Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with diethylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro studies are available on the registered substance to evaluate the genotoxicity potential. The HPRT assay (OECD 476) and the Ames test (OECD 471) showed positive results. However the in vitro micronucleus test (OECD 487) is negative. Based on these results, Diethylamine modified ethoxylated trimethylolpropane triacrylate was considered to be mutagenic in vitro, therefore further in vivo testing was performed.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 August 2012 to 12 February 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)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Mouse lymphoma L5178Y cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) 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:

mammalian liver post-mitochondrial fraction (S 9), prepared 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 S-9 present.
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, S-9 present.

Range finding test: Six concentrations tested both in the presence and absence of S-9 ranging from 156.3 to 5000 µg/mL.

Main study:
Experiment 1: Twelve concentrations ranging from 0.625 to 150 µg/mL were tested in the absence of S-9 and eleven concentrations ranging from 50 to 625 µg/mL were tested in the presence of S-9.
Experiment 2: Twelve concentrations ranging from 15 to 130 µg/mL were tested in the absence of S-9 and ten concentrations were tested ranging from 50 to 450 µg/mL 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 diluted 100-fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Remarks:

4-nitroquinoline-N-oxide without metabolic activation and benzo(a)pyrene with metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In suspension

DURATION
- Preincubation period: NA
-Incubation period: 3 Hours
- Exposure duration: 7 Days
- Expression time (cells in growth medium): Not reported

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.

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.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In Experiment 1 twelve concentrations, ranging from 0.625 to 150 µg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 50 to 625 µg/mL, were tested in the presence of S-9. No precipitate was observed in the absence and presence of S-9. Seven days after treatment, the highest three concentrations tested in the absence of S 9 (110 to 150 µg/mL) and the highest four concentrations in the presence of S-9 (450 to 625 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, the lowest three concentrations in the absence of S-9 (0.625 to 2.5 µg/mL) were not selected as there were sufficient concentrations to define the toxicity profile. All other concentrations were analysed in the absence and presence of S-9. The highest concentrations analysed were 90 µg/mL in the absence of S-9 and 400 µg/mL in the presence of S 9, which gave 17% and 13% RS, respectively.
In Experiment 2 twelve concentrations, ranging from 15 to 130 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 50 to 450 µg/mL, were tested in the presence of S-9. Seven days after treatment, the highest three concentrations tested in the absence of S-9 (100 to 130 µg/mL) and in the presence of S-9 (410 to 450 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 selected were 95 µg/mL in the absence of S-9 and 390 µg/mL in the presence of S-9, which gave 17% and 15% RS, respectively

Conclusions:

It is concluded that Diethylamine modified ethoxylated trimethylolpropane triacrylate (CAS Number 159034-91-0) induced mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in the absence of a rat liver metabolic activation system (S-9), but did not induce increases in mutant frequency in the same test system when tested up to toxic concentrations in the presence of S-9.

Executive summary:

Diethylamine modified ethoxylated trimethylolpropane triacrylate (CAS Number 159034-91-0) 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 156.3 to 5000 µg/mL (an acceptable maximum concentration for in vitro genetic toxicology studies according to current regulatory guidelines). The highest concentration to provide >10% relative survival (RS) in the presence of S-9 was 312.5 µg/mL, which gave 40% RS. In the absence of S-9 all concentrations analysed gave < 10% RS, with the lowest concentration tested (156.3 µg/mL) giving 1% RS.

In Experiment 1 twelve concentrations, ranging from 0.625 to 150 µg/mL, were tested in the absence of S-9 and eleven concentrations ranging from 50 to 625 µg/mL, in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 90 µg/mL in the absence of S-9 and 400 µg/mL in the presence of S-9, which gave 17% and 13% RS, respectively.

In Experiment 2 twelve concentrations, ranging from 15 to 130 µg/mL, were tested in the absence of S 9 and ten concentrations, ranging from 50 to 450 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 95 µg/mL in the absence of S-9 and 390 µg/mL in the presence of S-9, which gave 17% and 15% RS, respectively.

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

In the absence of S-9, significant increases in mutant frequency over the concurrent control were observed following treatment with Diethylamine modified ethoxylated trimethylolpropane triacrylate (CAS Number 159034-91-0) at the highest concentrations analysed in Experiments 1 and 2 (90 and 95 µg/mL, respectively) and statistically significant linear trends were observed in both experiments. Although the increases were observed towards the upper limit of cytotoxicity (10-20% RS) and there was some variation between experiments in the magnitude of the induced mutagenic response, the criteria for a positive result were fulfilled in both experiments.

In the presence of S-9, no significant increases in mutant frequency were observed in Experiments 1 and 2. A statistically significant linear trend was observed in Experiment 1 but as there were no significant increases in mutant frequency at any concentration analysed in this experiment, the observation was considered not biologically relevant.

It is concluded that Diethylamine modified ethoxylated trimethylolpropane triacrylate (CAS Number 159034-91-0) induced mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in the absence of a rat liver metabolic activation system (S-9), but did not induce increases in mutant frequency in the same test system when tested up to toxic concentrations in the presence of S-9.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 June 2012 - 31 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

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:

Without S9 : 312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments.
With S9 mix :
.         312.5, 625, 1250, 2500 and 5000 µg/plate for the first and second mutagenicity experiments,
.         625, 1250, 2500, 3750 and 5000 µg/plate in the third mutagenicity experiment.
 

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: test item was soluble in the vehicle at 100 mg/mL.

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:

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

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five 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.

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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered to be valid.
Since the test item was found freely soluble and non-cytotoxic in the preliminary test, the highest dose-level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.
No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels in any experiments, either with or without S9 mix.

Experiments without S9 mix
No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any dose-levels towards the five strains used. The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in any experiments without S9.
 
Experiments with S9 mix
No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any dose-levels towards the five strains used with S9.
Using the pre-incubation method, a noteworthy increase in the number of revertants was noted in the TA 98 strain at 5000 µg/plate in the second experiment. This increase was reproduced at 5000 µg/plate in the third experiment performed under the same experimental conditions (pre-incubation method). These increases exceeded the threshold of 2-fold the vehicle control (up to 2.9-fold). Moreover, the corresponding means and individual revertant colony counts obtained in both experiments were outside the historical data range of the vehicle control. Consequently, these increases were considered to be biologically relevant.
The test item did not induce any other biologically significant increase in the number of revertants in the other tested strains.

Conclusions:

The test item, showed a mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium in the presence of a metabolic activation system. In the absence of metabolic activation, the test item did not show any mutagenic activity in this bacterial mutation test.

Executive summary:

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

The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice.

 

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, 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. A third mutagenicity experiment was performed with S9 mix to check the reliability of the increase in the number of revertants observed in the TA 98 strain in the second experiment.

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

 

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five 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 was as specified in the acceptance criteria. The study was therefore considered to be valid.

Since the test item was found freely soluble and non-cytotoxic in the preliminary test, the highest dose-level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels in any experiments, either with or without S9 mix.

Experiments without S9 mix

The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments.

No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any dose-levels towards the five strains used.

