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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The three in vitro recommended tests were performed on 3-methyl-1,5-pentanediyl diacrylate. The Ames test, HPRT test and in vitro micronucleus test showed negative results in prese nce and in absence of metabolic activation. Based on these studies, 3-methyl-1,5-pentanediyl diacrylate is not considered as mutagen.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 April 2013 -- 04 June 2013
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
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Main test:
- 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains in the first experiment both with and without S9 mix, for the TA 1535, TA 98, TA 100 and TA 102 strains in the second experiment with and without S9 mix, and in TA 1537 strain with S9 mix only in the second experiment,
- 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for TA 1537 strain without S9 mix in the second experiment.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO), batch No. K42474850 145.
- Justification for choice according to solubility assays performed, the highest dose-level of 5000 µg/plate was achievable using a test item solution at 100 mg/mL under a treatment volume of 50 µL/plate.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.
Statistics:
no
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Experiments without S9 mix
In the first experiment, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels superior or equal to 1250 µg/plate (TA 1537 and TA 102 strains) and at dose-levels superior or equal to 2500 µg/plate (TA 1535, TA 98 and TA 100 strains).
 
In the second experiment, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels superior or equal to 1250 µg/plate (TA 98 and TA 100 strains), and at dose-levels superior or equal to
2500 µg/plate (TA 1535, TA 1537 and TA 102 strains).
 
The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains used.
 
Experiments with S9 mix
In the first experiment, a moderate to strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted at 312.5 µg/plate and at dose-levels superior or equal to 1250 µg/plate (TA 1537 strain), at dose-levels superior or equal to 1250 µg/plate (TA 98 strain), and at dose-level of 5000 µg/plate (TA 1535, TA 100 and TA 102 strains).
 
In the second experiment, a strong toxicity (thinning of the bacterial lawn and decrease in the number of revertants) was noted at tested dose-levels
superior or equal to 1250 µg/plate in the five strains.
 
The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains used.
Conclusions:
Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium either in the presence or in the absence of a rat liver metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium.

This study was conducted in compliance with OECD No. 471 and the principles of Good Laboratory Practices.

Methods

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity study. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

 

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

 

The number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were six analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

 

Since the test item was found to be cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

 

The treatment-levels were:

- 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains in the first experiment both with and without S9 mix, for the TA 1535, TA 98, TA 100 and TA 102 strains in the second experiment with and without S9 mix, and in TA 1537 strain with S9 mix only in the second experiment,

- 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for TA 1537 strain without S9 mix in the second experiment.

 

No precipitate was observed in the Petri plates when scoring the revertants whatever the strain and the tested dose-level, either with or without S9 mix.

Experiments without S9 mix

In the first experiment, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels superior or equal to 1250 µg/plate (TA 1537 and TA 102 strains) and at dose-levels superior or equal to 2500 µg/plate (TA 1535, TA 98 and TA 100 strains).

 

In the second experiment, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels superior or equal to 1250 µg/plate (TA 98 and TA 100 strains), and at dose-levels superior or equal to

2500 µg/plate (TA 1535, TA 1537 and TA 102 strains).

 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains used.

 

Experiments with S9 mix

In the first experiment, a moderate to strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted at 312.5 µg/plate and at dose-levels superior or equal to 1250 µg/plate (TA 1537 strain), at dose-levels superior or equal to 1250 µg/plate (TA 98 strain), and at dose-level of 5000 µg/plate (TA 1535, TA 100 and TA 102 strains).

 

In the second experiment, a strong toxicity (thinning of the bacterial lawn and decrease in the number of revertants) was noted at tested dose-levels

superior or equal to 1250 µg/plate in the five strains.

 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains used.

Conclusion

 

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium either in the presence or in the absence of a rat liver metabolizing system.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 April 2013 - 24 June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline No. 487 (In vitro mammalian cell micronucleus test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable (not a gene mutation assay).
Species / strain / cell type:
other: L5178Y TK+/-mouse lymphoma cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing 10% (v/v) heat-inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for the main treatments were as follows:
- 1.19, 1.91, 3.05, 4.88, 7.81 and 12.5 µg/mL in the first experiment without S9 mix,
- 1.36, 1.90, 2.66, 3.72, 5.21 and 10.2 µg/mL in the second experiment without S9 mix,
- 3.72, 5.21, 7.29, 10.2, 14.3 and 20 µg/mL in the first experiment with S9 mix,
- 3.72, 5.21, 7.29, 10.2, 14.3, 20, 40 and 80 µg/mL in the second experiment with S9 mix.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO), batch No. K42474850 145.
- Justification for choice according to solubility assays performed at CiToxLAB France, the highest recommended dose-level of 5000 µg/mL was achievable using a test item solution at 500 mg/mL under a treatment volume of 1% (v/v) in the culture medium.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
see Executive summary

