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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 1976
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines (with deviations).
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Recommended set of five strains not used; duplicate/triplicate plating not used with all strains
GLP compliance:
no
Remarks:
pre-GLP
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
Saccharomyces cerevisiae
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: strain D4
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver
Test concentrations with justification for top dose:
0.1, 1, 5 and 10 µL/plate
Vehicle / solvent:
No data
Untreated negative controls:
yes
Remarks:
solvent
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Anthramine, 2-Acetylaminofluorene, 8-Aminoquinoline, Dimethylnitrosamine
Remarks:
with metabolic activation
Untreated negative controls:
yes
Remarks:
solvent
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methylnitrosoguanidine, 2-Nitrofluorene, Quinacrine mustard
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48-72 h at 37 °C


NUMBER OF CELLS EVALUATED: 10^9


NUMBER OF REPLICATIONS: Single plate/dose, no replication
Evaluation criteria:
- Dose-related increases in mutant counts were considered as an indication of mutagenicity
- Mutant increases at only one or two doses may be significant if they occur at the higher doses
- Increases at low or intermediate concentrations followed by reduced mutant counts at higher doses may indicate that the test chemical has a narrow activity range or that the high dose levels were toxic and the induced revertant cells were killed
Statistics:
No data
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10 µL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
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
Remarks:
at 10 µL for all strains except TA-100
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Saccharomyces cerevisiae
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
INTERPRETATION OF RESULTS AND CONCLUSIONS

The test substance, was examined for mutagenic activity in a series of in vitro microbial assays employing Salmonella and Saccharomyces indicator organisms. The test substance was tested directly and in the presence of liver microsomal enzyme preparations from Aroclor-induced rats.

The following results were obtained:

A. Toxicity

The test substance was tested over a series of concentrations such that there was evidence of some chemically-induced physiological effects at the high dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The dose range employed for the evaluation of this test substance was 0.1 µL to 10 µL per plate. A considerably high toxicity level was observed at the high dose level for all strains except TA-100 and D4.

B. Non-activation Test Results

The results of the tests conducted on the test substance in the absence of a metabolic system were all negative.

C. Activation Test Results

The results of the tests conducted on the test substance in the presence of the rat liver activation system were all negative.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The substance was negative for mutagenicity (with or without metabolic activation) under the conditions of this Ames test.
Executive summary:

An Ames test was performed to determine the mutagenicity potential of SN-1738 pentaerythritol triacrylate.

Saccharomyces cerevisiae, strain D4 and Salmonella typhimurium strains TA1535, 1537, 1538, 98 and 100 were treated with the substance using the plate incorporation method at four dose levels (0.1, 1, 5 and 10 µL/plate) both with and without metabolic activation (1254 -Aroclor-induced rat liver). A considerably high toxicity level was observed at the high dose level for all strains except TA-100 and D4. Concurrent strain-specific positive and solvent controls, both with and without metabolic activation, were included in each assay.

The results of the tests conducted in the absence and presence of a metabolic activation system were negative.

In conclusion , the substance was not mutagenic under the conditions of this Ames test.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 30, 1979 to April 09, 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines (with deviations).
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Recommended set of five strains not used; duplicate/triplicate plating not used with all strains
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9
Test concentrations with justification for top dose:
0.5, 1.0, 10, 100, 500 and 1,000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Anthramine
Remarks:
with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide, 9-Aminoacridine, 2-Nitrofluorene
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h at 37 °C


NUMBER OF REPLICATIONS: Repeat test was conducted only with TA-100


NUMBER OF CELLS EVALUATED: 10^8 cells
Evaluation criteria:
Criteria for mutagenicity:
- For strains TA1535, 1537 and 1538: If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic
- For strains TA 98 and 100: If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the highest increase equal to twice-the solvent control value for TA 100 and 2-3 times the solvent control value for strain TA 98 is considered to be mutagenic

Reproducibility:
- If a chemical produces a response in a single test that cannot be reproduced in one or more additional runs and the initial positive test data loses significance.
Statistics:
No data
Key result
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
Remarks:
at 1,000 µL to TA 1537 in activation assay
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance was examined for mutagenic activity in a series of in vitro microbial assays employing Salmonella indicator organisms. The test substance was tested directly and in the presence of liver microsomal enzyme preparations from Aroclor-induced rats.

The test substance was tested over a series of concentrations such that there was either quantitative or qualitative evidence of some chemically-induced physiological effects at the high dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The dose range employed for the evaluation of this test substance was from 0.5 µg to 1,000 µg per plate. The test substance exhibited slight toxicity with the strain TA-1537 at 1,000 µg per plate dose level in the activation assay.

The results of the tests conducted on the test substance in the absence of a metabolic activation system were all negative.

The results of the tests conducted on the test substance in the presence of a rat liver activation system were all negative. A repeat test was conducted with TA-100 because of the overall increase in the number of revertants. The repeat test was negative.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The substance was negative for mutagenicity (with and without metabolic activation) under the conditions of this Ames test.
Executive summary:

An Ames test was performed to determine the mutagenic potential of pentaerythritol triacrylate.

Salmonella typhimurium strains TA1535, 1537, 1538, 98 and 100 were treated with the substance using the plate incorporation method at six dose levels (0.5, 1.0, 10, 100, 500 and 1,000 µL/plate) both with and without metabolic activation (1254 -Aroclor-induced rat liver). A considerably high toxicity level was observed at 1,000 µL for TA-1537 in the activation assay. Concurrent strain-specific positive and solvent controls, both with and without metabolic activation, were included in each assay.

 

The results of the tests conducted in the absence and presence of a metabolic activation system were negative. A repeat test was conducted with TA100 because of the overall increase in the number of revertants. The repeat test was also negative.

In conclusion, then substance was not mutagenic under the conditions of this Ames test.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines (with deviations).
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
all five recommended strains not used
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
33 - 10,000 μg/plate
Vehicle / solvent:
No data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (TA100 and TA1535), 9-aminoacridine (TA1537), and 4-nitro-o-phenylenediamine (TA98)
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Exposure duration: 20 min at 37 °C


NUMBER OF REPLICATIONS: Triplicate
Evaluation criteria:
- A positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination.
- An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity.
- A negative response is obtained when no increase in revertant colonies is observed following chemical treatment.
- There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.
Statistics:
No data
Key result
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The substance was not mutagenic (with or without metabolic activation) under the conditions of this Ames test.
Executive summary:

An Ames test was performed to determine the mutagenic potential of pentaerythritol triacrylate.

Salmonella typhimurium strains TA1535, 1537, 98 and 100 were treated with the substance using the plate incorporation method at concentration range of 33 - 10,000 µL/plate both with and without metabolic activation (S9 mix). Concurrent strain-specific positive and solvent controls, both with and without metabolic activation, were included in each assay.

The results of the tests conducted in the absence and presence of a metabolic activation system were negative.

 

In conclusion, the substance was not mutagenic under the conditions of this Ames test.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 19, 2014 to October 03, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, 10% FBS (except during 4 hour treatment), neomycin (5 μg/mL) and amphotericin B (1%). For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1:
4 h (without S9 mix): 0.0078, 0.016, 0.031*, 0.063*, 0.13*, 0.25*, 0.5*, 0.75 μg/mL
4 h (with S9 mix): 0.94, 1.9, 3.8*, 7.5*, 15.0*, 22.5*, 30.0* μg/mL

Experiment 2:
24 h (without S9 mix): 0.016, 0.031, 0.063*, 0.13*, 0.25*, 0.5*, 0.75*, 1.0 μg/mL
4 h (with S9 mix): 4.0, 8.0, 16.0*, 20.0*, 24.0*, 28.0*, 32.0* μg/mL

* these concentrations were chosen for the mutation rate analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
2-3 days after sub-cultivation stock cultures were trypsinized at 37°C for 5 minutes. Approximately 1.5×106 (single culture) and 5×102 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 h prior to treatment. After 24 h, the medium was replaced with serum-free medium containing the test substance, either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. In the second experiment, the cells were exposed to the test substance for 24 h in complete medium, supplemented with 10% FBS, in the absence of metabolic activation.

