tert-butyl methacrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 01 to July 22, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl methacrylate

EC Number:

209-548-7

EC Name:

tert-butyl methacrylate

Cas Number:

585-07-9

Molecular formula:

C8H14O2

IUPAC Name:

tert-butyl 2-methylprop-2-enoate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF
- Batch number of test material: 010365eda0
- Purity: 99.86 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the refrigerator protected from light
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability of the test substance under storage conditions over the test period was guaranteed by
the sponsor, and the sponsor holds this responsibility; Homogeneity: given

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid: 11.1, 22.2, 44.4, 88.9, 177.8, 355.5, 711.0 and 1422.0 μg/ml

FORM AS APPLIED IN THE TEST
- suspension

OTHER SPECIFICS
- Physical state: liquid, colorless, clear

Method

Target gene:

Hprt

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cell line, Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line.

For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: Large stocks of the V79 cell line are stored in liquid nitrogen in the cell bank of the testing facility allowing the repeated use of the same cell culture batch in experiments.
- Cell cycle length, doubling time or proliferation index : 12 - 16 h in stock cultures
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL), 10% FBS, and amphotericin B (1 %).
- During 4 hours treatment no FBS was added to the medium. During 24 hours treatment the medium was supplemented with 10% FBS.
- For the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine.
- All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital/ß-naphthoflavone induced rat liver S9 was used as metabolic activation system.
- method of preparation of S9 mix: The S9 was prepared and stored according to the currently valid version of the ICCR SOP for rat liver S9 preparation.
- concentration or volume of S9 mix and S9 in the final culture medium: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- The protein concentration of the S9 preparation was 33.8 mg/ml in the pre- and main experiment.
- 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:

The highest applied concentration in the pre-test on toxicity (1422 µg/mL) was chosen corresponding to 10 mM, with respect to the OECD guideline 476 (2016).
Based on the results of the pre-experiment the maximal concentration of the main experiment was 1422 µg/mL as well.

- Doses applied in the gene mutation assay: 11.1, 22.2, 44.4, 88.9, 177.8, 355.5, 711.0 and 1422.0 μg/ml
- concentrations chosen for the mutation rate analysis: 88.9, 177.8, 355.5, 711.0 and 1422.0 μg/ml

Vehicle / solvent:

- Vehicle/solvent used: DMSO (purity: c≥ 99.9)
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
- Percentage of solvent in the final culture medium: The final concentration of DMSO in the culture medium will not exceed 0.5% (v/v).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

positive controls: 7,12-dimethylbenz[a]anthracene (DMBA) was tested with S9 Mix, ethyl methanesulfonate (EMS) without S9 Mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: approx. 0.7 to 1.2x10^7 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h

FOR GENE MUTATION:
- Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0x10^6 cells per experimental point (concentration series plus controls) were subcultivated in 175 cm² flasks containing 30 ml medium. Two additional 25 cm² flasks were seeded per experimental point with approximately 500 cells each to determine the relative survival (RS) as measure of test item induced cytotoxicity. The colonies used to determine the relative survival (RS) are fixed and stained approximately 6 to 8 days after treatment as described below.Three or four days after the first sub-cultivation, at least 2.0x10^6 cells per experimental point are again sub-cultivated in 175 cm² flasks containing 30 ml medium. Following the expression time of approximately 7 days five 75 cm² cell culture flasks are seeded with about 4 - 5x10^5 cells each in medium containing 6-TG (11 µg/ml). Two additional 25 cm² flasks are seeded with approx. 500 cells each in non-selective medium to determine the viability. After approx. 8 days (evaluation for viability) and approx. 9 ± 2 days (mutation analysis) the colonies were stained with 10% methylene blue in 0.01% KOH solution. Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS FOR MEASUREMENT OF CYTOTOXICITY AND GENOTOXICIY:

Pre-test:
- cloning efficiency, absolute = mean number of cells per ml divided by the number of cells seeded x 100
- cloning efficiency, relative = (mean number of cells per ml divided by the mean number of cells per ml of the corresponding control) x 100