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells/dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling
Evaluation criteria:
A test item was considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was observed,
- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels. The biological relevance of the results was considered first. If the criteria of a positive response are only partially met, results will be evaluated on a case by case basis, taking into account other parameters such as reproducibility between experiments. If results remain inconclusive, or when the highest analyzable dose-level does not exhibit about 55% toxicity (in case of toxic items), additional confirmatory experiments may be needed.
Statistics:
no
Species / strain:
other: L5178Y TK+/- mouse lymphoma cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Experiments without S9 mix

Cytotoxicity
Following the first experiment, a severe toxicity was induced at tested dose-levels = 7.81 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 4.88 µg/mL induced a slight but acceptable toxicity, as shown by a 29% decrease in the PD.
Following the second experiment, a marked toxicity was induced at the highest tested dose-level of 10.2 µg/mL, as shown by a 61% decrease in the PD. The immediately lower dose-level of 5.21 µg/mL induced no noteworthy toxicity, as shown by no noteworthy decrease in the PD.
 
Micronucleus analysis
The dose-levels selected for micronucleus analysis were as follows:
- 1.91, 3.05 and 4.88 µg/mL for the 3-hour treatment, the higher being too cytotoxic,
- 2.66, 3.72 and 5.21 µg/mL for the 24-hour treatment, the higher being too cytotoxic.
 
In the first experiment, an increase in the frequency of micronucleated cells was noted at 3.05 µg/mL. However, this increase was neither dose-related nor statistically significant. Moreover the corresponding frequency of micronucleated cells remained within the historical data range of the corresponding vehicle control. In the second experiment, no statistically significant increase in the frequency of micronucleated cells was noted at the three analyzed dose-levels. Consequently, this effect did not meet the criteria for a positive response and was thus considered as non-biologically relevant.
 
Experiments with S9 mix

Cytotoxicity
Following the first experiment, a marked toxicity was induced at the highest tested dose-level of 20 µg/mL, as shown by a 71% decrease in the PD. The immediately lower dose-level of 14.3 µg/mL induced a moderate but acceptable toxicity, as shown by a 40% decrease in the PD.
Following the second experiment, a moderate but acceptable toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 43% decrease in the PD.
 
Micronucleus analysis
The dose-levels selected for micronucleus analysis were as follows:
- 7.29, 10.2 and 14.3 µg/mL for the first experiment, the higher being too cytotoxic,
- 20, 40 and 80 µg/mL for the second experiment (the highest tested dose-level was achieved).
 
In the first experiment, an increase in the frequency of micronucleated cells was noted at 10.2 µg/mL. However, this increase was neither dose-related nor statistically significant. Moreover the corresponding frequency of micronucleated cells remained within the historical data range of the corresponding vehicle control. In the second experiment performed in the same experimental conditions, some increases in the frequency of micronucleated cells were noted. However, no dose-response relationship was noted and these increases were not statistically significant. Moreover, the corresponding frequencies of micronucleated cells remained within the historical data range of the corresponding vehicle control. Consequently, these increases did not meet the criteria for a positive response and were thus considered not biologically relevant.
 
Conclusions:
Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cellsin the mouse cell line L5178Y TK+/-.

This study was conducted in compliance with OECD Guideline No. 487 and the principles of Good Laboratory Practices.

Methods

After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),

-Second experiment : 24 h treatment + 20 h recovery (without S9 mix), and 3 h treatment + 24 h recovery (with S9 mix).

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account.

For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per concentration).

 

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

 

The mean PD and mean frequencies of micronucleated cells for the vehicle control cultures were as specified in the acceptance criteria. Positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Since the test item was found to be cytotoxic and poorly soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of emulsion and cytotoxicity, according to the criteria specified in the international guidelines.

 

With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for the main treatments were as follows:

- 1.19, 1.91, 3.05, 4.88, 7.81 and 12.5 µg/mL in the first experiment without S9 mix,

- 1.36, 1.90, 2.66, 3.72, 5.21 and 10.2 µg/mL in the second experiment without S9 mix,

- 3.72, 5.21, 7.29, 10.2, 14.3 and 20 µg/mL in the first experiment with S9 mix,

- 3.72, 5.21, 7.29, 10.2, 14.3, 20, 40 and 80 µg/mL in the second experiment with S9 mix.