DETERMINATION OF CYTOTOXICITY
- toxicity was indicated by a reduction of the cloning efficiency.
Evaluation criteria:
A test substance was classified as positive if it induced either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test substance producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points was considered non-mutagenic in this system.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A trend was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological and statistical significance were considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentrations of the test substance. No precipitation or phase separation occurred up to the maximum concentration with and without metabolic activation following 4 and 24 h treatment.

RANGE-FINDING/SCREENING STUDIES:
The dose range of the first experiment was set according to the data generated in the pre-experiments. The dose range of the second experiment was adjustedaccording to cytotoxicity data generated in the pre-experiments (without metabolic activation) and in the first experiment with metabolic activation. The highest concentration used in the first pre-experiment was 4,240 μg/mL limited by the solubility of the test substance in DMSO and aqueous medium. Test substanceconcentrations between 33.1 μg/mL and 4,240 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation (4 and 24 h treatment). The pre-experiment was not analysable due to exceedingly severe cytotoxicity down to the lowest concentration. Therefore, a second pre-experiment was performed using concentrations from 0.5 to 60 μg/mL.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency 1 or cell density below 50% occurred in experiment 1 at 0.50 μg/mL without metabolic activation and at 22.5 μg/mL and above with metabolic activation. In experiment 2, cytotoxic effects were observed at 0.25 μg/mL and above without metabolic activation and at 20.0 μg/mL and above with metabolic activation.

The relative cloning efficiency in experiment I was within a range from 0 to 10% at the maximum analysable concentration in all experimental parts the data was judged as valid as the relative cell density at the first sub-cultivation following treatment remained at or above 10%.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.0 up to 20.7 mutants per 106 cells; the range of the groups treated with the test item was from 7.7 up to 34.5 mutants per 106 cells.

EMS (150 μg/mL) and DMBA (2.2 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:
Interpretation of results:
negative

Under the conditions of the study, the test substance did not induce gene mutations at the HPRT locus in V79 cells.
Executive summary:

A study was conducted to assess the mutagenic potential of PETIA at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments. The cells were exposed to the test substance for 4 h in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 h with and 24 h without metabolic activation.

The maximum concentration of the pre-experiment (4,240 μg/mL) was based on the solubility properties of the test substance in DMSO and aqueous medium. The concentration range of the main experiments was limited by cytotoxic effects. DMSO was used as solvent. Test concentrations were as follows:

Experiment 1:

4 h (without S9 mix): 0.0078, 0.016, 0.031*, 0.063*, 0.13*, 0.25*, 0.5*, 0.75 μg/mL

4 h (with S9 mix): 0.94, 1.9, 3.8*, 7.5*, 15.0*, 22.5*, 30.0* μg/mL

Experiment 2:

24 h (without S9 mix): 0.016, 0.031, 0.063*, 0.13*, 0.25*, 0.5*, 0.75*, 1.0 μg/mL

4 h (with S9 mix): 4.0, 8.0, 16.0*, 20.0*, 24.0*, 28.0*, 32.0* μg/mL

* These concentrations were chosen for the mutation rate analysis

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Under the conditions of the study, the test substance did not induce gene mutations at the HPRT locus in V79 cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
From August 03, 1978 to December 29, 1978
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK (Thymidine Kinase) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were maintained in Fischer's mouse leukemia medium supplemented with L-glutamine, sodium pyruvate, and horse serum (10 % by volume). Cloning medium consisted of the preceding growth medium with the addition of agar to a final concentration of 0.35 % to achieve a semisolid state. Selection medium was cloning medium containing 50 or 100 µg/mL of BrdU.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically "cleansed" against high spontaneous background: Yes
Additional strain / cell type characteristics:
other: TK+/-
Metabolic activation:
with and without
Metabolic activation system:
S9 mix. (2.4 mg NADP/mL; 4.5 mg isocitric acid/mL and 50 µL S9/mL)
Test concentrations with justification for top dose:
Experiment 1:
- Activation assay: 0.125, 0.25, 0.5, 1.0 and 2.0 nL/mL
- Nonactivation assay: 0.0156, 0.0313, 0.0625, 0.125 and 0.250 nL/mL

Experiment 2:
- Activation assay: 0.25, 0.5, 2.5, 5.0 and 25.0 nL/mL
- Nonactivation assay: 0.0156, 0.0625, 0.25, 0.375 and 0.500 nL/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation Migrated to IUCLID6: 0.5 µL/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
with metabolic activation Migrated to IUCLID6: 0.3 µL/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)


DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 or 3 d
- Selection time (if incubation with a selection agent): 10 d


SELECTION AGENT (mutation assays): 5-bromo-2'-deoxyuridine (BrdU)


NUMBER OF REPLICATIONS: Triplicate but one plate was contaminated


NUMBER OF CELLS EVALUATED: 10^6 cells/plate


DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth
Evaluation criteria:
A compound was considered mutagenic in the assay if:
- A dose-response relationship was observed over 3 of the 5 dose levels employed
- The minimum increase at the low level of the dose-response curve was at least 2.5 times greater than the solvent and/or negative control values
- The solvent and negative control data were within the normal range of the spontaneous background for the TK locus
Statistics:
Results were analysed using computer program
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 0.313 nL/mL (in non-activation assay)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Test material was tested for cytotoxicity at concentration range of 0.078-5,000 nL/mL. Toxicity was observed at 0.313 nL/mL (in non-activation assay) and concentrations ≥ 0.625 nL/mL were completely lethal to the cells within 24 h of treatment.
Remarks on result:
other: strain/cell type: TK+/-
Remarks:
Migrated from field 'Test system'.

The percent relative growth in the treated cultures was found to range from 82.3% to 16.1% without activation and from 155.8% to 13.7% with activation.

Without activation, the test substance did not induce a significant increase in mutant frequency for four of the five tested doses in the first trial. The minimum increase considered necessary to demonstrate mutagenesis at a given dose level is 2.5-fold over the average of the solvent and untreated negative control values (background frequency). An increase of approximately 3.1-fold over background was observed at the highest tested dose of 0.25 nL/mL. In order to determine the repeatability of this positive response, another nonactivation assay was performed for applied concentrations of 0.50 nL/mL to 0.0156 nL/mL. An elevated mutant frequency (about 3.5-fold over background) was observed for the two highest test concentrations (0.375 nL/mL and 0.50 nL/mL). The toxicity associated with a given applied concentration was variable between the two trials, but the mutagenic activity in both trials was asssociated with treatments that gave a percent relative growth in the 16.1% to 23.1% range. Therefore, the test substance was mutagenic for highly toxic treatments.

With activation, the first assay showed no evidence for mutagenic activity for treatments that did not cause a percent relative growth less than about 75% (1.0 to 2.0 nL/mL dose range). The second trial extended the applied concentration range to 25.0 nL/mL, which reduced the percent relative growth to 13.7%. At this highly toxic dose, the mutant frequency was substantially elevated (about 7.2-fold) over the background frequency. The other applied concentrations of 5.0 nL/mL and less were not demonstrably toxic and caused no increase in mutant frequency. Thus, the test substance was mutagenic only for a highly toxic treatment, as in the nonactivation assay, but higher applied concentrations were necessary to achieve the same toxic action in the presence of the S9 metabolic activation mix.