Main test:
- cloning efficiency I (survival) = cloning efficiency determined immediately after treatment to measure toxicity.
- cloning efficiency II (viability) = cloning efficiency determined after the expression period to measure viability of the cells without selective agent.
- cloning efficiency I (survival, absolute)= mean number of colonies per flask divided by the number of cells seeded per test point
- cloning efficiency I (survival, relative) = cloning efficiency I absolute divided by the cloning efficiency I absolute of the corresponding control x 100
- relative cell density % of control = (cell density at 1st subcultivation divided by the cell density at 1st subcultivation of the corresponding control) x 100
- cloning efficiency II (viability, absolute) = mean number of colonies per flask divided by the number of cells seeded
- cloning efficiency II (viability, relative) = cloning efficiency II absolute divided by the cloning efficiency II absolute of the corresponding control x 100
- cells survived (after plating in TG containing medium) = number of cells seeded x cloning efficiency II absolute
- mutant colonies / 10^6 cells = mean number of mutant colonies per flask found after plating in TG medium x 10^6 divided by the number of cells survived
- relative adjusted cloning efficiency I = relative cloning efficiency I x relative cell density at first subcultivation / 100

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:

a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:

a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b)there is no concentration-related increase when evaluated with an appropriate trend test,
c)all results are inside the distribution of the historical negative control data (based 95% control limits).

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.

In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.

Statistics:

The statistical analysis was performed on the mean values of culture I and II for the main experiments.

A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

A t-test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics to evaluate a significant increase of the mutation frequency at all test points where the mutation frequency is above the corresponding solvent control. A t-test is judged as significant if the p-value is <0.05.

Results and discussion

Test results

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: The osmolarity and the pH value were determined in culture medium of the solvent control and of the highest concentration in the pre-experiment without metabolic activation.
- Osmolarity [mOsm] test substance (1422 µg/mL): 380
- Osmolarity [mOsm] solvent control: 403
- pH-value test substance (1422 µg/mL): 7.42
- pH-value solvent control: 7.43
- Precipitation and time of the determination: Pre-experiment: The test medium was checked for phase separation and precipitation at the end of the treatment period (4 h) before the test item was removed. Phase separation occurred at 711 µg/mL and above in the presence of S9 mix.
- Main Experiment: Phase separation occurred at 1422 µg/mL after four hours treatment.

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the toxicity of the test item. In this pre-test approx. 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test substance was added and the treatment proceeds for 4 hours (with and without metabolic activation) (duplicate cultures per concentration level).

STUDY RESULTS
- Concurrent vehicle negative and positive control data: EMS (300 µg/ml) and DMBA (2.3 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies. The MF of the positive control with S9 mix (52.8) was slightly below the historical control data (54.5 – 347.1). This minor deviation was judged to represent fluctuations. A clear significancy was observed and it was above the 95% control limit of the solvent control and therefore acceptable and the test was considered valid.

Pre-Experiment
- No relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed either with or without metabolic activation. There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test item measured in the pre-experiment.

Main Experiment
- No cytotoxicity was observed up the highest evaluated concentration, which showed phase separation.The observed mean mutant frequency (MF) of the solvent control and all evaluated concentrations was within the 95% control limits of the solvent historical control data. Linear regression analysis showed no statistically significant trend. A t-test was performed for all concentrations with a MF above the MF of the solvent control and did not show a statistical significance.

For more detailed information please refer to table 2 in section "Any other information on results incl. tables".

HISTORICAL CONTROL DATA
- The historical control data were generated in accordance with the OECD Guideline 476. For the solvent controls data range (min-max) and data distribution (standard deviation) are calculated for each experimental part of at least 20 experiments. The calculated 95% control limit of the solvent controls (realized as 95% confidence interval) are applied for the evaluation of acceptability and interpretation of the data. Control charts of the corresponding experiments are added as quality control method. For the positive controls data range (min-max) and data distribution (standard deviation) are calculated for each experimental part of at least 20 experiments. The min-max range of the positive controls are applied for the evaluation of acceptability. Control charts of the corresponding experiments are added as quality control method.
For more detailed information please refer to table 1 in section "Any other information on materials and methods incl. tables".