 

No precipitate was observed in the culture medium at the end of the treatment periods.

Experiments without S9 mix

Cytotoxicity

Following the first experiment, a severe toxicity was induced at tested dose-levels = 7.81 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 4.88 µg/mL induced a slight but acceptable toxicity, as shown by a 29% decrease in the PD.

Following the second experiment, a marked toxicity was induced at the highest tested dose-level of 10.2 µg/mL, as shown by a 61% decrease in the PD. The immediately lower dose-level of 5.21 µg/mL induced no noteworthy toxicity, as shown by no noteworthy decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows:

- 1.91, 3.05 and 4.88 µg/mL for the 3-hour treatment, the higher being too cytotoxic,

- 2.66, 3.72 and 5.21 µg/mL for the 24-hour treatment, the higher being too cytotoxic.

 

In the first experiment, an increase in the frequency of micronucleated cells was noted at 3.05 µg/mL. However, this increase was neither dose-related nor statistically significant. Moreover the corresponding frequency of micronucleated cells remained within the historical data range of the corresponding vehicle control. In the second experiment, no statistically significant increase in the frequency of micronucleated cells was noted at the three analyzed dose-levels. Consequently, this effect did not meet the criteria for a positive response and was thus considered as non-biologically relevant.

 

Experiments with S9 mix

Cytotoxicity

Following the first experiment, a marked toxicity was induced at the highest tested dose-level of 20 µg/mL, as shown by a 71% decrease in the PD. The immediately lower dose-level of 14.3 µg/mL induced a moderate but acceptable toxicity, as shown by a 40% decrease in the PD.

Following the second experiment, a moderate but acceptable toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 43% decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows:

- 7.29, 10.2 and 14.3 µg/mL for the first experiment, the higher being too cytotoxic,

- 20, 40 and 80 µg/mL for the second experiment (the highest tested dose-level was achieved).

 

In the first experiment, an increase in the frequency of micronucleated cells was noted at 10.2 µg/mL. However, this increase was neither dose-related nor statistically significant. Moreover the corresponding frequency of micronucleated cells remained within the historical data range of the corresponding vehicle control. In the second experiment performed in the same experimental conditions, some increases in the frequency of micronucleated cells were noted. However, no dose-response relationship was noted and these increases were not statistically significant. Moreover, the corresponding frequencies of micronucleated cells remained within the historical data range of the corresponding vehicle control. Consequently, these increases did not meet the criteria for a positive response and were thus considered not biologically relevant.

 

Conclusion

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 April 2013 to 17 December 2013
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)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The master stock of L5178Y (3.7.2°C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5 ± 1% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Positive controls
4-nitroquinoline 1-oxide (NQO), stock solution: 0.015 and 0.020 mg/mL and final concentration: 0.15 and 0.20 µg/mL, no metabolic activation
Benzo[a]pyrene (B[a]P), stock solution: 0.200 and 0.300 mg/mL and final concentration: 2.00 and 3.00 µg/mL with metabolic activation