Validity Criteria:

The cloning efficiencies for the assays ranged from 88% to 114% without activation and from 78% to 118% with activation, which demonstrated excellent culturing conditions for the assays. The negative (solvent and untreated) control mutant frequencies were all within the normal ranges for the nonactivation and activation tests, and the positive control compounds yielded frequencies in the normal range that were greatly in excess of the negative control values.

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
positive without metabolic activation

Under the conditions of the study, the substance was active in the mouse lymphoma forward mutation assay both with and without metabolic activation.
Executive summary:

A study was conducted to assess the mutagenic potential of pentaerythritol triacrylate using the mouse lymphoma forward mutation assay.

 

L5178Y TK +/- mouse lymphoma cells were treated at concentration range of 0.078 - 5,000 nL/mL. Toxicity was observed at 0.313 nL/mL (in non-activation assay) and concentrations ≥ 0.625 nL/mL were completely lethal to the cells within 24 h of treatment. The mutation assay was performed at concentration range of 0.0039 - 2 nL/mL with vehicle (solvent, DMSO) and positive controls (ethylmethane sulfonate ordimethylnitrosamine)using 4-h exposure groups in the presence or absence of metabolic activation. Thymidine analog 5-bromo-2'-deoxyuridine (BrdU) was used as the selection agent. Two additional mutation assays were performed: one to extend the activation conditions to higher concentrations of the test compound and the other to confirm the results of the non-activation assay in the first trial.

 

The substance induced an increase in mutations at the TK locus in L5178Y mouse lymphoma cells at concentrations of 0.25 - 0.5 nL/mL without activation and at 25.0 nL/mL with activation. The mutagenic activity was associated with highly toxic treatments and higher applied concentrations were required to achieve the same toxicity in the presence of microsomal activation mix.

 

In conclusion, under the conditions of the study, the test substance was active in the mouse lymphoma forward mutation assay.

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996 to 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines in compliance with GLP. Pentaerythritol triacrylate is a constituent of PETIA.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Method: MacGregor JT et al (1990)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Source: Taconic Laboratory Animals and Service (Germantown, NY)
- Age at study initiation: 6 weeks
- Assigned to test groups randomly: Yes
- Housing: Individually housed in polycarbonate cages
- Diet (e.g. ad libitum): NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum, changed weekly
- Water (e.g. ad libitum): Tap water (Columbus municipal supply), ad libitum
- Acclimation period: 11-15 d


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ± 3 °F
- Humidity (%): 50 ± 15 %
- Air changes (per h): 10/h
- Photoperiod (h dark / h light): 12 h dark / 12 h fluorescent light
Route of administration:
dermal
Vehicle:
- Vehicle(s)/solvent(s) used: Acetone
Details on exposure:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 mL/kg bw
Duration of treatment / exposure:
3 months
Frequency of treatment:
5 days per week for 14 weeks
Post exposure period:
No data
Remarks:
Doses / Concentrations:
0.75, 1.5, 3, 6 and 12 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control(s):
No data
Tissues and cell types examined:
Peripheral blood samples
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES: Peripheral blood samples were obtained from male and female mice at the end of the study

DETAILS OF SLIDE PREPARATION: Smears were immediately prepared and fixed in absolute methanol. The methanol fixed slides were stained with acridine orange and coded.

METHOD OF ANALYSIS: Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) per animal. In addition, the normochromatic/polychromatic erythrocyte (NCE/PCE) ratio in 1,000 total erythrocytes per animal was determined to provide a measure of chemical-induced bone marrow toxicity.
Evaluation criteria:
No data
Statistics:
- Results were analyzed by a statistical software package
- Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed Cochran-Armitage trend test (significant at P ≤ 0.025), followed by Pairwise comparison with the vehicle controls (significant at P ≤ 0.005)
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): See Table 1
- Ratio of NCE/PCE (for Micronucleus assay): See Table 1

Table 1: Frequency of micronuclei in peripheral blood normochromatic erythrocytes of B6C3F1 mice following dermal application of pentaerythritol triacrylate for 3 monthsa

 

Compound

Dose (mg/kg)

Number of Mice with Erythrocytes Scored

Micronucleated NCEs/ 1,000 NCEsb

P Valuec

NCEsb(%)

Male

Acetoned

 

10

1.20 ± 0.21

 

98.2 ± 0.1

Pentaerythritol triacrylate

 

 

 

 

 

0.75

10

0.75 ± 0.15

0.9253

98.1 ± 0.1

1.5

10

1.00 ± 0.15

0.7269

98.4 ± 0.1

3

10

0.90 ± 0.19

0.8229

98.3 ± 0.1

6

10

1.05 ± 0.22

0.6727

98.4 ± 0.1

12

10

1.05 ± 0.16

0.6727

98.2 ± 0.1

 

 

P=0.394e

 

 

Female

Acetone

 

9

0.72 ± 0.15

 

98.2 ± 0.1

Pentaerythritol triacrylate

 

 

 

 

 

0.75

10

0.65 ± 0.15

0.606

98.1 ± 0.1

1.5

9

0.78 ± 0.19

0.4237

98.3 ± 0.1

3

10

0.65 ± 0.17

0.606

98.3 ± 0.1

6

10

0.90 ± 0.18

0.2722

98.3 ± 0.1

12

10

0.85 ± 0.11

0.329

98.4 ± 0.1

 

 

P=0.210

 

 

  

aStudy was performed at SITEK Research Laboratories, Inc.

NCE=normochromatic erythrocyte

bMean ± standard error

cPairwise comparison with the vehicle controls, significant at P ≤ 0.005

dVehicle control

eSignificance of micronucleated NCEs/1,000 NCEs tested by the one-tailed Cochran-Armitage trend test, significant at P ≤ 0.025

 

 

 

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this mouse micronucleus assay, the test substance was negative for mutagenic activity.
Executive summary:

A study was conducted to determine the mutagenicity potential of PETIA in the mouse peripheral blood micronucleus test.

The substance (0.75 - 12 mg/kg bw) was administered dermally to male and female B6C3F1 mice for 3 months. Peripheral blood samples were obtained at the end of the study and smears slides were immediately prepared and scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) per animal. In addition, the normochromatic erythrocytes/polychromatic erythrocyte (NCE/PCE) ratio in 1,000 total erythrocytes per animal was determined to provide a measure of chemical-induced bone marrow toxicity.

 

No increase in the frequency of micronucleated NCEs and NCE/PCE ratios were observed in peripheral blood samples from the male or female B6C3F1 mice.

Under the conditions of the study, the test substance was considered to be negative for mutagenic activity.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
1996-1999
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines in compliance with GLP. Pentaerythritol triacrylate is a constituent of PETIA.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Method: MacGregor JT et al (1990)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Tg.AC hemizygous mice, genetically modified for sensitive skin (specific phenotype for the induction of papillomas by 12-O-tetradecanoyl-phorbol-13-acetate)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Source: Taconic Laboratory Animals and Service (Germantown, NY)
- Age at study initiation: 6 weeks
- Assigned to test groups randomly: Yes
- Housing: Individually housed in polycarbonate cages
- Diet (e.g. ad libitum): NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum, changed weekly
- Water (e.g. ad libitum): Tap water (Columbus municipal supply), ad libitum
- Acclimation period: 11-15 d


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ± 3 °F
- Humidity (%): 50 ± 15 %
- Air changes (per h): 10/h
- Photoperiod (h dark / h light): 12 h dark / 12 h fluorescent light
Route of administration:
dermal
Vehicle:
- Vehicle(s)/solvent(s) used: Acetone
Details on exposure:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 3.3 mL/kg bw
Duration of treatment / exposure:
6 months
Frequency of treatment:
5 days per week for 27 weeks
Post exposure period:
No data
Remarks:
Doses / Concentrations:
0.75, 1.5, 3, 6 and 12 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Positive control(s):
No data
Tissues and cell types examined:
Peripheral blood samples
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES: Peripheral blood samples were obtained from male and female mice at the end of the study.

