2-(2-butoxyethoxy)ethyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Available in vitro studies:

- Ames test: +/-S9 5000 µg/plate negative, TA98, TA100, TA1535, TA1537 and TA102 of Salmonella typhimurium, guideline study OECD 471, GLP, Klimisch score 1 (Arkema, 1999)

- HPRT test: negative, guideline study OECD 476, GLP, Klimisch score 1 (HMRTF, 2020)

- Mammalian Cell Micronucleus Test: negative guideline study OECD 487, GLP, Klimisch score 1 (HMRTF, 2021)

Based on the results of several reliable in vitro studies, Butyldiglycol methacrylate shows no potential to induce gene mutations in bacterial cells nor does it induce micronuclei in human lymphocytes.

There were no in vivo genetic toxicity studies identified for Butyldiglycol methacrylate.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15.12.1998-01.09.1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 471, GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

EEC Annex V Test B14 (1993)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from male Sprage Dawley rats

Test concentrations with justification for top dose:

range finder: 8, 40, 200, 1000 and 5000 µg/plate
Experiment 1: 8, 40, 200, 1000 and 5000 µg/plate
Experiment 2: : 51.2; 128; 320; 800; 2000 and 5000 µg/plate

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

9-aminoacridine

Positive controls:

yes

Positive control substance:

other: Glutaraldehyd

Positive controls:

yes

Positive control substance:

other: 2-amonianthracene

Details on test system and experimental conditions:

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.
Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.

Range-finder:
TA100 using final concetrations of Butyldiglycol methacrylate at 8, 40, 200, 1000 and 5000 µg/plate, plus negative (solvent) and positive controls with and without metabolic activation.

No evidence of toxicity (as would normally be indicated by a thinning of the background bacteriallawn and/or areduction in revertant numbers) was observed following any of these range-finder treatments. These results were considered acceptable for mutagenicity assessment and
were therefore used to provide the TAl00 mutagenicity data for Experiment 1.

Main Experiment:
Experiment 1
TA98, TA102, TA1535, TA1537 inal concetrations of Butyldiglycol methacrylate at 8, 40, 200, 1000 and 5000 µg/plate, plus negative (solvent) and positive controls with and without metabolic activation. Following this experiment, evidence of toxicity (manifest as slight thinning of the
background bacterial lawn) was observed solely with strains TA98 and TA102 in the absence of S-9 at the maximum per dose.

Experiment 2:
TA98, TA100, TA1535, TA1537, TA102
Test concentrations: 51.2; 128; 320; 800; 2000 and 5000 µg/plate plus negative (solvent) and positive controls with and without metabolic activation.
Treatments in the presence of S9 in experiment 2 included a pre-incubation step for 1 hour at 37 °C before addition of molten agar at 46 °C.

Evaluation criteria:

Acceptance criteria
The assay was considered valid if the following criteria were met:
1) the mean negative control counts fell within the normal ranges as defined in Appendix 3
2) the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an
active S-9 preparation
3) no more than 5% of the plates were lost through contamination or some other unforeseen event.

On one experimental occasion, the solvent control counts for one strain were outside of the laboratory historical control ranges. Although these counts failed to meet Acceptance criteria 1), these data were considered valid .

Evaluation criteria:
The test article was considered to be mutagenic if:
1) the assay was valid (see acceptance criteria)
2) a dose related and reproducible increase in the number of revertants was observed, or a significant* and reproducible increase in the
number of revertants was induced at one or more test concentration.
* An increase in revertant numbers was considered to be significant if the number of revertant colonies was at least two times the mean negative control counts in strains TA98, TAl00 and TA 102 , or three times in strains TA1535 and TA1537.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Butydiglycol methacrylate did not induce mutation in the Salmonella typhimurium strains TA98, TAl00, TA1535, TA1537 and
TA 102 up to a concentration of 5000 µg/plate, in the absence and in the presence of a rat liver metabolic activation system (S-9).

