 24, 48 and 72 hours after treatment, respectively.

DETAILS OF SLIDE PREPARATION:

Preparation of the Animals:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal
calf serum, using a syringe. The cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. A small
drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Kindler, D-79110 Freiburg,
Germany/Giemsa. Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg, Germany). At least one slide was made from each bone
marrow sample.

METHOD OF ANALYSIS:
Analysis of Cells:
Evaluation of the slides was performed using NlKON microscopes with 100x oil immersion objectives. At least 1000 polychromatic erythrocytes (PCE)
were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was
determined in the same sample and expressed in normochromatic erythrocytes per 1000 erythrocytes. The analysis was performed with coded
slides.
Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining animal of each test group was evaluated in case an
animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated
polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a
statistically significant and reproducible positive response at anyone of the test points is considered nonmutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.
mntire

Statistics:

Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration
is the biological relevance of the results.

Key result

Sex:

male/female

Genotoxicity:

negative

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg bw Dodecylmethacrylate suspended in CMC (1%). The volume administered was 10 mL/kg bw. All treated animals expressed toxic reactions: reduction of spontaneous activity, eyelid closure and apathy. One female expressed pilo erection.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July 09, 1989 - September 18, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Micronucleus Assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm, Füllinsdorf, Switzerland
- Number of animals. 84 (42 males/42 females)
- Age at study initiation: minimum 11 weeks
- Weight at study initiation: approximately 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: 18 hours before treatment
- Housing: single
- Diet: pelleted standard diet,ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±3°C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs artifical light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Polyethylene glycol, PEG
- Justification for choice of solvent/vehicle: vehicle was chosen to its relative non-toxicity for animals. All animals received a single standard volume
of 10 mL/kg body weight orally.
- Supplier: Merck KGaA, Darmstadt, Germany

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test article was suspended in PEG. The vehicle was chosen to its nontoxicity for the animals. All animals received a
single standard dose volume of 10 mL/kg body weight orally.

Duration of treatment / exposure:

24 h, 48 h and 72 h

Frequency of treatment:

single dose

Dose / conc.:

5 000 other: mg/kg

Remarks:

nominal conc

No. of animals per sex per dose:

5

Positive control(s):

CPA; cyclophosphamide
- Supplier: SERVA, Heidelberg, Germany
- Purity: commercial grade
- Dissolved in: physiological saline
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg bw
- Volume administered: 10 mL/kg bw

The stability of CPA at room temperature is sufficient. At 20 °C only 1% of CPA is hydrolysed per day in aqueous solution.

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or
the highest dose that can be formulated and administered reproducibly.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 72
hours.
The volume to be administered should be compatible with physiological space available.

TREATMENT AND SAMPLING TIMES:
Treatment:
Approximately 18 hours before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including
the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time.
Sampling of bone marrow was done 24, 48 and 72 hours after treatment, respectively.

DETAILS OF SLIDE PREPARATION:
Preparation of the Animals:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal
calf serum, using a syringe. The cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. A small
drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Kindler, D-79110 Freiburg,
Germany/Giemsa. Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg, Germany). At least one slide was made from each bone
marrow sample.

METHOD OF ANALYSIS:
Analysis of Cells:
Evaluation of the slides was performed using NlKON microscopes with 100x oil immersion objectives. At least 1000 polychromatic erythrocytes (PCE)
were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was
determined in the same sample and expressed in normochromatic erythrocytes per 1000 erythrocytes. The analysis was performed with coded
slides.Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining animal of each test group was evaluated in case an
animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated
polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a
statistically significant and reproducible positive response at anyone of the test points is considered nonmutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.

Statistics:

Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration
is the biological relevance of the results.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

All treated animals expressed toxic reactions: reduction of spontaneous activity, apathy.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Pre-Experiment for Toxicity
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg bw. Octadecylmethacrylate suspended in PEG. The volume administered was 10 mL/kg bw. All treated animals expressed toxic reations: reduction of spontaneous activity, apathy.

Summary of results

test group	dose mg/kg bw	sampling time (h)	PCEs with micronuclei	range	PCE/NCE
suspending agent	0	24	0.05%	0 – 1	1000/632
test article	5000	24	0.05%	0 – 2	1000/626
cyclo- phosphamide	40	24	0.61%	1 – 18	1000/714
suspending agent	0	48	0.03%	0 – 1	1000/634
test article	5000	48	0.07%	0 – 4	1000/561
suspending agent	0	72	0.10%	0 – 2	1000/539
test article	5000	72	0.09%	0 – 2	1000/527

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In Vitro

Bacterial reverse mutation assay

The test substance Docosyl methacrylate was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. Strains tested TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA, dose range 33 μg - 5000 μg/plate (SPT) and 33 μg - 5000 μg/plate (PIT).

Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found in the SPT from about 1000 μg/plate onward and from 333 μg/plate in the PIT both with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 1000 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance Docosyl methacrylate is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (BASF SE, 2018) .

In a further Ames test according to OECD TG 471 Salmonella typhimurium TA98, TA1535, TA1537 and TA100 strains were exposed to Behenyl alcohol at concentrations up to 1000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background (Iglesias et al 2001).

In a supporting reverse gene mutation assay in bacteria according to OECD TG 471 Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 strains were exposed to Isodecyl methacrylate in THF at concentrations up to 5000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background (Evonik Röhm GmbH UNTER 08-037).

In a supporting reverse gene mutation assay in bacteria according to OECD TG 471 Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 strains were exposed to monomer mix in THF at concentrations up to 3000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background (Evonik Oil Additives GmbH UNTER 05-021).

In vitro Chromosome aberration test

No study is available on Docosyl methacrylate but there are studies available on the structurally related substance Isodecyl methacrylate, monomer mix and the metabolite Docosan-1-ol (Behenyl alcohol).

The potential of Behenyl alcohol to induce structural chromosome aberrations in Chinese hamster V79 cells was evaluated in vitro, with and without metabolic activation. The following concentrations were tested: Cultures prepared at 7 and 24 h were treated with 20 μg/mL behenyl alcohol, while cultures prepared at 18 h were treated with 0.6, 10.0, and 20.0 μg/mL behenyl alcohol. In the absence and presence of S9 mix neither a statistically nor a biologically relevant increase in the number of cells carrying structural chromosome aberrations were observed after treatment with the test item (Iglesias et al 2001).

In a further chromosome aberration test in human lymphocytes according to OECD TG 473, human cultures were exposed to Isodecyl methacrylate in THF with and without metabolic activation (S9 mix). The following concentrations were evaluated:

Experiment I: 22 hours prep. Interval, 4 h treatment without metabolic activation: 14.5, 25.4, 44.4, 77.7, 136.0, 238.1, 416.7, 729.1, 1276.0, 2233 µg/mL

22 hours prep. interval, 4 h treatment with metabolic activation: 14.5, 25.4, 44.4, 77.7, 136.0, 238.1, 416.7, 729.1, 1276.0, 2233 µg/mL

Experiment II: 22 hours prep. Interval, 22 h treatment without metabolic activation: 44.4, 77.7, 136.0, 238.1, 416.7, 729.1, 1276.0, 2233 µg/mL

22 hours prep. interval, 4 h treatment with metabolic activation: 44.4, 77.7, 136.0, 238.1, 416.7, 729.1, 1276.0, 2233 µg/mL

In Experiment I, visible precipitation of the test item in the culture medium was observed at 136 µg/mL and above in the absence and presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix, at 136 µg/mL and above and in the presence of S9 mix at 416.7 µg/mL and above. No relevant increase in the osmolarity or pH value was observed. In Experiment I, in the absence and presence of S9 mix, and in Experiment II, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II, in the absence of S9 mix, a single clearly reduced mitotic index was observed at the highest dose evaluated for cytogenetic damage. Isodecyl methacrylate was tested up to cytotoxic concentration. In the absence and presence of S9 mix neither a statistically nor a biologically relevant increase in the number of cells carrying structural chromosome aberrations were observed after treatment with the test item (Evonik Röhm UNTER 08-038).

In a supporting chromosome aberration test in Chinese hamster V79 cells according to OECD TG 473, cultures were exposed to monomer mix dissolved in THF with and without metabolic activation (S9 mix). The following concentrations were evaluated: Experiment I: 18 hours prep. Interval, 4 h treatment without and with metabolic activation: 5.9, 11.7, 23.4, 46.9, 93.8, 187.5 (p), 375.0 (p), 750.0 (p), 1500.0 (p), 3000.0 (p) µg/mL (p): Precipitation occurred

Experiment II: 18 hours prep. Interval, 18 h treatment without metabolic activation: 46.9, 93.8, 187.5 (p), 375.0 (p), 750.0 (p), 1500.0 (p), 3000.0 (p) µg/mL