DETAILS OF SLIDE PREPARATION: Smears were immediately prepared and fixed in absolute methanol. The methanol fixed slides were stained with acridine orange and coded.

METHOD OF ANALYSIS: Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) per animal. In addition, the normochromatic/polychromatic erythrocyte (NCE/PCE) ratio in 1,000 total erythrocytes per animal was determined to provide a measure of chemical-induced bone marrow toxicity.
Evaluation criteria:
No data
Statistics:
- Results were analyzed by a statistical software package
- Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed Cochran-Armitage trend test (significant at P ≤ 0.025), followed by Pairwise comparison with the vehicle controls (significant at P ≤ 0.005)
Sex:
female
Genotoxicity:
other: Significant increase in frequency of micronucleated NCEs in peripheral cell blood samples of genetically modified Tg.AC female mice
Toxicity:
no effects
Sex:
male
Genotoxicity:
other: Equivocal response in genetically modified Tg.AC male mice
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): See Table 1
- Ratio of NCE/PCE (for Micronucleus assay): See Table 1

Table 1: Frequency of micronuclei in peripheral blood normochromatic erythrocytes of Tg.AC hemizygous mice following dermal application of pentaerythritol triacrylate for 6 monthsa

  

Compound

Dose (mg/kg)

Number of Mice with Erythrocytes Scored

Micronucleated NCEs/ 1,000 NCEsb

P Valuec

PCEs(%)

Male

Acetoned

 

12

0.58 ± 0.17

 

3.3

Pentaerythritol triacrylate

 

 

 

 

 

0.75

 

 

0.6585

 

1.5

14

0.50 ± 0.14

0.4076

2.2

3

15

0.63 ± 0.16

0.2511

2.1

6

15

0.73 ± 0.16

0.0694

2.1

12

12

0.96 ± 0.17

0.0206

11.7*

 

10

P=0.001e

 

 

Female

Acetone

 

12

0.75 ± 0.21

 

2.2

Pentaerythritol triacrylate

 

 

 

 

 

0.75

 

 

0.7344

2

1.5

14

0.61 ± 0.16

0.3154

2.2

3

12

0.88 ± 0.21

0.1428

2.5

6

12

1.04 ± 0.13

0.0001

9.8*

12

13

1.96 ± 0.31

0.7344

7.3*

 

 

P ≤ 0.001

 

 

  

*Significantly increased over the vehicle control.

aStudy was performed at SITEK Research Laboratories, Inc.

NCE=normochromatic erythrocyte

bMean ± standard error

cPairwise comparison with the vehicle controls, significant at P ≤ 0.005

dVehicle control

eSignificance of micronucleated NCEs/1,000 NCEs tested by the one-tailed Cochran-Armitage trend test, significant at P ≤ 0.025

Conclusions:
Interpretation of results (migrated information): ambiguous
The outcome of the present study is equivocal. The positive results seen in female Tg.AC mice treated for 6 months indicate a potential for a genotoxic mode of action. This was however not clearly confirmed in male mice. Therefore, the available evidence does not permit classification of the substance as either genotoxic or non-genotoxic.
Executive summary:

A study was conducted to determine the mutagenicity potential of PETIA in the mouse peripheral blood micronucleus test.

The substance (0.75 - 12 mg/kg bw) was administered dermally to male and female genetically modified Tg.AC hemizygous mice for 6 months. Peripheral blood samples were obtained at the end of the study and smears slides were immediately prepared and scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) per animal. In addition, the normochromatic erythrocytes/polychromatic erythrocyte (NCE/PCE) ratio in 1,000 total erythrocytes per animal was determined to provide a measure of chemical-induced bone marrow toxicity.

 

Treatment of Tg.AC hemizygous female mice induced a significant increase in micronucleated NCEs. In Tg.AC hemizygous male mice, a small increase in micronucleated NCEs was detected; however, the response was judged to be equivocal, due to a positive trend (p = 0.001) without any one dose group being significantly elevated over the vehicle control frequency. The NCE/PCE ratios in peripheral blood of Tg.AC mice were significantly altered at 6 and 12 mg/kg bw. Male and female mice in these dosed groups had markedly elevated levels of immature PCEs in their blood, implying a stimulation of erythropoiesis, which was maybe in response to pentaerythritol triacrylate-induced toxicity.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 31, 2017 to May 29, 2018
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 (incl. QA statement)
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test system
Wistar WI (Han) rats (SPF) were used as the test system. These rats are recommended by international guidelines (e.g. OECD, EC). Females were nulliparous and non-pregnant. The animals were provided by Charles River, Sulzfeld, Germany. Young adult animals were selected (6-8 weeks old at the start of treatment). The total number of animals used in the dose-range finding study was 13 and in the main study 84. In the main study 5 male or 5 female rats were treated per sampling time in each treatment group. At the highest dose 3 additional animals were used to compensate for possible deaths. The body weights of the rats at the start of the treatment on day 1 in the main study were within 20% of the sex mean. The mean body weights were for males 142 ± 5 g and 164 ± 9 g (initials and repeat assay) and for females 120 ± 7 g and the range for males 133 – 152 g and 143 – 174 g (initial and repeat assay) and for females 109 - 136 g. The rats were identified by a unique number on the tail written with a marker pen. The animals were allocated at random to the treatment groups. The acclimatization period was at least 6 days before the start of treatment under laboratory conditions. On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.

Husbandry

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 19 to 21°C with an actual daily mean relative humidity of 48 to 65%. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Accommodation
Group housing of maximum 5 animals per sex in labeled Macrolon cages (type MIV height 180 mm, length 600 mm and width 330 mm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

Diet
The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.

Water
The animals had free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived. Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.
Route of administration:
oral: gavage
Vehicle:
The vehicle of the test substance was Arachid oil (Sigma Aldrich, Steinheim, Germany). The solubility of the test substance in arachid oil was in the range of 25 to 75 mg/mL. The test substance stability for at least 6 hours at room temperature protected from light confirmed over the concentration range 25 to 75 mg/mL.
Details on exposure:
The rats were dosed for three consecutive days (once daily) with the test substance by oral gavage (oral intubation with a plastic gavage needle) of a maximum tolerated (high), an intermediate and a low dose of the test substance. The rats were dosed twice with the positive control EMS by oral gavage. A limited quantity of food was supplied during the night before dosing (approximately 7 g/rat). The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use. The first dose of the test substance and vehicle was administered at t=0 h. The second and third dose were administered at approximately t=24 h (± 2 h) and t=45 h (± 2 h), respectively. The positive control was administered at t=24 h (± 2 h) and t=45 h (± 2 h). The animals were sacrificed 3-6 hours after the second dose for the positive control EMS and after the third dose of the vehicle and test substance. The animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia.
Duration of treatment / exposure:
Three consecutive days
Frequency of treatment:
Once daily
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
males
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
males
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
males
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
females
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
females
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
females
No. of animals per sex per dose:
In total 5 treatment groups were used, each consisting of 5 animals per sexes.
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl Methane Sulfonate (EMS)
Tissues and cell types examined:
Approximately 3-4 hours after the third treatment with the test compound or vehicle and second treatment with EMS Liver, Duodenum and Stomach were collected/isolated and examined for DNA damage with the alkaline Comet assay.
Details of tissue and slide preparation:
Preparation of Slides
To 20 µL of the cell suspension, 280 µL melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added. The cells were mixed with the LMAgarose and 50 µL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Two slides per tissue were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 19 – 54 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

Sampling, fixation and storage of tissue for histotechnology and histopathology
Part of the liver, stomach and duodenum from the animals (with exception of the positive control) used in the repeat experiment (in case of duodenum after isolation of a part for the comet assay) were collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was needed.