Any other information on results incl. tables

  Summary of results

				relative	relative	rel. adjusted	(MF)	95%	statistical
	conc.	PS	S9	cloning	cell	cloning	mutant	control	analysis*
	µg/mL		mix	efficiency I	density	efficiency I	colonies/	limit	t-test	linear
				%	%	%	10^6cells			regression
Column	1	2	3	4	5	6	7	8	9	10
Main Experiment / 4 h treatment				mean values of culture I and II
Solvent control with DMSO			-	100.0	100.0	100.0	9.9	2.9 – 22.4
Positive control (EMS)	300		-	73.3	103.5	74.6	271.6		0.000^S
Test item	11.1	-	-	98.6	95.7	94.4	#	2.9 – 22.4
Test item	22.2	-	-	100.7	97.7	98.4	#	2.9 – 22.4		0.266
Test item	44.4	-	-	95.8	88.2	84.2	#	2.9 – 22.4
Test item	88.9	-	-	104.2	80.4	84.0	4.4	2.9 – 22.4	n.c.
Test item	177.8	-	-	104.4	81.6	85.1	9.2	2.9 – 22.4	n.c.
Test item	355.5	-	-	99.8	75.6	75.5	7.8	2.9 – 22.4	n.c.
Test item	711.0	-	-	95.4	72.7	70.2	8.3	2.9 – 22.4	n.c.
Test item	1422.0	PS	-	88.7	74.3	65.3	11.3	2.9 – 22.4	0.623
Solvent control with DMSO			+	100.0	100.0	100.0	7.9	2.9 – 23.7
Positive control (DMBA)	2.3		+	90.4	107.7	98.5	52.8		0.011^S
Test item	11.1	-	+	87.2	98.5	85.8	#	2.9 – 23.7
Test item	22.2	-	+	87.3	106.1	92.7	#	2.9 – 23.7		0.148
Test item	44.4	-	+	89.9	95.5	86.3	#	2.9 – 23.7
Test item	88.9	-	+	94.4	101.0	96.0	12.7	2.9 – 23.7	0.034^S
Test item	177.8	-	+	88.6	105.4	93.3	7.6	2.9 – 23.7	n.c.
Test item	355.5	-	+	93.2	115.3	108.3	7.9	2.9 – 23.7	n.c.
Test item	711.0	-	+	78.8	96.0	75.8	8.6	2.9 – 23.7	0.392
Test item	1422.0	PS	+	54.4	102.2	55.7	4.7	2.9 – 23.7	n.c.

 

*       statistical analysis based on the mean values of culture I and II

n.c.   not calculated (mean MF below MF of the solvent control)

PS =  Phase separation at the end of treatment

S =    significant trend (p < 0.05)

MF   Mutant frequency

#       cultures not continued as a minimum of only four analyzable concentrations are required

Applicant's summary and conclusion

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

This GLP compliant study (according to OECD TG 476) was performed to investigate the potential of the test substance, dissolved in DMSO, to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in one experiment using two parallel cultures. The main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The highest applied concentration in the pre-test on toxicity (1422 µg/mL) was chosen corresponding to 10 mM, with respect to the OECD guideline 476 (2016). Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 11.1, 22.2, 44.4, 88.9, 177.8, 355.5, 711, and 1422 µg/ml.

In the main experiment in the absence and presence of S9 mix, no relevant cytotoxicity was observed up to the highest applied concentration, which showed phase separation.

In the main experiment in the absence and presence of S9 mix, no relevant increases in the numbers of mutant colonies were observed after treatment with the test substance.

Appropriate reference mutagens, 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.