Conclusions:

Butydiglycol methacrylate did not induce mutation in the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and
TA 102 up to a concentration of 5000 µg/plate, in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 Salmonella . typhimurium TA98, TA100, TA1535, TA1537 and TA102 strains were exposed to Butyldiglycol methacrylate in DMSO at concentrations up to 5000 µg/plate in the presence and absence of mammalian metabolic activation. The test was performed as pre-incubation test with 1 hour pre-incubation.  The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.  It was concluded that Butyldiglycol methacrylate did not induce mutation in the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102, under the conditions employed in this study with test concentrations up to 5000 µg/plyte, in the absence and presence of rat liver metabolic activation system (S-9).

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09/12/2020 until 08/02/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 487, GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

yes

Remarks:

A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls (Bohnenberger et al., 2011). To achieve such response the test design, specifically for the

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

TEST MATERIAL:
- Name of Test material: Butyldiglycol methacrylate
- CAS No. 7328-22-5
- EC No. 230-813-8

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Blood samples were drawn from healthy non-smoking donors with no known illness or recent exposures to genotoxic agents (e.g.chemicals, ionising radiation) at levels that would increase the background incidence of micronucleate cells. For this study, blood was collected from a female donor (19 years old) for Experiment I and from a male donor (30 years old) for Experiment II.

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 was used as the metabolic activation system. 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 used for this study was 31.0 mg/mL (Lot no. 100920).

Test concentrations with justification for top dose:

Pre-Test:
Experiment I (Preparation intervall 40h, exposure time 4 h, without S9 Mix): 13.2/ 23.2/40.6/71.0/124/217/380/666/1165 PS/2039PS

Experiment II (Preparation intervall 40h, exposure time 20 h, without S9 Mix):
23.2/40.6/71.0/124/217/380/666/1165 PS/2039PS

Experiment I (Preparation intervall 40h, exposure time 4 h, withS9 Mix): 13.2/ 23.2/40.6/71.0/124/217/380/666/1165 PS/2039PS

PS-Phase separation

With regard to the molecular weight and the purity (98.11%) of the test item, 2039 µg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 13.2 to 2039 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 1165 µg/mL and above in the absence and presence of S9 mix. No cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 2039 µg/mL were chosen as top treatment concentration for Experiment II.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

1) Culture conditions
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA 1.5% (v/v) as extract, 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.


2) Pre-experiment
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterised by the percentages of reduction in the CBPI in comparison to the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.
This preliminary test was designated Experiment I since the cultures fulfilled the acceptability criteria and appropriate concentrations could be selected for cytogenetic evaluation.

3) Cytogenetic Experiment
a) Pulse exposure
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL culture medium was added. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period. After this period, Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation (Clare et al, 2006, Lorge et al, 2006).

b) Continuous exposure (without S9 mix)
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in and washed with "saline G". The washing procedure was repeated once as described. After washing, the cells were re-suspended in complete culture medium containing 10 % FBS (v/v). Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation (Whitwell et al, 2019).

4) Preparation of cells
The cultures were harvested by centrifugation 40 hrs after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa, mounted after drying and covered with a coverslip.

Evaluation criteria:

Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
(MONC x 1) + (BINC x 2) + (MUNC x 3)
CBPI = ------------------------------------------------
n
CBPI Cytokinesis-block proliferation index
n Total number of cells
MONC Mononucleate cells
BINC Binucleate cells
MUNC Multinucleate cells

Cytostasis % = 100 – 100 [(CBPIT – 1) / (CBPIC – 1)]
T Test item
C Solvent control
Acceptability Criteria:
The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% control limit realized as 95% confidence interval)
The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase compared with the concurrent solvent control
Cell proliferation criteria in the solvent control are considered to be acceptable
All experimental conditions were tested unless one exposure condition resulted in a clearly positive result
The quality of the slides must allow the evaluation of an adequate number of cells and concentrations
The criteria for the selection of top concentration are consistent with those described in section ‘Dose Selection’

Statistics:

Statistical significance was confirmed by the Chi Square Test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

A linear regression was performed using a validated test script of “R”, to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated 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.

Both, biological and statistical significance were considered together.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: The substance was determined not to be mutagenic.

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, Butyldiglycol methacrylate is considered to be non-mutagenic in this in vitro micronucleus test according to OECD 487, when tested up to phase separating concentrations.