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

 

Experiments with S9 mix

The selected treatment-levels were:

.         312.5, 625, 1250, 2500 and 5000 µg/plate for the first and second mutagenicity experiments,

.         625, 1250, 2500, 3750 and 5000 µg/plate in the third mutagenicity experiment.

 

No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any dose-levels towards the five strains used.

Using the pre-incubation method, a noteworthy increase in the number of revertants was noted in the TA 98 strain at 5000 µg/plate in the second experiment. This increase was reproduced at 5000 µg/plate in the third experiment performed under the same experimental conditions (pre-incubation method). These increases exceeded the threshold of 2-fold the vehicle control (up to 2.9-fold). Moreover, the corresponding means and individual revertant colony counts obtained in both experiments were outside the historical data range of the vehicle control. Consequently, these increases were considered to be biologically relevant.

The test item did not induce any other biologically significant increase in the number of revertants in the other tested strains.

 

Conclusion

The test item showed a mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium in in the presence of a metabolic activation system. In the absence of metabolic activation, the test item did not show any mutagenic activity in this bacterial mutation test.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 June 2012 - 16 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2010

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: mouse lymphoma L5178Y TK+/- cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 10% 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:

The test concentrations ranged from 3.13 to 50 µg/mL in the absence of S9 mix and from 25 to 200 µg/mL in the presence of S9 mix.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide
- Justification for choice: vehicle compatible with the test system, and allowing the obtention of a solution at 500 mg/mL.
Therefore, using a test item concentration at 500 mg/mL and a treatment volume of 1% in culture medium, the highest recommended dose-level of 5000 µg/mL was achievable.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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
Without S9 mix:
- 3 h treatment + 24 h recovery (first experiment),
- 24 h treatment + 20 h recovery (second ahd third experiments).

With S9 mix:
- 3 h treatment + 24 h recovery (first and second experiments).

NUMBER OF CELLS EVALUATED: 2000/dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling

Evaluation criteria:

The biological relevance of the results should be considered first. Statistical methods are used as an aid in evaluating the test results but should not be the only determinant of a positive response. A result is considered as positive if at least a 2.5-fold increase in the number of micronucleated cells in comparison to the concurrent control is observed, with a statistically significant difference, at one or more concentrations. Concentration-related increases in the frequency of micronucleated cells and comparison to the vehicle control historical data will also be taken into account.

Statistics:

yes

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Following the 3-hour treatment in the first experiment (without S9 mix), a moderate to severe toxicity was noted at dose-levels = 25 µg/mL, as shown by a 44 to 100% decrease in the PD. Following the 24-hour treatment in the second experiment (without S9 mix), a slight to severe toxicity was noted at dose-levels = 25 µg/mL, as shown by a 37 to 100% decrease in the PD. Following the 24-hour treatment in the third experiment (without S9 mix) , a slight to severe toxicity was noted at all tested dose-levels, as shown by a 29 to 100% decrease in the PD.
No biologically relevant increase in the frequency of micronucleated cells was noted after the 3- or 24-hour treatments in either experiment without S9 mix.
 
In the first experiment with S9 mix, a slight to severe toxicity was noted at dose-levels = 100 µg/mL as shown by a 30 to 98% decrease in the PD. In the second experiment with S9 mix, a moderate to severe toxicity was noted at dose-levels >= 125 µg/mL as shown by a 56 to 100% decrease in the PD. No significant increase in the frequency of micronucleated cells reaching or exceeding the threshold of 2.5 fold the vehicle control value was noted after the 3-hour treatment in either experiment.

Conclusions:

Diethylamine modified ethoxylated trimethylolpropane triacrylate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or in the absence of a rat metabolizing 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 cells,in L5178Y TK+/- mouse lymphoma cells (OECD 487).

After two preliminary toxicity tests, 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 for " h (and 24h recovery) with and without S9 mix. In the second experiment, cells were treated for 24 hours (and 20h recovery) without S9, and for 3 hours (and 24 h recovery) with S9 mix. Since the highest analyzable dose-level did not exhibit about 55% toxicity in both assays performed without S9 mix and since an increase in the frequency of micronucleated cells has been observed in the first experiment, a third experiment in the absence of S9 mix was undertaken using a treatment duration of 24 hour treatment + 20 hour recovery to maximize the incubation of the cells with the test item.

The test item was dissolved in dimethylsulfoxide (DMSO).

The population doublings and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Positive controls 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 cytotoxic during the preliminary tests, 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 regulations.

 

Following the 3-hour treatment in the first experiment (without S9 mix), a moderate to severe toxicity was noted at dose-levels = 25 µg/mL, as shown by a 44 to 100% decrease in the PD. Following the 24-hour treatment in the second experiment (without S9 mix), a slight to severe toxicity was noted at dose-levels = 25 µg/mL, as shown by a 37 to 100% decrease in the PD. Following the 24-hour treatment in the third experiment (without S9 mix) , a slight to severe toxicity was noted at all tested dose-levels, as shown by a 29 to 100% decrease in the PD.

No biologically relevant increase in the frequency of micronucleated cells was noted after the 3- or 24-hour treatments in either experiment without S9 mix.

 

In the first experiment with S9 mix, a slight to severe toxicity was noted at dose-levels = 100 µg/mL as shown by a 30 to 98% decrease in the PD. In the second experiment with S9 mix, a moderate to severe toxicity was noted at dose-levels >= 125 µg/mL as shown by a 56 to 100% decrease in the PD. No significant increase in the frequency of micronucleated cells reaching or exceeding the threshold of 2.5 fold the vehicle control value was noted after the 3-hour treatment in either experiment.

 

Diethylamine modified ethoxylated trimethylolpropane triacrylate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK +/- mouse lymphoma cells, either in the presence or in the absence of a rat metabolizing system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A Comet Assay (OECD 489) was conducted using liver, stomach and duodenum, which returned negative results in all three tissues. The study was considered valid based on acceptability criteria. Therefore, it can be concluded that the test substance was not mutagenic under the conditions of the test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start Date: 07 March 2022
Experimental Completion Date: 28 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Justification for Selection
The comet assay is recommended by various regulatory authorities as an appropriate test to determine the genotoxic potential of a compound in vivo (OECD, 2016). This study has been designed to provide data at the request of the European chemical agency (ECHA), based on Article 40 of the REACH regulation (Regulation (EC) No 1907/2006).

The rat was selected as there is a large volume of background data in this species and has been specifically requested by the authorities.

The liver is recommended as the primary site of xenobiotic metabolism and is often highly exposed to both the test article and any metabolites. The stomach and duodenum are recommended tissues to examine for site of contact effects after oral exposure. Male gonadal cells will be collected for potential analysis if induction of DNA strand breaks is determined in any somatic tissue. The analysis of gonadal cells may be relevant for the overall assessment of possible germ cell mutagenicity including classification and labelling according to CLP Regulation. As such, this study will be performed in male rats only.

Sex:

male

Details on test animals or test system and environmental conditions:

Species, Strain and Supplier
33 male young adult out-bred Sprague Dawley rats (HSD:Sprague DawleySD) were obtained from Envigo, Blackthorn, UK.