Cytotoxicity Range-Finder Experiment: 70.63, 141.3, 282.5, 565.0, 1130 and 2260 µg/mL
Experiment 1: 2.5, 5, 10, 15 and 30 µg/mL in the absence of S-9 and 10, 20, 40, 60, 80, 100, 120 and 140 µg/mL in the presence of S-9
Experiment 2: 2.5, 5, 10, 15, 17.5, 20 and 22.5 µg/mL in the absence of S-9 and 20, 40, 60, 80, 100, 120, 130, 140 and 150 µg/L in the presence of S-9
Vehicle / solvent:
DMSO diluted 100-fold in the treatment medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
DURATION
- Preincubation period: Not applicable
- Exposure duration: 3-hour exposure period, followed by 7-day expression period
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p < 0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p < 0.05).
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RS lower than 10%.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS Guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 70.63 to 2260 µg/mL (equivalent to 10 mM at the highest concentration tested). Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations tested in the absence and presence of S-9 (565 to 2260 µg/mL). Following the 3-hour treatment incubation period, no precipitate was observed in the absence or presence of S-9, therefore all cultures were retained. The highest concentration to provide >10% RS in the presence of S-9 was 141.3 µg/mL, which gave 16% RS. In the absence of S-9, complete toxicity was observed at the lowest concentration (70.63 µg/mL) and at the highest four concentrations tested (282.5 µg/mL and above), but cultures treated at 141.3 µg/mL gave 89% RS.
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (2260 µg/mL), compared to the concurrent vehicle controls (individual data not reported).
In Experiment 1, thirteen concentrations, ranging from 2.5 to 280 µg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 10 to 225 µg/mL, were tested in the presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the highest concentration tested (280 µg/mL) in the absence of S-9 only. Following the 3-hour treatment incubation period, no precipitate was observed in the absence or presence of S-9, therefore all cultures were retained. Seven days after treatment, the highest eight concentrations in the absence of S-9 (50 to 280 µg/mL) and the highest three concentrations in the presence of S-9 (160 to 225 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 30 µg/mL in the absence of S-9 and 140 µg/mL in the presence of S-9, which gave 1% and 19% RS, respectively. In the absence of S-9, no concentration gave 10-20% RS due to steep concentration-related toxicity. Cultures treated at 15 and 30 µg/mL gave 30% and 1% RS, respectively, therefore both concentrations were analysed for viability and 6TG resistance.
In Experiment 2, eleven concentrations, ranging from 2.5 to 40 µg/mL in the absence of S-9 and from 20 to 200 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest four concentrations in the absence of S-9 (25 to 40 µg/mL) and the highest two concentrations in the presence of S-9 (175 and 200 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 22.5 µg/mL in the absence of S-9 and 150 µg/mL in the presence of S-9, both of which gave 15%.

Table 1 RS Values -Range-Finder Experiment

Treatment

(µg/mL)

-S-9

% RS

+S-9

% RS

0

100

100

70.63

0

63

141.3

89

16

282.5

0

0

565.0 P

0

0

1130 P

0

0

2260 P

0

0

% RS                      Percent relative survival adjusted by post treatment cell counts

P                             Precipitation observed at time of treatment

 

Table 2 Summary of Mutation Data

Experiment 1 (3-hour treatment in the absence and presence of S-9)

Treatment

(mg/mL)

-S-9

Treatment

(mg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

1.64

 

0

 

100

1.90

 

2.5

 

89

1.52

NS

10

 

103

1.99

NS

5

 

63

1.32

NS

20

 

109

2.62

NS

10

 

44

2.14

NS

40

 

102

3.03

NS

15

 

30

1.47

NS

60

 

80

1.84

NS

30

 

1

4.80

*

80

 

49

4.62

NS

 

 

 

 

 

100

 

34

1.46

NS

 

 

 

 

 

120

 

25

3.10

NS

 

 

 

 

 

140

 

19

5.11

NS

Linear trend

*

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.15

 

58

22.23

 

2

 

82

39.11

 

0.2

 

57

32.78

 

3

 

36

58.99

 

Experiment 2 (3-hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

2.40

 

0

 

100

5.81

 

2.5

 

71

0.71

NS

20

 

94

3.20

NS

5

 

62

1.65

NS

40

 

92

3.52

NS

10

 

38

2.82

NS

60

 

69

4.91

NS

15

 

26

1.01

NS

80

 

55

5.72

NS

17.5

 

19

2.25

NS

100

 

40

4.47

NS

20

 

19

1.99

NS

120

 

25

4.26

NS

22.5

 

15

3.61

NS

130

 

24

3.73

NS

 

 

 

 

 

140

 

22

8.99

NS

 

 

 

 

 

150

 

15

3.55

NS

Linear trend

*

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.15

 

61

46.87

 

2

 

71

29.67

 

0.2

 

58

50.91

 

3

 

59

66.58

 

§                             6TG resistant mutants /106 viable cells 7 days after treatment.

% RS                      Percent relative survival adjusted by post treatment cell counts.

NS                          Not significant.

*                             Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level.

*, **, ***               Test for linear trend: c2 (one-sided), significant at 5%, 1% and 0.1% level respectively.

 

 

Conclusions:
It is concluded that 3-methyl-1,5-pentanediyl diacrylate did not induce biologically relevant increases in mutant frequency at the hprt locus of L5178Y mouse lymphoma cells when tested up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolising system (S-9).
Executive summary:

3-methyl-1,5-pentanediyl diacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3-hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 70.63 to 2260 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide >10% relative survival (RS) in the presence of S-9 was 141.3 µg/mL, which gave 16% RS. In the absence of S-9, complete toxicity was observed at the lowest concentration (70.63 µg/mL) and at the highest four concentrations tested (282.5 µg/mL and above), but cultures treated at 141.3 µg/mL gave 89% RS.