Executive summary:

The test item Butyldiglycol methacrylate, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I
Stimulation period	48 hrs	48 hrs	48 hrs
Exposure period	4 hrs	20 hrs	4 hrs
Recovery	16 hrs	-	16 hrs
Cytochalasin B exposure	20 hrs	20 hrs	20 hrs
Total culture period	88 hrs	88 hrs	88 hrs

In each experimental group, two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2039 µg/mL of the test item) was chosen with regard to the molecular weight and the purity (98.11%) of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487. The rationale for the dose selection is reported in section 3.5.1. The chosen treatment concentrations are reported in Table 1 and the results are summarised in Table 2.

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II in the absence of S9 mix cytotoxic effects were observed at the highest evaluated concentration, which showed phase separation.

In Experiment I in the absence and presence of S9 mix and in Experiment II in the absence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Conclusion

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, Butyldiglycol methacrylate is considered to be non-mutagenic in this in vitro micronucleus test according to OECD 487, when tested up to phase separating concentrations.

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-10-29 to 2020-25-11

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 using the Hprt and xprt genes)

Version / remarks:

29 July 2016

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

Qualifier:

according to guideline

Guideline:

other: Kanpoan No. 287 -- Environmental Protection Agency Eisei No. 127 -- Ministry of Health & Welfare Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry

Deviations:

no

GLP compliance:

yes

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:

TEST MATERIAL:
- Name of test material: Butyldiglycol methacrylate or 2-(2-butoxyethoxy)ethyl methacrylate
- CAS No. 7328-22-5
- EC No. 230-813-8
- Storage conditions: at room temperature
- Physical state: colourless liquid

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

According to the OECD Guideline for Cell Gene Mutation Tests at least 4 analysable concentrations should be used in two parallel cultures.
The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2.0.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

Vehicle / solvent:

DMSO (purity >= 99.9%)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.7 to 1.2 x 10^7 cells
- After 24 h, the medium was replaced with serum-free medium containing the test item, either without S9 ix or with 50 µl/ml S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was prelaced with complete medium following two washing steps with PBS. Immediately after the end of treatment, the cells were trypsinised and subcultivated. At least 2.0x10^6 cells per experimental point (concentration series plus controls) were subcultivated in 175 cm² flasks containing 30 ml medium. Three to four days after the first subcultivation, at least 2.0x10^6 cells per experimental point were again, subcultivated in 175 cm² flasks containing 30 ml medium. Following the expression time of approx. 7 days, five 75 cm² cell culture flasks were seeded with about 4-5x10^5 cells each in medium containing 6-TG (11 µg/ml). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 +/- 1.5°C in humidified atmosphere with 1.5 +/- 0.5 CO2.
After approx. 8 days (evaluation for viability) and approx. 9 days +/- 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 sized was checked with a preparation microscope.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survial (RS) as measure of test item induced cytotoxicity. The cultures were incubated at 37 +/- 1.5°C in a humidified atmosphere with 1.5% +/- 0.5 CO2. The colonies to determine the relative survival (RS) were fixed and stained approx. 8 +/- 2 days after treatment.

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 conditions exhibits a statistically significant increase compared with the concurrent negative control;
b) the increase is dose-related when evaluated with the 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 any of the 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 the appropriate trend test;
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-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 rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies (mean values) obtained for the groups treated 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 not performed since all mean mutant frequencies of the groups treated with the test item were well within the 95% confidence interval of our laboratory's historical negative control data.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

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, Butyldiglycol methacrylate is considered to be non-mutagenic in this HPRT assay.

Executive summary:

This study was performed to investigate the potential of Butyldiglycol methacrylate to induce gene mutations at the HPRT locus in V79 cells of Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration in the pre-experiment (2039 µg/ml) was chosen with respect to the OECD guideline regarding the purity of the test item. The concentration range of the main experiment was chosen according to the observed cytotoxicity and phase separation in the pre-experiment. No substantial and dose dependent increase of the mutation frequency was observed in the main experiment. The test concentrations are described in chapter 3.5.2. The evaluated experimental points and the results are summarised in Table 3. 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. 

Conclusion: 

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, Butyldiglycol methacrylate is considered to be non-mutagenic in this HPRT assay.

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the reliable available data, Butyldiglycol methacrylate does not need to be classified for mutagenicity according to the criteria given in regulation (EC) 1272/2008 or the former European directive on classification and labelling 67/548/EEC. Thus, no labelling is required.