Animals not dosed in this study were transferred to Labcorp Early Development Laboratories Ltd. stock.

Specification
3 animals were dosed during the Range-Finder Experiment. They were approximately 7 to 8 weeks old and 194-221 g on the first day of dosing.

27 animals were dosed during the Main Experiment. They were approximately 7 to 8 weeks old and 194-235 g on the first day of dosing.


Environment
Animals were housed in wire topped, solid bottomed cages, with three animals per cage.
The animals were housed in rooms air-conditioned to provide a minimum of 15 air changes/hour and set to maintain temperature and relative humidity in the range 19-25°C and 40-70%, respectively. Fluorescent lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800) and 12 hours dark. The animals were routinely kept under these conditions except for short periods of time where experimental procedures dictated otherwise.

Diet, Water, Bedding and Environmental Enrichment
Throughout the study the animals had ad libitum access to 5LF2 EU Rodent Diet. Each batch of diet was analysed for specific constituents and contaminants.

Mains water was provided ad libitum via water bottles. The water supply was periodically analysed for specific contaminants.

Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd., Manchester). The bedding was analysed for specific contaminants.

In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and rodent retreats.

No contaminants were present in any of the above at levels that might interfere with achieving the objective of the study. Results of any analyses performed are held centrally at Labcorp Early Development Laboratories Ltd.

Allocation to Treatment Group
On arrival, animals were randomly allocated to cages. Range-Finder animals were allocated to groups of three and Main Experiment animals were randomised to groups of six (three for the positive control).

Checks were made to ensure the weight variation of Main Experiment animals prior to dosing was minimal and did not exceed ±20% of the mean weight.

Identification of the Test System
The animals were individually identified by uniquely numbered tail mark (Range-Finder) or subcutaneous electronic transponder (Main Experiment). Cages were appropriately identified (using a colour-coded procedure) with study information including study number, study type, start date, number and sex of animals, together with a description of the dose level and proposed time of necropsy.

Acclimatisation and Health Procedures
All animals were given a clinical inspection for ill health on arrival. They were acclimatised for at least 5 days and a health inspection was performed before the start of dosing to ensure their suitability for the study.

Route of administration:

oral: gavage

Vehicle:

Corn oil

The vehicle was selected as it has been used in previous in vivo studies with this compound.

Details on exposure:

Test Article Formulation
Preparation
Formulations were freshly prepared prior to each dosing occasion for the Range-Finder and prepared once for each dose group in the Main Experiment. Acrylated amine synergist (CN3715) was formulated in corn oil as follows:

The test article was weighed out into a pre-labelled bottle and vehicle was added to achieve the final volume. Formulations were stirred on a magnetic stirrer to homogenise and aliquoted (as required).

Stability
No stability information was provided by the Sponsor prior to the start of the study. Consequently, formulations for the Range-Finder were stored at 15-25°C, protected from light, and used within 2 hours of preparation. The formulations were assumed stable for this period as a non-GLP study determined test article formulations at 5 and 200 mg/mL were stable when stored for 4 days at +4°C, protected from light.

Stability data generated in this study prior to the Main Experiment confirmed formulations of Acrylated amine synergist (CN3715) in corn oil at 50 and 200 mg/mL were stable for 3 days when stored at 15-25°C, protected from light.

Consequently, all test article formulations for the Main Experiment were stored at 15-25°C, protected from light, and used within 24 hours of preparation.

Homogeneity
To ensure homogeneity, dose formulations were stirred continuously (on a magnetic stirrer) before and throughout dosing.

Formulations Analysis
Samples were taken from each article formulation used in the Main Experiment together with concurrent vehicle controls.

Duplicate (2 x 1 mL) samples were taken from the top, middle and bottom of each test article formulation together with a single 1 mL sample (taken from the middle) of the vehicle control and all samples were analysed for achieved concentration and a determination of homogeneity.

Samples were stored at 15-25°C, prior to analysis by Labcorp Early Development Laboratories Ltd.
The analytical method is detailed in the Formulation Analysis Contributory Report.

Stability Assessment
Test article formulations at 50 and 200 mg/mL were assessed for stability.

Duplicate (2 x 1 mL) samples were taken from the top, middle and bottom of each test article formulation and all samples were analysed for achieved concentration and a determination of homogeneity.

The remaining bulk formulation was split into 2 aliquots; one aliquot was stored at room temperature (15-25°C) and triplicate samples were taken and analysed for achieved concentration on Day 1 and Day 3 and the remaining aliquot was stored at 2 8ºC. As stability was confirmed at room temperature no sampling or analysis of the refrigerated aliquot was performed.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

The test article and vehicle control were given as two administrations, at 0 and 21 hours; the positive control was administered once only at 21 hours.

Main Experiment animals were dosed in replicate cage order i.e. cage 1 of Groups 1-4 dosed in ascending group order then cage 2 of Groups 1-4 in ascending group order. Group 5 was dosed at a time that allowed necropsy of these animals after Group 4 necropsy. Animals were not fasted prior to administration and were sampled at 24 hours.

Post exposure period:

Observation times were as follows:

Animals Day Approximate Observation Time
Range-Finder Experiment 1 Prior to dose, immediate, 0.5, 1, 2 and 4-6 hours post dose
2 Prior to dose, immediate, 0.5, 1, 2 and 4-6 hours post dose

Main Experiment 1* Prior to dose, immediate, 1, 2 and 4 hours post dose
2 Prior to dose, immediate and prior to necropsy
* Excluding positive control group

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Range-finder experiment

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Group 1 - Vehicle control

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Group 4

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Group 5 - Positive cntrol

No. of animals per sex per dose:

6 males per dose group, including vehicle control
3 males - positive control

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate 200 mg/kg, single oral administration at 21 hours (Day 2)

Tissues and cell types examined:

Liver, stomach, duodenum, gonad*
* Left testis for Comet; Right for Histopathology

The liver, stomach, duodenum and gonad were removed from each control (vehicle and positive) and test article treated animal.
For histopathology, a sample of liver, stomach, duodenum and gonad from vehicle control and test article treated animals only was removed. Liver, stomach and duodenum samples were immediately preserved in neutral buffered formalin and stored at room temperature. Gonad samples were immediately preserved in modified Davidson’s fluid and stored at room temperature. No histopathology samples were preserved for the positive control animals.

Details of tissue and slide preparation:

Justification for Dose Selection
The following information on the in vivo toxicity of Acrylated amine synergist (CN3715) was provided:

In an acute toxicity study, CN3715 was administered once by oral route (gavage) to two groups of three fasted female Sprague-Dawley rats at the dose-level of 2000 mg/kg (in corn oil). No unscheduled deaths occurred during the study. Piloerection was observed in 4/6 females within 4 hours after treatment. No clinical signs persisted from day 2. The test item administration did not induce any macroscopic changes.