In Experiment 1, thirteen concentrations, ranging from 2.5 to 280 µg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 10 to 225 µg/mL, were tested in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 30 µg/mL in the absence of S-9 and 140 µg/mL in the presence of S-9, which gave 1% and 19% RS, respectively. In the absence of S-9, no concentration gave 10-20% RS due to steep concentration-related toxicity. Cultures treated at 15 and 30 µg/mL gave 30% and 1% RS, respectively, therefore both concentrations were analysed for viability and 6TG resistance.

In Experiment 2, eleven concentrations, ranging from 2.5 to 40 µg/mL in the absence of S-9 and from 20 to 200 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 22.5 µg/mL in the absence of S-9 and 150 µg/mL in the presence of S-9, both of which gave 15% RS.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

In the absence of S-9 in Experiment 1, a statistically significant increase in MF, compared to the concurrent vehicle control, was observed at the highest concentration analysed (30 µg/mL) However, the increase was observed only at a highly toxic concentration (giving 1% RS) and the MF value at 30 µg/mL (4.80 mutants/106 viable cells) was small in magnitude and certainly less than 3-fold higher than the historical mean vehicle control MF at the time of this experiment (3.74). In Experiment 2 (in which concentrations of 17.5, 20 and 22.5 µg/mL gave 10-20% RS), no statistically significant increases in MF, compared to the concurrent vehicle control, were observed at any concentration analysed. As the increase in MF in Experiment 1 was small in magnitude, observed only at an extremely toxic concentration and not reproduced in a further experiment in which acceptable levels of toxicity were achieved, this observation was considered not biologically relevant.

Weak but statistically significant linear trends were observed in Experiments 1 and 2 but as there were no biologically relevant increases in MF at any concentration analysed in either experiment, these observations were also considered not biologically relevant.

In the presence of S-9, no statistically significant increases in MF, compared to the concurrent vehicle controls, were observed at any concentration analysed in Experiments 1 and 2 and there were no significant linear trends,indicating a negative result.

It is concluded that 3-methyl-1,5-pentanediyl diacrylate did not induce biologically relevant increases in mutant frequency at the hprt locus of L5178Y mouse lymphoma cells when tested up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolising system (S-9).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay (Brient 2013):

The objective of this study was to evaluate the potential of the test item to induce reverse mutations inSalmonella typhimurium (OECD 471).

In the main test, the test item was tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimuriumwere used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). The test item was dissolved in dimethylsulfoxide (DMSO).

Experiments without S9 mix : In the first experiment, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels superior or equal to 1250 µg/plate (TA 1537 and TA 102 strains) and at dose-levels superior or equal to 2500 µg/plate (TA 1535, TA 98 and TA 100 strains). In the second experiment, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels superior or equal to 1250 µg/plate (TA 98 and TA 100 strains), and at dose-levels superior or equal to 2500 µg/plate (TA 1535, TA 1537 and TA 102 strains). The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains used.

Experiments with S9 mix : In the first experiment, a moderate to strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted at 312.5 µg/plate and at dose-levels superior or equal to 1250 µg/plate (TA 1537 strain), at dose-levels superior or equal to 1250 µg/plate (TA 98 strain), and at dose-level of 5000 µg/plate (TA 1535, TA 100 and TA 102 strains). In the second experiment, a strong toxicity (thinning of the bacterial lawn and decrease in the number of revertants) was noted at tested dose-levels superior or equal to 1250 µg/plate in the five strains. The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains used. 

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium either in the presence or in the absence of a rat liver metabolizing system.

In vitro mammalian cell micronucleus test (Brient 2013):

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells n the mouse cell line L5178Y (OECD 487).

After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. In the First experiment, cells were treated during 3h with a 24h-recovery (without and with S9 mix),and in the second experiment, the cells were treated during 24 h with a 20h-recovery (without S9 mix) or during 3h with a 24h-recovery (with S9 mix).The test item was dissolved in dimethylsulfoxide (DMSO).

Since the test item was found to be cytotoxic and poorly soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of emulsion and cytotoxicity, according to the criteria specified in the international guidelines. No precipitate was observed in the culture medium at the end of the treatment periods.

Experiments without S9 mix: Following the first experiment, a severe toxicity was induced at tested dose-levels = 7.81 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 4.88 µg/mL induced a slight but acceptable toxicity, as shown by a 29% decrease in the PD.

Following the second experiment, a marked toxicity was induced at the highest tested dose-level of 10.2 µg/mL, as shown by a 61% decrease in the PD. The immediately lower dose-level of 5.21 µg/mL induced no noteworthy toxicity, as shown by no noteworthy decrease in the PD. 