28 hrs prep. interval, 28 h treatment without metabolic activation: 23.4, 46.9, 93.8, 187.5 (p), 375.0 (p), 750.0 (p), 1500.0 (p), 3000.0 (p) µg/mL

Experiment II: 28 hrs prep. interval, 4 hrs exposure period: with S9 mix: 23.4, 46.9, 93.8, 187.5 (p), 375.0 (p), 750.0 (p) µg/mL

(p): Precipitation occurred

No clear toxic effects indicated by reduced mitotic indices or cell numbers of below 50% were observed up to the highest required test item concentration. Therefore, concentrations at the border of test item solubility in culture medium were scored for cytogenetic damage. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiments I and II, in the absence of S9 mix, statistically significant increases (2 % - 2.5 % aberrant cells, exclusive gaps) were observed, but were within the historical control range (0.0 - 4.0 % aberrant cells, exclusive gaps) and are regarded as being biologically irrelevant. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. In the absence and presence of S9 mix neither a statistically nor a biologically relevant increase in the number of cells carrying structural chromosome aberrations were observed after treatment with the test item (Evonik Oil Additives GmbH UNTER 05-022).

HPRT-test in mammalian cells

No study is available with Docosyl methacrylate but there are studies with the structurally related substance Isodecyl methacrylate and the metabolite Behenyl alcohol available.

The potential for behenyl alcohol to induce gene mutations, in vitro, at the HPRT locus was evaluated in Chinese hamster V79 cells, with and without metabolic activation. The following concentrations were tested: 2.0, 7.5, 15.0, and 20.0 μg/mL. EMS and DMBA were used as positive controls and showed a distinct increase in induced mutant colonies. This showed the sensitivity of the test system and the activity of the S9 mix. Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells (Iglesias et al 2001).

Isodecyl methacrylate was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD TG 476.

The assay was performed in two independent experiments with identical experimental procedures, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix:  0.1; 0.3; 0.5; 1.0; and 2.0 µg/mL

with S9 mix: 37.5; 75; 150; 300; and 1200 µg/mL

Experiment II:

without S9 mix: 18.8 ;37.5; 75.0; 150; and 600 µg/mL

with S9 mix: 37.5; 75.0; 150; 300; and 600 µg/mL

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.7 up to 24.0 mutants per 10E6cells; the range of the groups treated with the test item was from 3.3 up to 34.1 mutants per 10E6cells. EMS (150 µg/mL in experiment I and 75 µg/mL in experiment II) and DMBA (2.0 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. This showed the sensitivity of the test system and the activity of the S9 mix. Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells (Evonik Röhm GmbH UNTER 08-039).

In vivo

Micronucleus test

No study is available with docosyl methacrylate but there are studies with the structurally related substances Dodecyl methacrylate and Octadecyl methacrylate available.

In a NMRI mouse bone marrow micronucleus assay, 5 animals/male/female/dose were treated orally with structural analogue Dodecyl methacrylate at a dose of 0, 5000 mg/kg bw according to OECD 474. The test article was suspended in carboxymethyl cellulose (1%). This suspending agent was used as negative control. The volume administered orally was 10 mL/kg bw. At 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects.

In comparison with the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. Therefore, Dodecyl methacrylate is considered to be non-mutagenic in the micronucleus assay (Evonik Röhm GmbH UNTER 89-004).

In a second NMRI mouse bone marrow micronucleus assay, 5 animals/male/female/dose were treated orally with Octadecyl methacrylate at a dose of 0, 5000 mg/kg bw according to OECD 474. The test article was suspended in PEG. This suspending agent was used as negative control. The volume administered orally was 10 mL/kg bw. At 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects.

In comparison with the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency. Octadecyl methacrylate is considered to be non-mutagenic in the micronucleus assay (Evonik RohMax UNTER 89-005).

Conclusion:

The absence of a mutagenic potential for the target substance was demonstrated in a bacterial reverse mutation assay. In addition, studies with structural analogues Isodecyl methacrylate and Monomer Mix as well as with the metabolite Behenyl alcohol showed non-mutagenicity in the Ames test, Chromosome Aberration or HPRT test.

These results are also representative for Docosyl methacrylate as Isodecyl methacrylate represents a worst-case in terms of bioavailability due to its molecular size, lipophilicity and water solubility.

A negative mouse micronucleus tests with Dodecyl methacrylate and Octadecyl methacrylate supports the absence of a mutagenic potential in vivo.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the negative bacterial reverse mutation test for the target substance as well as the negative in vitro mammalian genotoxicity and Chromosome aberration assay for the metabolite, the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.