A preliminary study was performed to evaluate the potential toxicity of the test article, Acrylated amine synergist (CN3715), following daily oral administration (5 mL/kg via gavage) to Sprague Dawley rats for 2 weeks. Groups of three male and three female rats were dosed at 100, 300 or 1000 mg/kg/day. There were no unscheduled deaths and clinical observations were limited to hypersalivation. All dose levels were well tolerated with a maximum tolerated dose considered to exceed 1000 mg/kg/day in males and females.

A Range-Finder Experiment was performed in this study to confirm that there is no impact on dose tolerability for unfasted male animals (compared to fasted female animals in the previous acute toxicity study) under the dosing regimen of this study (two administrations). An initial dose of 2000 mg/kg/day (the regulatory recommended maximum dose) was administered in the Range-Finder Experiment (OECD, 2016).

From the results of the Range-Finder Experiment dose levels of 500, 1000 and 2000 mg/kg Acrylated amine synergist (CN3715) (equivalent to 25%, 50% of the maximum dose and the maximum dose respectively) were tested in the Main Experiment.

Histopathology
Preserved liver, stomach, duodenum and gonad samples were embedded in wax blocks and sectioned at 5 µm nominal. Liver, stomach and duodenum slides were stained with haematoxylin and eosin and examined by the Study Pathologist. Gonad samples were not examined as the somatic tissues did not show genotoxic potential.

Preparation of Cell Suspensions
The comet liver samples were washed thoroughly in Merchants solution and placed in fresh buffer. The samples were cut into small pieces in Merchants solution and the pieces of liver were then pushed through bolting cloth (pore size of 150 µm) with approximately 4 mL of ice cold Merchants solution to produce single cell suspensions.

The comet stomach samples were washed in ice cold Merchants solution and then incubated on ice for 15 minutes prior to processing. After incubation the stomach samples were removed from the Merchants solution and the inner surface gently scraped twice (released material discarded) using the back of a scalpel blade. Cells were gently scraped from the inside surface of the stomach using the back of a scalpel blade in 200 µL of fresh Merchants solution to produce single cell suspensions.

The comet duodenum samples were washed thoroughly in ice cold Merchants solution; each sample was vortexed in ice cold Merchants solution for approximately 15 seconds. The tissue was removed from the Merchants solution and the inner surface gently scraped twice (released material discarded) using the back of a scalpel blade. The tissue was vortexed in ice cold Merchants solution for a further 15 seconds prior to gently scraping the inside of the duodenum three times with the back of a scalpel blade in 150 µL of fresh Merchants solution to produce single cell suspensions.

The comet gonad samples were prepared by making an incision along the length of a single gonad, removing the contents from the membrane and discarding the membrane. The remaining tissue was cut into small pieces and gently pushed through bolting cloth (pore size of 150 µm) with approximately 10 mL of Merchants solution to produce single cell suspensions.

All cell suspensions were held on ice prior to slide preparation.

Slide Preparation
Three slides, labelled ‘A’, ‘B’ and ‘C’ were prepared per single cell suspension per tissue. Slides were labelled with the study number, appropriate animal tag number and tissue. Slides were dipped in molten normal melting point agarose (NMA) such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 40 µL of each single cell suspension was added to 400 µL of 0.7% low melting point agarose (LMA) at approximately 37°C. 100 µL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on ice.

Cell Lysis
Once gelled the coverslips were removed and all slides placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH adjusted to pH 10 with NaOH, 1% Triton X 100, 10% DMSO) overnight at 2-8°C, protected from light.

Unwinding and Electrophoresis
Following lysis, slides were washed in purified water for 5 minutes, transferred to electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH>13) at 2-8°C and the DNA unwound for 20 minutes (stomach and duodenum) or 30 minutes (liver and gonad). At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 20 minutes (stomach and duodenum) or 40 minutes (liver and gonad). As not all slides could be processed at the same time a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run; i.e. for all animals the same number of triplicate slides was processed at a time.

Neutralisation
At the end of the electrophoresis period, slides were neutralised in 0.4 M Tris, pH 7.0 (3 x 5 minute washes). After neutralisation the slides were dried and stored at room temperature prior to scoring.

Staining
Prior to scoring, the slides were stained with 100 µL of 2 µg/mL ethidium bromide and coverslipped.

Slide Analysis
Scoring was carried out using fluorescence microscopy at an appropriate magnification and with suitable filters.

A slide from a vehicle and positive control animal were checked for quality and/or response prior to analysis. All slides were allocated a random code by an individual not connected to scoring of the study.

All animals per group were analysed.

Measurements of tail intensity (%DNA in tail) were obtained from 150 cells/ animal/tissue. In general, this was evenly split over three slides.

The number of ‘hedgehogs’ (a morphology indicative of highly damaged cells often associated with severe cytotoxicity, necrosis or apoptosis) observed during comet scoring was recorded for each slide. To avoid the risk of false positive results ‘hedgehogs’ were not used for comet analysis. Each slide was scanned starting to the left of the centre of the slide.

The following criteria were used for analysis of slides:
1. Only clearly defined non overlapping cells were scored
2. Hedgehogs were not scored
3. Cells with unusual staining artefacts were not scored.

Comet slides were retained until report finalisation; at this time the slides were discarded with SD approval. Due to the nature of the slides, long term storage is not recommended as comet integrity cannot be assured.

Evaluation criteria:

For valid data, the test article was considered to induce DNA damage if:
1. A least one of the test doses exhibited a statistically significant increase in tail intensity, in any tissue, compared with the concurrent vehicle control
2. The increase was dose related in any tissue
3. The increase exceeded the laboratory’s historical control data for that tissue.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met and target tissue exposure was confirmed.
Results which only partially satisfied the criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example comparison of the response against the historical control data, consistency of response within and between dose levels and any confirmatory experiments.

Statistics:

Please refer to "Any other information on materials and methods"

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDER RESULTS
A group of three male rats was dosed at 0 hours and approximately 21 hours with Acrylated amine synergist (CN3715) at 2000 mg/kg/day. No clinical signs of toxicity were observed for any animal for the duration of the observation period. All 3 males increased in body weight between Day 1 and Day 3.

From these results the regulatory limit dose of 2000 mg/kg/day was tolerated. This was selected as the maximum dose for the Main Experiment. Two lower doses of 500 and 1000 mg/kg/day (equivalent to 25% and 50% of the maximum dose) were also selected.


Post Dose Observations
.There were no clinical observations of toxicity for any animal dosed with the vehicle, Acrylated amine synergist (CN3715) (at 500, 1000 or 2000 mg/kg/day) or the positive control (EMS).

Body Weights
There was no clear test article related impact on animal bodyweights between Day 1 and Day 2 with group mean body weight change values of +1.0%, +2.1% and +2.0% for 500, 1000 and 2000 mg/kg/day, respectively, compared to +1.1% for the concurrent vehicle control group.

Bioanalysis
Plasma was processed from whole blood samples as a contingency for systemic exposure confirmation. Analysis of these samples was not performed.

Clinical Pathology
Clinical Chemistry

There were no Acrylated amine synergist (CN3715)-related clinical chemistry changes recorded. Any differences in individual clinical pathology parameters observed for animals administered the test article were considered not test article related due to the negligible magnitude of the change, individual animal variability, and overlap of values for test article treated animals with concurrent control values.