In-Life Procedures, Observations and Measurements
Animals were checked for mortality at least twice a day. The time of death was recorded as precisely as possible. The systemic toxic signs were recorded at least once a day from start of treatment onwards. All signs were recorded. The animals were weighed just prior to dosing.

Lysis, Electrophoresis and Staining of the Slides
The cells on the slides were overnight (approximately 20 h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 10 – 49 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 1 Volt/cm. The electrophoresis was performed for 20 – 30 minutes under constant cooling (actual temperature 5 – 6 °C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in 70% ethanol and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark.

Comet Scoring
To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample. On a few slides, one of the agarose circles was damaged, therefore an agarose circle from the second backup slide was used for scoring.

The following criteria for scoring of Comets were used:
• Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.
In the repeat assay, in addition the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal in the repeat experiment. The occurrence of hedgehogs was noted in all treatment groups and the control.
Evaluation criteria:
Acceptability criteria
The in vivo comet is considered acceptable if it meets the following criteria:
a) The percentage tail intensity of the solvent control should reasonably be within the laboratory historical control data range.
b) The positive control EMS should produce at least a statistically significant (one-sided, p< 0.05) increase in the percentage Tail Intensity compared to the negative control treated animals.
c) Adequate numbers of cells and doses have been analysed as indicated in the OECD guideline.
d) The criteria for the selection of highest dose are consistent with those described OECD guideline.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis. All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
Statistics:
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data:
A test substance is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test substance is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Dose-range Finding Study

In the dose range finding test, daily dosing was done for 3 consecutive days. For the first trial, one male and one female animal were dosed with 2000 mg PETIA /kg body weight. Both animals died on day 3. Consequently, one male and one female animal were dosed with 1500 mg/kg body weight. The male animal died on day 3 and the female animal died the day after the third dosing. Therefore one male and one female animal were dosed with 1000 mg/kg body weight. The male animal died on day 3 and the female animal showed clinical symptoms from the second dosing on but was recovered on the day after the third dosing. Additionally, one male and one female animal were dosed with 500 mg/kg body weight. It appeared that only slight clinical symptoms were observed after the third dosing. Two additional female animals were dosed with 1000 mg/kg body weight to verify if this could be used as high dose. Clinical signs were observed, but all animals survived. For the male animals one additional animal was dosed with 750 mg/kg body weight, this animal showed clinical symptoms after the second dosing. Two additional male animals were dosed with 750 mg/kg body weight but these animals died on day 2. A dose of 750 mg/kg body weight was therefore considered to be higher than the maximum tolerated dose for male animals. Since substantial differences were observed between sexes, the main study has been performed with male and female animals.

In vivo Comet Main Study

Based on the results of the dose range finding study a dose level of 500 and 1000 mg/kg body weight were the highest dose level tested for male and female animals, respectively. In the main study male and female animals were dosed three times by oral gavage with vehicle or with 125, 250 and 500 mg or 250, 500 and 1000 mg PETIA per kg body weight in males and females, respectively. Five animals were used in each treatment group.

Mortality and Clinical Signs  

Males

The animals of the groups treated with 250 and 125 mg test substance/kg bw and the animals of the negative and positive control groups showed no clinical signs or mortality. In the initial assay, within the first hour after the third dosing all animals of the group treated with 500 mg/kg bw showed a hunched posture, two animals suffered of diarrhea and one of these two animals also had a rough coat and was lethargic. In the repeat assay, after the third dosing all animals of the group treated with 500 mg/kg bw showed diarrhea, 4 animals showed a hunched posture and one of these two animals also had a rough coat.  

Females

The animals of the groups treated with 500 and 250 mg test substance/kg bw and the animals of the negative and positive control groups showed no clinical signs or mortality. In the high dose group (1000 mg test substance/kg bw), one animal died approximately 5 hours after the first dose. One animal died within 1 day after the first dose, the remaining animals did not completely eat the provided food. Within two hours after the second dosing all animals were lethargic and had a rough coat, four animals showed also an hunched posture and one of these animals showed ataxia in addition (this animal died 5 hours after the second dose). Two additional animals died within one day after the second dose. The surviving three animals showed lethargy, a rough coat and hunched posture one day after the second dose, two of these animals had also diarrhea. In the group of female rats dosed with 1000 mg/kg test substance per kg/bw only 3 out of the 8 animals survived. This indicates that this dose was above the maximum tolerated dose and therefore these animals were excluded from analysis. This was different from the dose-range finding study since no animals died at this dose. Values obtained during scoring are not presented in the report, but are kept in the raw data. Consequently the MTD in male and female animals was comparable and no additional experiment in female animals was needed.

Comet Slide Analysis

After treatment, single cell suspensions from the Liver, Duodenum and Stomach were prepared. Comet slides were prepared and analyzed.

Liver

The mean Tail Intensity in Liver cells of vehicle-treated rats was 7.1% and 0.61% in male and female animals respectively and within the historical control data range. The positive control EMS induced the Tail Intensity significantly in both sexes. Hence, both criteria for an acceptable assay were met. No statistically significant increase in the mean Tail Intensity (%) was observed in Liver cells of test substance treated male animals compared to the vehicle treated animals. In female animals a statistically significant increase was present. However, as this was inside the historical control data of the negative control this was not biologically relevant.

Duodenum

In a first assay, the mean Tail Intensity in Duodenum cells of male vehicle-treated rats was outside the historical control range and indicates that the DNA from Duodenum in vehicle-treated rats was already damaged. Therefore the assay with males was repeated. For the female animals, the mean Tail Intensity in Duodenum cells vehicle-treated rats was 18.5%. This mean value is close to the historical negative control data and no significant increases were observed in Duodenum cells of the test substance treated female animals compared to the vehicle treated animals. The positive control EMS was significantly increased. Hence, both criteria for an acceptable assay were met. In the second assay with male animals, the mean Tail Intensity in Duodenum cells vehicle-treated rats was 1.59%. This mean value is within the historical negative control data. The positive control EMS was significantly increased. Hence, both criteria for an acceptable assay were met. No statistically significant increase in the mean Tail Intensity (%) was observed in Duodenum cells of the test substance treated animals compared to the vehicle treated female animals in the first assay and male animals in the second assay.

Stomach

The mean Tail Intensity in Stomach cells of vehicle-treated rats was 22% and 18% in male and female animals respectively. The positive control EMS was significantly increased. Hence, both criteria for an acceptable assay were met. No statistically significant increase in the mean Tail Intensity (%) was observed in Stomach cells of the test substance treated animals compared to the vehicle treated animals.

Chemical analysis

Since initially substantial differences were observed between sexes, the study was performed with male (Groups A, B, C and D; analysis date 28 Nov 2017) and female (Groups F, G, H and I; analysis date 09 Jan 2018) animals.