In the first experiment, an increase in the frequency of micronucleated cells was noted at 3.05 µg/mL (intermediate dose). However, this increase was neither dose-related nor statistically significant. Moreover the corresponding frequency of micronucleated cells remained within the historical data range of the corresponding vehicle control. In the second experiment, no statistically significant increase in the frequency of micronucleated cells was noted at the three analyzed dose-levels. Consequently, this effect did not meet the criteria for a positive response and was thus considered as non-biologically relevant.

Experiments with S9 mix: Following the first experiment, a marked toxicity was induced at the highest tested dose-level of 20 µg/mL, as shown by a 71% decrease in the PD. The immediately lower dose-level of 14.3 µg/mL induced a moderate but acceptable toxicity, as shown by a 40% decrease in the PD. Following the second experiment, a moderate but acceptable toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 43% decrease in the PD. In the first experiment, an increase in the frequency of micronucleated cells was noted at 10.2 µg/mL (intermediate dose). However, this increase was neither dose-related nor statistically significant. Moreover the corresponding frequency of micronucleated cells remained within the historical data range of the corresponding vehicle control. In the second experiment performed in the same experimental conditions, some increases in the frequency of micronucleated cells were noted. However, no dose-response relationship was noted and these increases were not statistically significant. Moreover, the corresponding frequencies of micronucleated cells remained within the historical data range of the corresponding vehicle control. Consequently, these increases did not meet the criteria for a positive response and were thus considered not biologically relevant.

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

 

 

In vitro mammalian cell gene mutation assay / HPRT (Lloyd 2013)

3-methyl-1,5-pentanediyl diacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3-hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 70.63 to 2260 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide >10% relative survival (RS) in the presence of S-9 was 141.3 µg/mL, which gave 16% RS. In the absence of S-9, complete toxicity was observed at the lowest concentration (70.63 µg/mL) and at the highest four concentrations tested (282.5 µg/mL and above), but cultures treated at 141.3 µg/mL gave 89% RS.

In Experiment 1, thirteen concentrations, ranging from 2.5 to 280 µg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 10 to 225 µg/mL, were tested in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 30 µg/mL in the absence of S-9 and 140 µg/mL in the presence of S-9, which gave 1% and 19% RS, respectively.In the absence of S-9, no concentration gave 10-20% RS due to steep concentration-related toxicity. Cultures treated at 15 and 30 µg/mL gave 30% and 1% RS, respectively, therefore both concentrations were analysed for viability and 6TG resistance.

In Experiment 2, eleven concentrations, ranging from 2.5 to 40 µg/mL in the absence of S-9 and from 20 to 200 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 22.5 µg/mL in the absence of S-9 and 150 µg/mL in the presence of S-9, both of which gave 15% RS.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

In the absence of S-9 in Experiment 1, a statistically significant increase in MF, compared to the concurrent vehicle control, was observed at the highest concentration analysed (30 µg/mL) However, the increase was observed only at a highly toxic concentration (giving 1% RS) and the MF value at 30 µg/mL (4.80 mutants/10^6 viable cells) was small in magnitude and certainly less than 3-fold higher than the historical mean vehicle control MF at the time of this experiment (3.74). In Experiment 2 (in which concentrations of 17.5, 20 and 22.5 µg/mL gave 10-20% RS), no statistically significant increases in MF, compared to the concurrent vehicle control, were observed at any concentration analysed. As the increase in MF in Experiment 1 was small in magnitude, observed only at an extremely toxic concentration and not reproduced in a further experiment in which acceptable levels of toxicity were achieved, this observation was considered not biologically relevant.

Weak but statistically significant linear trends were observed in Experiments 1 and 2 but as there were no biologically relevant increases in MF at any concentration analysed in either experiment, these observations were also considered not biologically relevant.

In the presence of S-9, no statistically significant increases in MF, compared to the concurrent vehicle controls, were observed at any concentration analysed in Experiments 1 and 2 and there were no significant linear trends,indicating a negative result.

It is concluded that 3-methyl-1,5-pentanediyl diacrylate did not induce biologically relevant increases in mutant frequency at the hprt locus of L5178Y mouse lymphoma cells when tested up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolising system(S-9).


Justification for classification or non-classification

Based on the negative results in all three regulatory in vitro genotoxicity tests, no classification for 3-methyl-1,5-pentanediyl diacrylate is required for genotoxicity according to the Regulation EC n°1272/2008.