Histopathology
No macroscopic or microscopic changes were considered related to Acrylated amine synergist (CN3715).

Examined tissues were considered macroscopically or microscopically unremarkable, or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

Data Analysis
There were no dose-related increases in %hedgehogs in liver, stomach or duodenum, thus demonstrating that treatment with Acrylated amine synergist (CN3715) did not cause excessive DNA damage that could have interfered with comet analysis.

Animals treated with Acrylated amine synergist (CN3715) at all doses exhibited group mean tail intensities in the liver that fell within the 95% reference range of the laboratory’s historical vehicle control data. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, and the statistical assessment for dose response was not significant (p > 0.05).

There were two individual animal liver %tail intensity values at 2000 mg/kg/day (R0301, 2.39% and R0303, 3.52%) that exceeded the upper limit of the 95% reference range (1.80%). However, these increases above the concurrent vehicle control group and the 95% reference range were not reproduced in all animals at 2000 mg/kg/day, did not increase the group mean above the 95% reference range and did not contribute to a statistically significant increase for the group compared to the vehicle control group. None of the evaluation criteria for a positive result were met and therefore, these isolated data were considered of no biological relevance.

Animals treated with Acrylated amine synergist (CN3715) at all doses exhibited group mean tail intensities in the stomach that fell within the laboratory's the 95% reference range of the laboratory’s historical vehicle control data. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control. There was a statistically significant dose response noted, however, this response was driven predominantly by an isolated animal response at 2000 mg/kg/day (R0301). All individual animal stomach %tail intensity values fell within the 95% reference range, however the %tail intensity for animal R0301 (5.31%) was elevated compared to all other vehicle and test article dosed animals (0.08-1.01%). As it was within the 95% reference range, it fell within the normal variation of the assay and was likely induced by the mechanical processing of the tissue.
Overall, it was considered that this isolated animal value, which increased the group mean and contributed to the statistically significant dose response, but was not reproduced in other animals and did not contribute to a statistically significant increase in tail intensity at 2000 mg/kg/day compared to the vehicle control group, was of no biological relevance.

Animals treated with Acrylated amine synergist (CN3715) at all doses exhibited group mean tail intensities in the duodenum that fell within, or marginally below, the 95% reference range of the laboratory’s historical vehicle control data. There was a statistically significant increase in tail intensity at the intermediate dose (1000 mg/kg/day) group only, compared to the concurrent vehicle control; both the low dose (500 mg/kg/day) and high dose (2000 mg/kg/day) groups were not statistically significantly increased compared to the concurrent vehicle control. As all individual animal and group mean %tail intensity values fell within, or marginally below, the 95% reference range (moreover all values fell below the mean value of the laboratory’s historical vehicle control data indicate value fell towards the lower end of normal variation within the assay), and there was no evidence of a dose response, the statistical significance associated with the intermediate dose group was considered of no biological relevance.

These data were considered negative in the liver, stomach and duodenum. Therefore, no analysis of gonad slides is required.

Formulations Analysis

Analyses demonstrated that the test article formulations at 50, 100 and 200 mg/mL were homogeneous (0.67-1.17% relative standard deviation (RSD), which fell within target criteria of ≤5%) and met criteria (100±15% of the nominal test article concentrations) for acceptable achieved concentration (mean values of 99-100%). The formulations were therefore considered acceptable. No test article was detected in the vehicle sample.

 

Validity of Data

The data generated in this study confirm that:

1. The vehicle control data were comparable to laboratory historical control data for each tissue


2. The positive control induced responses that were compatible with the laboratory historical control data and produces are statistically significant compared to the concurrent vehicle control


3. Adequate numbers of cells and doses were analysed


4. The high dose was considered to be the maximum recommended dose.

As dosing was via oral gavage, exposure to the stomach and duodenum was assured. With regards to the liver, there were no clinical observations of toxicity and no clinical chemistry changes or histopathological changes related to the test article to provide indirect evidence of systemic exposure. Publically available data suggest Acrylated amine synergist (CN3715) is a UVCB whose constituents have a molecular weight ranging from 369 to 820 g/mol. At 20°C, the substance is soluble in water at ca. 59 g/L and has a octanol:water partition coefficient (Log Kow) ranging from 1.05 to 4.08 (GRL, 2012 and GRL, 2013). According to ECHA (2017), these properties are favourable for absorption following exposure via the oral route. Therefore, while this study did not show effects that could only be attributed to indirect systemic exposure to the test article and/or its metabolites (exposure does not have to be confirmed concurrently within the study, OECD, 2016), absorption is considered very likely by the sponsor based on the physicochemical properties of Acrylated amine synergist (CN3715).

The assay data were therefore considered valid.

 

Text Table 1: Acrylated amine synergist (CN3715): Summary of Group Mean Data – Liver

Group/Dose Level (mg/kg/day)	Tail Intensity						Mean % Hedgehogs
	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
1/ Vehicle (0)	0.32	0.07	-	-	-	-	0.75
2/ Acrylated amine synergist (CN3715) (500)	0.42	0.09	1.08	R	0.7354	NS	0.62
3/ Acrylated amine synergist (CN3715) (1000)	0.22	0.06	0.59	R	0.9794	NS	1.07
4/ Acrylated amine synergist (CN3715) (2000)	1.31	0.59	1.69	R	0.8279	NS	1.01
5/ EMS (200)	19.16	0.65	67.56	U	<0.0001	***	1.20
Dose response: (groups 1,2,3,4 )				R	0.7333	NS	N/A

 

Text Table 2: Acrylated amine synergist (CN3715): Summary of Group Mean Data – Stomach

Group/Dose Level (mg/kg/day)	Tail Intensity						Mean % Hedgehogs
	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
1/ Vehicle (0)	0.22	0.05	-	-	-	-	5.25
2/ Acrylated amine synergist (CN3715) (500)	0.39	0.07	1.55	U	0.3765	NS	7.73
3/ Acrylated amine synergist (CN3715) (1000)	0.59	0.12	2.48	U	0.0946	NS	6.74
4/ Acrylated amine synergist (CN3715) (2000)	1.21	0.83	2.37	U	0.1117	NS	6.89
5/ EMS (200)	12.91	4.04	67.17	U	<0.0001	***	6.50
Dose response: (groups 1,2,3,4 )				U	0.0319	*	N/A

 

EMS	Ethyl Methanesulfonate
SEM	Standard Error of Mean
N/A	Not applicable
NS	Not significant (P>0.05)
*	P≤0.05
***	P≤0.001
R	Ranked
U	Unranked

 

Text Table 3: Acrylated amine synergist (CN3715): Summary of Group Mean Data – Duodenum