Accuracy

The concentrations analyzed in the formulations of Group B, Group D, Group G, Group, Group H and Group I were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). For the formulation of Group C, the mean accuracy was below the target concentration (i.e. 65% of target). As only the middle dose did not fulfil the criteria, the required top-dose was reached and study outcomes were not affected. A small response at the retention time of the test item was observed in the chromatograms of the Group F formulation. The maximum contribution to the formulation with the lowest analysed concentration was 0.078%. It was considered that it has no significant effect on the results of the formulation. A small response at the retention time of the test item was also observed in the chromatograms of the Group A formulation. The maximum contribution to the formulation with the lowest analysed concentration was 0.095%. It was considered that it has no significant effect on the results of the formulation.

Homogeneity

The formulations of Group B, Group D, Group G and Group I were homogeneous (i.e. coefficient of variation ≤ 10%).

Stability

The formulation of Group G was not stable when stored at room temperature protected from light for at least 4 hours, however this group was excluded from the analysis and the study results were therefore not affected. The formulations of Group I was stable when stored at room temperature protected from light for at least 4 hours.

Mortality and Toxic Signs after Treatment with PETIA in the Dose-range Finding Study

 

 

 

 

 

 

Toxic signs*

 

 

 

Group

Sex

AnimalNumber

Dose mg/kg

day 1 within

4 hours after dosing

 

day 2

day 2within

4 hours afterdosing

 

day 3within

4 hours afterdosing

 

 

 

day 3

day 4

 

A

 

Male

 

1

 

2000

 

B

 

B

 

FNJ

 

A

 

 

A

Female

2

2000

B

B

FNJ

A

 

 

B

Male

3

1500

B

B

N

A

 

 

B

Female

4

1500

B

B

NJ

NJ

FNJ

A

C

Male

5

1000

B

B

FJ

A

 

 

C

Female

6

1000

B

B

FJ

J

NJ

B

D

Male

7

500

B

B

B

B

NJ

 

D

Female

8

500

B

B

B

B

J

 

E

Male

9

750

B

B

J

NJ

FCNJ

 

C

Female

10

1000

B

B

J

NJ

FCNJ

 

C

Female

11

1000

B

B

J

FCNJ

FCNJ

 

E

Male

12

750

B

A

 

 

 

 

E

Male

13

750

B

A

 

 

 

 

* Legend 'Mortality and toxic signs':

A = died; B = showed no abnormalities; C= Ataxia, F = lethargy; J = hunched posture; N = rough coat

Mortality and Toxic Signs after Treatment with PETIA in Main Study

Toxic signs*

Group

Sex

AnimalNumber

Dose mg/kg

day 1 within

4 hours after dosing

 

day 2

day 2 within

4 hours after dosing

 

 

 

day 3

 

B

 

Male

 

19

 

500

 

B

 

B

 

B

 

J

B

Male

20

500

B

B

B

J

B

Male

21

500

B

B

B

F, N, J, U

B

Male

22

500

B

B

B

J

B

Male

23

500

B

B

B

U, J

B1

Male

39

500

B

B

B

J

B1

Male

40

500

B

B

B

J

B1

Male

41

500

B

B

B

J

G

Female

44

1000

B

B

F, N, J

F, N, J

G

Female

45

1000

B

B

F, N, J

A

G

Female

46

1000

B

B

F, N, J, C

A

G

Female

47

1000

B

A

 

 

G

Female

48

1000

B

A

 

 

G1

Female

64

1000

B

B

F, N, J

A

G1

Female

65

1000

B

B

F, N

F, N, J, U

G1

Female

66

1000

B

B

F, N

F, N, J, U

Repeat test

B

Male

69

500

B

B

B

U

B

Male

70

500

B

B

B

U

B

Male

71

500

B

B

B

J, U

B

Male

72

500

B

B

B

J, U

B

Male

73

500

B

B

B

N, J, U

B1

Male

89

500

B

B

B

U

B1

Male

90

500

B

B

B

J, U

B1

Male

91

500

B

B

B

U

Only animals showing clinical signs are presented

* Legend 'Mortality and toxic signs':

A = died; B = showed no abnormalities; C= Ataxia, F = lethargy; J = hunched posture; N = rough coat, U = diarrhea

1Additional animals to compensate for possible deaths. It was not needed to use these animals. They may have the same numbers as animals that were part of the evaluated animals. As the 3 experiments were run separately, no mixing was possible.

Mean Body Weight of Male Rats Immediately Prior to Dosing with PETIA and EMS

 

Group code

Dose (mg/kg/bw)

Day 1

Body weight (Mean ± S.D.)

Day 2

Body weight (Mean ± S.D.)

Day 3

Body weight (Mean ± S.D.)

A

0

139

±

4.1

142

±

3.9

149

±

4.1

B

500

141

±

3.8

144

±

6.9

145

±

7.0

C

250

144

±

4.5

148

±

2.4

153

±

3.5

D

125

143

±

7.0

145

±

4.7

150

±

6.3

E

200 (EMS)

1

±

 

147

±

7.2

147

±

7.8

Not dosed on day 1. Dosing with EMS was started on Day 2

 

Mean Body Weight of Female Rats Immediately Prior to Dosing with PETIA and EMS

 

Group code

Dose (mg/kg/bw)2

Day 1

Body weight (Mean ± S.D.)

Day 2

Body weight (Mean ± S.D.)

Day 3

Body weight (Mean ± S.D.)

F

0

119

±

5.3

123

±

5.0

125

±

5.4

H

500

116

±

6.0

122

±

7.3

121

±

4.7

I

250

121

±

6.4

125

±

7.5

126

±

9.1

J

200 (EMS)

1

±

 

120

±

4.5

122

±

4.2

Not dosed on day 1. Dosing with EMS was started on Day 2

2 Group G is excluded from analysis.

Overview Tail Intensity in Liver Cells of Male Rats

 

Tail Intensity (%)

S.D.

Vehicle1

7.05

3.06

PETIA 125 mg/kg

1.67

0.27

PETIA 250 mg/kg

1.56

0.54

PETIA 500 mg/kg

2.07

0.76

EMS 200 mg/kg

87.89

1.80

1 Based on 4 rats due to an outlier.


Overview Tail Intensity in Duodenum Cells of Male Rats

 

Tail Intensity (%)

S.D.

Vehicle

1.59

0.54

PETIA 125 mg/kg

1.74

1.16

PETIA 250 mg/kg

2.30

0.46

PETIA 500 mg/kg

1.98

0.82

EMS 200 mg/kg

46.37

6.49

 

Overview Tail Intensity in Stomach Cells of Male Rats

 

Tail Intensity (%)

S.D.

Vehicle

21.92

8.31

PETIA 125 mg/kg

16.20

4.86

PETIA 250 mg/kg

16.72

4.60

PETIA 500 mg/kg

25.94

8.90

EMS 200 mg/kg

67.54

2.31

Overview Tail Intensity in Liver Cells of Female Rats

 

Tail Intensity (%)

S.D.

Vehicle

0.61

0.22

PETIA 250 mg/kg

1.51

0.42

PETIA 500 mg/kg

1.27

0.60

EMS 200 mg/kg

97.96

0.70

 

Overview Tail Intensity in Duodenum Cells of Female Rats

 

Tail Intensity (%)

S.D.

Vehicle

18.50

7.30

PETIA 250 mg/kg

28.12

15.60

PETIA 500 mg/kg

30.95

14.87

EMS 200 mg/kg

61.78

8.21

 

Overview Tail Intensity in Stomach Cells of Female Rats

 

Tail Intensity (%)

S.D.