Group/Dose Level (mg/kg/day)	Tail Intensity						Mean % Hedgehogs
	Mean	SEM	Back-Transformed Difference from Vehicle	Ranked	P-value	Significance
1/ Vehicle (0)	0.15	0.02	-	-	-	-	9.65
2/ Acrylated amine synergist (CN3715) (500)	0.20	0.03	1.38	U	0.2016	NS	11.42
3/ Acrylated amine synergist (CN3715) (1000)	0.38	0.05	2.62	U	0.0007	***	10.23
4/ Acrylated amine synergist (CN3715) (2000)	0.15	0.03	1.01	U	0.7271	NS	9.86
5/ EMS (200)	6.01	0.37	54.30	U	<0.0001	***	14.34
Dose response: (groups 1,2,3,4 )				U	0.1800	NS	N/A

 

EMS	Ethyl Methanesulfonate
SEM	Standard Error of Mean
N/A	Not applicable
NS	Not significant (P>0.05)
***	P≤0.001
U	Unranked

 

Table 9.5: Liver: Animal Comet Data

Group/ Dose Level	Animal		Total	Tail Intensity (%)		Hedgehogs
(mg/kg/day)	Number		Comets	Mean	SD	(%)
1/ Vehicle (0)	R0001		150	0.62	0.23	0.57
	R0002		150	0.20	0.07	0.57
	R0003		150	0.39	0.03	1.71
	R0004		150	0.21	0.04	0.00
	R0005		150	0.35	0.16	0.55
	R0006		150	0.17	0.04	1.01
2/ Acrylated amine synergist (CN3715) (500)	R0101		150	0.33	0.19	1.69
	R0102		150	0.17	0.17	0.00
	R0103		150	0.77	0.24	1.18
	R0104		150	0.56	0.30	0.48
	R0105		150	0.46	0.22	0.00
	R0106		150	0.23	0.22	0.48
3/ Acrylated amine synergist (CN3715) (1000)	R0201		150	0.49	0.15	1.15
	R0202		150	0.11	0.11	2.91
	R0203		150	0.23	0.20	0.54
	R0204		150	0.12	0.04	0.49
	R0205		150	0.23	0.20	0.52
	R0206		150	0.14	0.05	1.01
4/ Acrylated amine synergist (CN3715) (2000)	R0301		150	2.39	0.97	0.55
	R0302		150	0.15	0.13	0.63
	R0303		150	3.52	0.67	0.59
	R0304		150	0.10	0.04	3.00
	R0305		150	1.62	0.45	0.00
	R0306		150	0.08	0.03	1.01
5/ EMS (200)	R0401		150	18.02	5.05	2.11
	R0402		150	19.18	1.47	0.48
	R0403		150	20.28	1.57	1.09
SD		Standard Deviation
EMS		Ethyl Methanesulfonate

 

 

Table 9.7: Stomach: Animal Comet Data

Group/ Dose Level	Animal		Total	Tail Intensity (%)		Hedgehogs
(mg/kg/day)	Number		Comets	Mean	SD	(%)
1/ Vehicle (0)	R0001		150	0.37	0.28	8.47
	R0002		150	0.36	0.10	1.72
	R0003		150	0.08	0.04	2.47
	R0004		150	0.19	0.16	5.92
	R0005		150	0.14	0.10	6.13
	R0006		150	0.19	0.11	6.56
2/ Acrylated amine synergist (CN3715) (500)	R0101		150	0.32	0.17	5.17
	R0102		150	0.27	0.33	10.98
	R0103		150	0.16	0.11	17.48
	R0104		150	0.54	0.22	4.40
	R0105		150	0.45	0.31	3.09
	R0106		150	0.61	0.69	3.70
3/ Acrylated amine synergist (CN3715) (1000)	R0201		150	0.96	0.59	6.74
	R0202		150	0.45	0.51	12.71
	R0203		150	0.36	0.24	3.01
	R0204		150	0.81	0.40	4.97
	R0205		150	0.23	0.12	5.95
	R0206		150	0.74	0.50	6.67
4/ Acrylated amine synergist (CN3715) (2000)	R0301		150	5.31	3.08	5.21
	R0302		150	0.18	0.12	5.92
	R0303		150	1.01	0.55	5.41
	R0304		150	0.16	0.11	8.54
	R0305		150	0.23	0.19	2.58
	R0306		150	0.36	0.09	14.13
5/ EMS (200)	R0401		150	7.37	0.54	5.08
	R0402		150	20.77	6.24	8.00
	R0403		150	10.59	2.53	6.43
SD		Standard Deviation
EMS		Ethyl Methanesulfonate

 

 

Table 9.9: Duodenum: Animal Comet Data

Group/ Dose Level	Animal		Total	Tail Intensity (%)		Hedgehogs
(mg/kg/day)	Number		Comets	Mean	SD	(%)
1/ Vehicle (0)	R0001		150	0.17	0.09	11.27
	R0002		150	0.14	0.04	15.32
	R0003		150	0.23	0.32	9.74
	R0004		150	0.06	0.03	5.68
	R0005		150	0.13	0.10	5.84
	R0006		150	0.19	0.13	10.26
2/ Acrylated amine synergist (CN3715) (500)	R0101		150	0.10	0.03	11.91
	R0102		150	0.21	0.02	7.85
	R0103		150	0.20	0.03	21.59
	R0104		150	0.19	0.16	10.97
	R0105		150	0.32	0.25	8.33
	R0106		150	0.19	0.14	9.46
3/ Acrylated amine synergist (CN3715) (1000)	R0201		150	0.24	0.17	8.51
	R0202		150	0.45	0.48	5.63
	R0203		150	0.22	0.06	12.87
	R0204		150	0.36	0.20	8.41
	R0205		150	0.45	0.40	11.84
	R0206		150	0.56	0.31	12.42
4/ Acrylated amine synergist (CN3715) (2000)	R0301		150	0.10	0.01	13.60
	R0302		150	0.23	0.19	7.14
	R0303		150	0.20	0.14	10.22
	R0304		150	0.08	0.08	9.06
	R0305		150	0.10	0.07	6.39
	R0306		150	0.18	0.09	11.27
5/ EMS (200)	R0401		150	6.68	1.88	8.94
	R0402		150	5.95	0.58	8.42
	R0403		150	5.41	1.14	22.71
SD		Standard Deviation
EMS		Ethyl Methanesulfonate

 

Conclusions:

It is concluded that, under the conditions of this comet assay, Acrylated amine synergist (CN3715) did not induce DNA strand breaks in the liver, stomach or duodenum of male Sprague Dawley rats administered up to 2000 mg/kg/day (the maximum recommended dose for in vivo comet studies).

Executive summary:

Acrylated amine synergist (CN3715) was tested for its potential to induce DNA strand breaks in the liver, stomach and duodenum of treated rats. As there was no strand break induction observed in any of the somatic tissues, the gonad was not assessed.

Strain / Species:	Sprague Dawley rats
Vehicle:	Corn oil
Administration route:	Oral by gavage
Dosing regimen:	Two administrations at 0 (Day 1) and 21 hours (Day 2)
Sex:	Male rats only due to the potential analysis of gonadal cells and to ethically minimise animal testing.
Dose levels:	500, 1000 and 2000 mg/kg/day
Maximum dose:	Maximum recommended dose for this type of study
Positive control:	Ethyl methanesulfonate 200 mg/kg, single oral administration at 21 hours (Day 2)
Animals per group:	Six (three for the positive control group)
Dose volume:	10 mL/kg
Clinical signs of toxicity:	There were no clinical observations of toxicity for any animal dosed and no apparent test article related impact on animal bodyweights between Day 1 and Day 2 (percentage change values of +1.0%, +2.1% and +2.0% for 500, 1000 and 2000 mg/kg/day, respectively, compared to +1.1% for the concurrent vehicle control group).
Tissues sampled:	Liver, stomach, duodenum and gonad were sampled on Day 2, equivalent to 24 hours.
Formulation analysis:	Analyses confirmed that 50, 100 and 200 mg/mL formulations were homogenous (0.67-1.17% RSD) and achieved intended concentrations (mean values of 99-100%). No test article was detected in the vehicle control sample.
Clinical Chemistry:	No Acrylated amine synergist (CN3715)-related clinical chemistry changes were recorded.
Histopathology:	No macroscopic or microscopic changes were considered related to Acrylated amine synergist (CN3715).
Exposure:	As dosing was via oral gavage, exposure to the stomach and duodenum was assured. With regards to the liver, there were no clinical observations of toxicity and no clinical chemistry changes or histopathological changes related to the test article to provide indirect evidence of systemic exposure. Publically available data suggest Acrylated amine synergist (CN3715) properties are favourable for absorption following exposure via the oral route. Therefore, while this study did not show effects attributable to indirect systemic exposure to the test article and/or its metabolites, absorption is considered very likely by the sponsor based on the physicochemical properties of Acrylated amine synergist (CN3715).
Assay validity:	The vehicle control data were comparable with the laboratory’s historical vehicle control data ranges. The positive control induced significant increases in %tail intensity in the liver, stomach and duodenum (over the current vehicle control group) that were comparable with the laboratory’s historical positive control data. The data were therefore accepted as valid.

 

There were no dose-related increases in %hedgehogs in liver, stomach or duodenum, thus demonstrating that treatment with Acrylated amine synergist (CN3715) did not cause excessive DNA damage that could have interfered with comet analysis.

Animals treated with Acrylated amine synergist (CN3715) at all doses exhibited group mean tail intensities in the liver that fell within the 95% reference range of the laboratory’s historical vehicle control data. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, and the statistical assessment for dose response was not significant (p > 0.05).

There were two individual animal liver %tail intensity values at 2000 mg/kg/day (R0301, 2.39% and R0303, 3.52%) that exceeded the upper limit of the 95% reference range (1.80%). However, these increases above the concurrent vehicle control group and the 95% reference range were not reproduced in all animals at 2000 mg/kg/day, did not increase the group mean above the 95% reference range and did not contribute to a statistically significant increase for the group compared to the vehicle control group. None of the evaluation criteria for a positive result were met and therefore, these isolated data were considered of no biological relevance.

Animals treated with Acrylated amine synergist (CN3715) at all doses exhibited group mean tail intensities in the stomach that fell within the laboratory's the 95% reference range of the laboratory’s historical vehicle control data. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control. There was a statistically significant dose response noted, however, this response was driven predominantly by an isolated animal response at 2000 mg/kg/day (R0301). All individual animal stomach %tail intensity values fell within the 95% reference range, however the %tail intensity for animal R0301 (5.31%) was elevated compared to all other vehicle and test article dosed animals (0.08-1.01%). As it was within the 95% reference range, it fell within the normal variation of the assay and was likely induced by the mechanical processing of the tissue. Overall, it was considered that this isolated animal value, which increased the group mean and contributed to the statistically significant dose response, but was not reproduced in other animals and did not contribute to a statistically significant increase in tail intensity at 2000 mg/kg/day compared to the vehicle control group, was of no biological relevance.

Animals treated with Acrylated amine synergist (CN3715) at all doses exhibited group mean tail intensities in the duodenum that fell within, or marginally below, the 95% reference range of the laboratory’s historical vehicle control data. There was a statistically significant increase in tail intensity at the intermediate dose (1000 mg/kg/day) group only, compared to the concurrent vehicle control; both the low dose (500 mg/kg/day) and high dose (2000 mg/kg/day) groups were not statistically significantly increased compared to the concurrent vehicle control. As all individual animal and group mean %tail intensity values fell within, or marginally below, the 95% reference range (moreover all values fell below the mean value of the laboratory’s historical vehicle control data indicate value fell towards the lower end of normal variation within the assay), and there was no evidence of a dose response, the statistical significance associated with the intermediate dose group was considered of no biological relevance.

These data were considered negative in the liver, stomach and duodenum. Therefore, no analysis of gonad slides is required.

It is concluded that, under the conditions of this comet assay, Acrylated amine synergist (CN3715) did not induce DNA strand breaks in the liver, stomach or duodenum of male Sprague Dawley rats administered up to 2000 mg/kg/day (the maximum recommended dose for in vivo comet studies).

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay = Ames test (Sarlang 2012)

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium (OECD 471). The test item was tested in two independent experiments, 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. A third mutagenicity experiment was performed with S9 mix to check the reliability of the increase in the number of revertants observed in the TA 98 strain in the second experiment. Experiments were performed according to the direct plate incorporation method except for the second and the third experiments with S9 mix, which were performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five 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).

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered to be valid. Since the test item was found freely soluble and non-cytotoxic in the preliminary test, the highest dose-level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines. No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels in any experiments, either with or without S9 mix.

In the experiments without S9 mix, no noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any dose-levels towards the five strains used. The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in any experiments without S9.

In the experiments with S9 mix, no noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any dose-levels towards the five strains used. Using the pre-incubation method, a noteworthy increase in the number of revertants was noted in the TA 98 strain at 5000 µg/plate in the second experiment. This increase was reproduced at 5000 µg/plate in the third experiment performed under the same experimental conditions (pre-incubation method). These increases exceeded the threshold of 2-fold the vehicle control (up to 2.9-fold). Moreover, the corresponding means and individual revertant colony counts obtained in both experiments were outside the historical data range of the vehicle control. Consequently, these increases were considered to be biologically relevant. The test item did not induce any other biologically significant increase in the number of revertants in the other tested strains with S9. 

The test item showed a mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium in in the presence of a metabolic activation system. In the absence of metabolic activation, the test item did not show any mutagenic activity in this bacterial mutation test.

 

In vitro mammalian cell gene mutation test = HPRT (Lloyd 2013)

Diethylamine modified ethoxylated trimethylolpropane triacrylate (CAS Number 159034-91-0) 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 Experiment 1 twelve concentrations, ranging from 0.625 to 150 µg/mL, were tested in the absence of S-9 and eleven concentrations ranging from 50 to 625 µg/mL, in the presence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 90 µg/mL in the absence of S-9 and 400 µg/mL in the presence of S-9, which gave 17% and 13% RS, respectively.

In Experiment 2 twelve concentrations, ranging from 15 to 130 µg/mL, were tested in the absence of S 9 and ten concentrations, ranging from 50 to 450 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 95 µg/mL in the absence of S-9 and 390 µg/mL in the presence of S-9, which gave 17% and 15% RS, respectively.