Vehicle

18.07

12.17

PETIA 250 mg/kg

38.17

18.32

PETIA 500 mg/kg

32.64

8.19

EMS 200 mg/kg

75.86

1.67

Historical data Comet assay Negative control

 

Liver Tail Intensity (%) Males and Females

Duodenum Tail Intensity (%)

Males and Females

Stomach Tail Intensity (%) Males and Females

Mean

6.10

7.11

9.42

SD

7.15

7.64

10.76

n

178

20

25

Lower control limit

(95% control limits)

-2.74

-3.95

-6.31

Upper control limit

(95% control limits)

14.94

18.17

25.16

SD = Standard deviation

n = Number of observations

 

Historical control data from experiments performed in Feb 2012 – May 2018

 

Historical data Comet assay (200 mg/kg EMS orally dosed for two consecutive days)

 

Liver Tail Intensity (%) Males and Females

Duodenum Tail

Intensity (%) Males and Females

Stomach Tail Intensity (%) Males and Females

Mean

86.73

43.65

53.65

SD

15.23

20.48

16.90

n

166

20

25

Lower control limit

(95% control limits)

72.38

13.07

36.64

Upper control limit (95% control limits)

101.09

74.24

70.65

SD = Standard deviation

n = Number of observations

 

Historical control data from experiments performed in Feb 2012 – May 2018

Conclusions:
Under the study conditions, the test substance is not genotoxic in the Comet assay in Liver, Duodenum and Stomach cells when sampled approximately 3-4 hours post dosing, of male and female rats that were dosed via oral gavage for three consecutive days up to the maximum tolerated dose.
Executive summary:

A study was conducted to determine the potential genotoxicity of the test substance when administered to rats at the maximum recommended dose, by measuring the increase in DNA strand breaks in Liver, Duodenum and Stomach according to OECD Guideline 489. The test substance was suspended in Arachid oil. Since, initially differences were observed between sexes, the study was performed with male and female animals. Based on the results of the dose range finding study a dose level of 500 and 1000 mg/kg bw were selected as the highest dose level tested for male and female animals, respectively. The concentrations analyzed in the formulations of all groups were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%), except for the formulation of Group C, for which the mean accuracy was below the target concentration (i.e. 65% of target). Group C represents the middle dose for males. As the required top-dose was well according to the target concentration, the study outcome was not affected. In the main study, male and female animals were dosed three times by oral gavage with vehicle or with 125, 250 and 500 mg or 250, 500 and 1000 mg kg/bw of the test substance in males and females, respectively, for three consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS)/kg bw. In total 5 treatment groups were used, each consisting of 5 animals per sexes. The female animals dosed with 1000 mg/kg showed the following toxic signs after dosing: diarrhea, lethargy, rough coat and a hunched posture. In this group only 3 out of the 8 animals survived. It was concluded that this dose was above the maximum tolerated dose. Therefore this group was excluded from the analysis. Consequently the MTD in male and female animals was comparable. Approximately 3-4 hours after the second dose of EMS and third dose of the vehicle or the test substance, Liver, Duodenum and Stomach were collected. The animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia and Liver, Duodenum and Stomach were isolated. Single cell suspensions from Liver, Duodenum and Stomach were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed. No statistically significant increase in the mean Tail Intensity (%) was observed in Liver cells of test substance treated male animals compared to the vehicle treated animals. In female animals a statistically significant increase was present. However, as this was inside the historical control data of the negative control this was not biologically relevant. No statistically significant increase in the mean Tail Intensity (%) was observed in Duodenum cells of the test substance treated animals compared to the vehicle treated female and male animals. No statistically significant increase in the mean Tail Intensity (%) was observed in Stomach cells of the test substance treated animals compared to the vehicle treated animals. Under the study conditions, the test substance is not genotoxic in the Comet assay in Liver, Duodenum and Stomach cells when sampled approximately 3-4 hours post dosing, of male and female rats that were dosed via oral gavage for three consecutive days up to the maximum tolerated dose (Eurlings, 2018).

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

Additional information

In vitro

In vitro gene mutation studies in bacteria

An Ames test was performed to determine the mutagenic potential of the test substance. Salmonella typhimurium strains TA1535, 1537, 98 and 100 were treated with the substance using the plate incorporation method at concentration range of 33 - 10,000 µL/plate both with and without metabolic activation (S9 mix). Concurrent strain-specific positive and solvent controls, both with and without metabolic activation, were included in each assay. The results of the tests conducted in the absence and presence of a metabolic activation system were negative. In conclusion, the substance was not mutagenic under the conditions of this Ames test (Zeiger et al., 1987).

An Ames test was performed to determine the mutagenic potential of C-78 / SN-2074. Salmonella typhimurium strains TA1535, 1537, 1538, 98 and 100 were treated with the substance using the plate incorporation method at six dose levels (0.5, 1.0, 10, 100, 500 and 1,000 µL/plate) both with and without metabolic activation (1254-Aroclor-induced rat liver). A considerably high toxicity level was observed at 1,000 µL for TA-1537 in the activation assay. Concurrent strain-specific positive and solvent controls, both with and without metabolic activation, were included in each assay. The results of the tests conducted in the absence and presence of a metabolic activation system were negative. A repeat test was conducted with TA100 because of the overall increase in the number of revertants. The repeat test was also negative. In conclusion, then substance was not mutagenic under the conditions of this Ames test (Jaganath, 1979).

An Ames test was performed to determine the mutagenicity potential of SN-1738. Saccharomyces cerevisiae strain D4 and Salmonella typhimurium strains TA1535, 1537, 1538, 98 and 100 were treated with the substance using the plate incorporation method at four dose levels (0.1, 1, 5 and 10 µL/plate) both with and without metabolic activation (1254-Aroclor-induced rat liver). A considerably high toxicity level was observed at the high dose level for all strains except TA-100 and D4. Concurrent strain-specific positive and solvent controls, both with and without metabolic activation, were included in each assay. The results of the tests conducted in the absence and presence of a metabolic activation system were negative. In conclusion, the substance was not mutagenic under the conditions of this Ames test (Brusick, 1976).

In vitro gene mutation studies in mammalian cells

A study was conducted to assess the mutagenic potential of the test substance using the mouse lymphoma forward mutation assay. L5178Y TK +/- mouse lymphoma cells were treated at concentration range of 0.078 - 5,000 nL/mL. Toxicity was observed at 0.313 nL/mL (in non-activation assay) and concentrations ≥ 0.625 nL/mL were completely lethal to the cells within 24 h of treatment. The mutation assay was performed at concentration range of 0.0039 - 2 nL/mL with vehicle (solvent, DMSO) and positive controls (ethylmethane sulfonate ordimethylnitrosamine) using 4-h exposure groups in the presence or absence of metabolic activation. Thymidine analog 5-bromo-2’-deoxyuridine (BrdU) was used as the selection agent. Two additional mutation assays were performed: one to extend the activation conditions to higher concentrations of the test compound and the other to confirm the results of the non-activation assay in the first trial. The substance induced an increase in mutations at the TK locus in L5178Y mouse lymphoma cells at concentrations of 0.25 - 0.5 nL/mL without activation and at 25.0 nL/mL with activation. The mutagenic activity was associated with highly toxic treatments and higher applied concentrations were required to achieve the same toxicity in the presence of microsomal activation mix. In conclusion, under the conditions of the study, the test substance was active in the mouse lymphoma forward mutation assay (Myrh, 1979). Experience with several similar types of acrylates has shown that these tend to produce positive results in the mouse lymphoma forward mutation assay when the TK locus is used as endpoint. However, PETIA and all other acrylates tested are negative in the same assay for the HPRT locus and are also negative in in vivo testing. The results from the mouse lymphoma forward mutation assay (TK locus) are therefore considered to be false positives.