In the absence of S-9, significant increases in mutant frequency over the concurrent control were observed following treatment with test item at the highest concentrations analysed in Experiments 1 and 2 (90 and 95 µg/mL, respectively) and statistically significant linear trends were observed in both experiments. Although the increases were observed towards the upper limit of cytotoxicity (10-20% RS) and there was some variation between experiments in the magnitude of the induced mutagenic response, the criteria for a positive result were fulfilled in both experiments.

In the presence of S-9, no significant increases in mutant frequency were observed in Experiments 1 and 2. A statistically significant linear trend was observed in Experiment 1 but as there were no significant increases in mutant frequency at any concentration analysed in this experiment, the observation was considered not biologically relevant.

It is concluded that Diethylamine modified ethoxylated trimethylolpropane triacrylate (CAS Number 159034-91-0) induced mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in the absence of a rat liver metabolic activation system (S-9), but did not induce increases in mutant frequency in the same test system when tested up to toxic concentrations in the presence of S-9.

 

In vitro mammalian cell micronucleus test (Sarlang 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,in L5178Y TK+/- mouse lymphoma cells (OECD 487).

After two preliminary toxicity tests, 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 for 3h (and 24h recovery) with and without S9 mix. In the second experiment, cells were treated for 24 hours (and 20h recovery) without S9, and for 3 hours (and 24 h recovery) with S9 mix. Since the highest analyzable dose-level did not exhibit about 55% toxicity in both assays performed without S9 mix and since an increase in the frequency of micronucleated cells has been observed in the first experiment, a third experiment in the absence of S9 mix was undertaken using a treatment duration of 24 hour treatment + 20 hour recovery to maximize the incubation of the cells with the test item.

The test item was dissolved in dimethylsulfoxide (DMSO).

Since the test item was found cytotoxic during the preliminary tests, 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 regulations.

Following the 3-hour treatment in the first experiment (without S9 mix), a moderate to severe toxicity was noted at dose-levels = 25 µg/mL, as shown by a 44 to 100% decrease in the PD. Following the 24-hour treatment in the second experiment (without S9 mix), a slight to severe toxicity was noted at dose-levels = 25 µg/mL, as shown by a 37 to 100% decrease in the PD. Following the 24-hour treatment in the third experiment (without S9 mix) , a slight to severe toxicity was noted at all tested dose-levels, as shown by a 29 to 100% decrease in the PD.

No biologically relevant increase in the frequency of micronucleated cells was noted after the 3- or 24-hour treatments in either experiment without S9 mix.

In the first experiment with S9 mix, a slight to severe toxicity was noted at dose-levels = 100 µg/mL as shown by a 30 to 98% decrease in the PD. In the second experiment with S9 mix, a moderate to severe toxicity was noted at dose-levels >= 125 µg/mL as shown by a 56 to 100% decrease in the PD. No significant increase in the frequency of micronucleated cells reaching or exceeding the threshold of 2.5 fold the vehicle control value was noted after the 3-hour treatment in either experiment.

Diethylamine modified ethoxylated trimethylolpropane triacrylate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK +/- mouse lymphoma cells, either in the presence or in the absence of a rat metabolizing system.

in vivo alkaline comet assay (Labcorp 2022)

Diethylamine modified ethoxylated trimethylolpropane triacrylate was tested for its potential to induce DNA strand breaks in the liver, stomach and duodenum of treated rats. Following a dose-range finding study, groups of male rats were treated with doses of 500, 1000 or 2000 mg/kg of test substance in corn oil, twice at 0 and 21 hours. Tissues were sampled at 24 hours for analysis.
There were no dose-related increases in %hedgehogs in liver, stomach or duodenum, thus demonstrating that treatment with Diethylamine modified ethoxylated trimethylolpropane triacrylate did not cause excessive DNA damage that could have interfered with comet analysis.
Animals treated with Diethylamine modified ethoxylated trimethylolpropane triacrylate at all doses exhibited group mean tail intensities in the liver that fell within the 95% reference range of the laboratory’s historical vehicle control data. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, and the statistical assessment for dose response was not significant (p > 0.05).
There were two individual animal liver %tail intensity values at 2000 mg/kg/day (R0301, 2.39% and R0303, 3.52%) that exceeded the upper limit of the 95% reference range (1.80%). However, these increases above the concurrent vehicle control group and the 95% reference range were not reproduced in all animals at 2000 mg/kg/day, did not increase the group mean above the 95% reference range and did not contribute to a statistically significant increase for the group compared to the vehicle control group. None of the evaluation criteria for a positive result were met and therefore, these isolated data were considered of no biological relevance.
Animals treated with Diethylamine modified ethoxylated trimethylolpropane triacrylate at all doses exhibited group mean tail intensities in the stomach that fell within the laboratory's the 95% reference range of the laboratory’s historical vehicle control data. There were no statistically significant increases in %tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control. There was a statistically significant dose response noted, however, this response was driven predominantly by an isolated animal response at 2000 mg/kg/day (R0301). All individual animal stomach %tail intensity values fell within the 95% reference range, however the %tail intensity for animal R0301 (5.31%) was elevated compared to all other vehicle and test article dosed animals (0.08-1.01%). As it was within the 95% reference range, it fell within the normal variation of the assay and was likely induced by the mechanical processing of the tissue. Overall, it was considered that this isolated animal value, which increased the group mean and contributed to the statistically significant dose response, but was not reproduced in other animals and did not contribute to a statistically significant increase in tail intensity at 2000 mg/kg/day compared to the vehicle control group, was of no biological relevance.
Animals treated with Diethylamine modified ethoxylated trimethylolpropane triacrylate at all doses exhibited group mean tail intensities in the duodenum that fell within, or marginally below, the 95% reference range of the laboratory’s historical vehicle control data. There was a statistically significant increase in tail intensity at the intermediate dose (1000 mg/kg/day) group only, compared to the concurrent vehicle control; both the low dose (500 mg/kg/day) and high dose (2000 mg/kg/day) groups were not statistically significantly increased compared to the concurrent vehicle control. As all individual animal and group mean %tail intensity values fell within, or marginally below, the 95% reference range (moreover all values fell below the mean value of the laboratory’s historical vehicle control data indicate value fell towards the lower end of normal variation within the assay), and there was no evidence of a dose response, the statistical significance associated with the intermediate dose group was considered of no biological relevance.
It is concluded that, under the conditions of this comet assay, Diethylamine modified ethoxylated trimethylolpropane triacrylate did not induce DNA strand breaks in the liver, stomach or duodenum of male Sprague Dawley rats administered up to 2000 mg/kg/day (the maximum recommended dose for in vivo comet studies).

Justification for classification or non-classification

The Ames test and the HPRT test showed positive results in presence of metabolic activation. However, the in vitro micronucleus test showed a negative response with and without metabolic activation. A Comet Assay (in vivo) was conducted, which returned negative in the liver, stomach and duodenum.

In accordance with Regulation (EC) No 1272/2008, since the registered substance returned negative results during in vivo genotixicity studies, it does not meet the criteria for classification.