A study was conducted to assess the mutagenic potential of PETIA at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments. The cells were exposed to the test substance for 4 h in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 h with and 24 h without metabolic activation. The maximum concentration of the pre-experiment (4,240μg/mL) was based on the solubility properties of the test substance in DMSO and aqueous medium. The concentration range of the main experiments was limited by cytotoxic effects. DMSO was used as solvent. Test concentrations were as follows

Experiment 1:

4 h (without S9 mix): 0.0078, 0.016, 0.031*, 0.063*, 0.13*, 0.25*, 0.5*, 0.75μg/mL

4 h (with S9 mix): 0.94, 1.9, 3.8*, 7.5*, 15.0*, 22.5*, 30.0*μg/mL

Experiment 2:

24 h (without S9 mix): 0.016, 0.031, 0.063*, 0.13*, 0.25*, 0.5*, 0.75*, 1.0μg/mL

4 h (with S9 mix): 4.0, 8.0, 16.0*, 20.0*, 24.0*, 28.0*, 32.0*μg/mL

(* These concentrations were chosen for the mutation rate analysis)

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Under the conditions of the study, the test substance did not induce gene mutations at the HPRT locus in V79 cells (Wollny, 2014).


In vivo

In vivo mammalian somatic cell studies: cytogenicity / erythrocyte micronucleus

A study was conducted to determine the mutagenicity potential of PETIA in the mouse peripheral blood micronucleus test. The substance (0.75 - 12 mg/kg bw) was administered dermally to male and female B6C3F1 mice for 3 months. Peripheral blood samples were obtained at the end of the study and smears slides were immediately prepared and scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) per animal. In addition, the normochromatic erythrocytes/polychromatic erythrocyte (NCE/PCE) ratio in 1,000 total erythrocytes per animal was determined to provide a measure of chemical-induced bone marrow toxicity. No increase in the frequency of micronucleated NCEs and NCE/PCE ratios were observed in peripheral blood samples from the male or female B6C3F1 mice. Under the conditions of the study, the test substance was considered to be negative for mutagenic activity (Hejtmancik et al., 2005).

A study was conducted to determine the mutagenicity potential of PETIA in the mouse peripheral blood micronucleus test. The substance (0.75 - 12 mg/kg bw) was administered dermally to male and female genetically modified Tg. AC hemizygous mice for 6 months. Peripheral blood samples were obtained at the end of the study and smears slides were immediately prepared and scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) per animal. In addition, the normochromatic erythrocytes/polychromatic erythrocyte (NCE/PCE) ratio in 1,000 total erythrocytes per animal was determined to provide a measure of chemical-induced bone marrow toxicity. Treatment of Tg. AC hemizygous female mice induced a significant increase in micronucleated NCEs. In Tg. AC hemizygous male mice, a small increase in micronucleated NCEs was detected; however, the response was judged to be equivocal, due to a positive trend (p = 0.001) without any one dose group being significantly elevated over the vehicle control frequency. The NCE/PCE ratios in peripheral blood of Tg. AC mice were significantly altered at 6 and 12 mg/kg bw. Male and female mice in these dosed groups had markedly elevated levels of immature PCEs in their blood, implying a stimulation of erythropoiesis, which was maybe in response to PETIA-induced toxicity (Hejtmancik et al., 2005). Furthermore, this assay is not considered appropriate for mutagenicity assessment as it uses a non-standard animal strain, not validated for this purpose. The corresponding carcinogenicity study of which it is part was not accepted by the National Toxicology Program Board of Scientific Councillors Subcommittee on Technical reports when presented in 2002

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

A study was conducted to determine the potential genotoxicity of the test substance when administered to rats at the maximum recommended dose, by measuring the increase in DNA strand breaks in Liver, Duodenum and Stomach according to OECD Guideline 489.The test substance was suspended in Arachid oil. Since, initially differences were observed between sexes, the study was performed with male and female animals. Based on the results of the dose range finding study a dose level of 500 and 1000 mg/kg bw were selected as the highest dose level tested for male and female animals, respectively. The concentrations analyzed in the formulations of all groups were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%), except for the formulation of Group C, for which the mean accuracy was below the target concentration (i.e. 65% of target). Group C represents the middle dose for males. As the required top-dose was well according to the target concentration, the study outcome was not affected. In the main study, male and female animals were dosed three times by oral gavage with vehicle or with 125, 250 and 500 mg or 250, 500 and 1000 mg kg/bw of the test substance in males and females, respectively, for three consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS)/kg bw. In total 5 treatment groups were used, each consisting of 5 animals per sexes. The female animals dosed with 1000 mg/kg showed the following toxic signs after dosing: diarrhea, lethargy, rough coat and a hunched posture. In this group only 3 out of the 8 animals survived. It was concluded that this dose was above the maximum tolerated dose. Therefore this group was excluded from the analysis. Consequently the MTD in male and female animals was comparable. Approximately 3-4 hours after the second dose of EMS and third dose of the vehicle or the test substance, Liver, Duodenum and Stomach were collected. The animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia and Liver, Duodenum and Stomach were isolated. Single cell suspensions from Liver, Duodenum and Stomach were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed. No statistically significant increase in the mean Tail Intensity (%) was observed in Liver cells of test substance treated male animals compared to the vehicle treated animals. In female animals a statistically significant increase was present. However, as this was inside the historical control data of the negative control this was not biologically relevant. No statistically significant increase in the mean Tail Intensity (%) was observed in Duodenum cells of the test substance treated animals compared to the vehicle treated female and male animals. No statistically significant increase in the mean Tail Intensity (%) was observed in Stomach cells of the test substance treated animals compared to the vehicle treated animals. Under the study conditions, the test substance is not genotoxic in the Comet assay in Liver, Duodenum and Stomach cells when sampled approximately 3-4 hours post dosing, of male and female rats that were dosed via oral gavage for three consecutive days up to the maximum tolerated dose (Eurlings, 2018).

Justification for classification or non-classification

PETIA was tested for in vitro genotoxicity in several bacterial reverse mutation (Ames) assays and found to be negative. An increase in mutations at the TK locus was seen in L5178Y mouse lymphoma cells with and without activation, however the substance did not induce gene mutations at the HPRT locus in V79 cells. The absence of mutagenic activity of PETIA was confirmed in the in vivo COMET assay performed in rats on liver, duodenum and stomach cells.

PETIA is considered as not clastogenic. Indeed, the substance did not cause an increase in the frequency of micronucleated NCEs and NCE/PCE ratios in peripheral blood samples from the male or female B6C3F1 mice exposed for 3 months (dermal route).

An extensive genotoxicity testing database has been developed for a large number of structurally similar esters of acrylic acid. Overall, the data obtained for PETIA is consistent with observations made for acrylates in general. With few exceptions, acrylates are non-mutagenic in point mutations assays such as the Ames test. In contrast, virtually all acrylates demonstrate a mutagenic response in the mouse lymphoma assay, which appears to be related to effects on chromosomal aberrations rather than direct induction of point mutation. Significant cytotoxicity is observed, generally with a non-linear dose-response both with and without activation. The results of in vitro mouse lymphoma assays, raising the concern of mutagenicity, are clarified by in vivo testing. The behavior of acrylates in the mouse micronucleus test (including that of PETIA in standard mice strains), the in vivo chromosomal aberration assay and the in vivo COMET assay indicate that these chemicals are non-genotoxic in whole animals. This is in line with observations of non-tumorigenic activity from a series of rodent bioassays conducted with acrylic acid and several acrylate esters.

In conclusion, the weight of evidence supports the conclusion that PETIA has no in vivo genotoxic potential. No classification of PETIA for mutagenicity is there required according to the Regulation EC 1272/2008.