Reaction mass of (2,4,6-trioxo-1,3,5-triazine-1,3,5(2H,4H,6H)-triyl)tri-2,1-ethanediyl triacrylate and 2-Propenoic acid, 1,1'-[[dihydro-5-(2-hydroxyethyl)-2,4,6-trioxo-1,3,5-triazine-1,3(2H,4H)-diyl]di-2,1-ethanediyl] ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No data is available for Reaction mass of THEICTA and THEICDA (T).

Mutagenicity, in vitro, Salmonella typhimurium: OECD 471 testing performed with THEICTA as a mono-consituent substance was negative in all strains of

Salmonella typhimurium except strain TA 1535 which showed positive response with metabolic activation.

OECD 471 testing for TMPTA andtris(2-hydroxyethyl) isocyanuratewere also showed mutagenic effects in this strain, however, only as a weak and non dose-related response.

Based on these data Reaction mass of THEICTA and THEICDA may be considered as having a mutagenic potential in bacteria.

Mutagenicity, in vitro, mutations mammalian cells: OECD 476 testing performed with TMPTA was positive for mutagenic effects, however, only at cytotoxic exposure levels. Due to the very close structural similarity to TMPTA, Reaction mass of THEICTA and THEICDA may have a mutagenic potential in mammalian cells.

Mutagenicity, in vitro, chromosome aberration mammalian cells:OECD 471 testing performed with TMPTA andtris(2-hydroxyethyl) isocyanurate was positive for TMPTA and negative fortris(2-hydroxyethyl) isocyanurate. Due to the very close structural similarity to TMPTA, Reaction mass of THEICTA and THEICDA should be considered as having a potential for a clastogenic effects in mammalian cellsin vitroas well.

See justification for read-across attached in section 13.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Data on target substance not available. Thus, read-across has been applied using data from the source substance THEICTA (S1), TMPTA (S2) and Tris(2-hydroxyethyl)isocyanurate (S3).
See further read-acoss justification in attached document to section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 100, TA 1537 and TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

weak positive reaction with above 2500µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 100, TA 1537 and TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight decrease for TA98

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: only positive in strain TA 1535

Conclusions:

No data is available for Reaction mass of THEICTA and THEICDA (T).
OECD 471 testing performed with THEICTA as a mono-constituent substance was negative in all strains of Salmonella typhimurium except for strain TA 1535 which was positive with metabolic activation. Similar results were obtained in OECD 471 testing for TMPTA but only with a rather weak positive and not dose-related response for strain TA 1535, whereas tris(2-hydroxyethyl) isocyanurate were negative for mutagenic effects in OECD 471 testing. Based on these data Reaction mass of THEICTA and THEICDA may be considered as having a mutagenic potential in bacteria

Executive summary:

No data is available for Reaction mass of THEICTA and THEICDA (T).

OECD 471 testing performed with THEICTA as a mono-constituent substance was negative in all strains of Salmonella typhimurium except for strain TA 1535 which was positive with metabolic activation. Similar results were obtained in OECD 471 testing for TMPTA but only with a rather weak positive and not dose-related response for strain TA 1535, whereas tris(2-hydroxyethyl) isocyanurate were negative for mutagenic effects in OECD 471 testing. Based on these data Reaction mass of THEICTA and THEICDA may be considered as having a mutagenic potential in bacteria.

See justification for read-across attached in section 13.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

13 September 2016 to 20 October 2016

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:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008, B.13/14. "Mutagenicity: Reverse Mutation Test Using Bacteria", 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

EPA Health Effects Test Guidelines, OPPTS 870.5100 "Bacterial Reverse Mutation Test", EPA 712-C-98-247, August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The purpose of this study was to evaluate the mutagenic potential of the test item by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli WP2 uvrA strain in the presence and absence of activated rat liver S9 fraction.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test).
In the Initial Mutation Test and Confirmatory Mutation Test different concentrations were used. Furthermore, a Complementary Confirmatory Mutation Test was also performed based on the results of the Confirmatory Mutation Test using a modified concentration range.
Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 µg/plate and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 µg/plate.
Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation were 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate.
Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1535 strain with metabolic activation were 5000, 2500, 1581, 1000, 500, 158.1, 50 and 15.81 µg/plate.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO):
Supplier: Sigma-Aldrich Co.
Batch No.: SZBG1310V
Expiry date: 25 April 2019

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-1,2-phenylenediamine (NPD); Methyl-methanesulfonate (MMS); 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

DESCRIPTION OF THE TEST PROCEDURE
The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test, a Confirmatory Mutation Test and a Complementary Confirmatory Mutation Test. In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test, the pre-incubation method was used.

Preliminary Compatibility Test
The solubility of the test item was examined using Distilled water, N,N-Dimethylformamide (DMF) and Dimethyl sulfoxide (DMSO). The test item was insoluble in Distilled water at 100 mg/mL concentrations. However, clear solution was observed at 100 mg/mL concentration using DMF and DMSO as vehicles. Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study. The obtained stock formulation (50 µL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility (Preliminary Compatibility Test).

Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the available information and the solubility and compatibility test, 100 mg/mL stock solution was prepared in DMSO, which was diluted in 6 steps by factors of 2, 2.5 and approximately v10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate of the test item. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test, Confirmatory Mutation Tests and Complementary Confirmatory Mutation Test)
Based on the results of the preliminary test, 100 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in at least five steps to obtain at least six dosing formulations for lower doses. The maximum test concentration was 5000 µg test item/plate.
Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 µg/plate.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 µg/plate.
Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation were 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate.
Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1535 strain with metabolic activation were 5000, 2500, 1581, 1000, 500, 158.1, 50 and 15.81 µg/plate.

Control Groups Used in the Tests
Strain-specific positive and negative (vehicle/solvent) controls, both with and without metabolic activation were included in each test. In addition, untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.

Procedure for Exposure in the Initial Mutation Test
A standard plate incorporation procedure was performed as an Initial Mutation Test.
Bacteria (cultured in Nutrient Broth No.2.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar: 2000 µL
vehicle (solvent) or test item solution (or reference controls): (50) µL
overnight culture of test strain: 100 µL
phosphate buffer (pH 7.4) or S9 mix: 500 µL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls.
After preparation, the plates were incubated at 37°C for 48 hours.

Procedure for Exposure in the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test
A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed. The same method was used in the Complementary Confirmatory Mutation Test.
For the pre-incubation method, bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, 50 µL of test item formulations or its vehicle (or positive reference controls or their solvent), 100 µL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (nonactivated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test item. These tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37°C in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.

Rationale for test conditions:

The experimental methods were conducted according to the methods described Ames et al. and Maron and Ames, Kier et al., Venitt and Parry, OECD Guideline No. 471, 1997, Commission Regulation (EC) No. 440/2008, 2008, EPA Guidelines, OPPTS 870.5100, 1998, 1996 and according to the relevant SOPs of CiToxLAB Hungary Ltd.

Evaluation criteria:

The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Statistics:

no

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test and Complementary Mutation Test, the pre-incubation method was used.
The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain in the presence and absence of a metabolic activation system (±S9 mix) with appropriate untreated, negative (vehicle/solvent) and positive controls.
The Complementary Confirmatory Mutation Test was carried out using Salmonella typhimurium TA98 and TA1535 strains in the presence and/or absence of a metabolic activation system (S9 mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the main tests, each sample (including the controls) was tested in triplicate.
Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 µg/plate and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 µg/plate.
Based on the results of the Confirmatory Mutation Test, the examined test concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation were 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate and in Salmonella typhimurium TA1535 strain with metabolic activation were 5000, 2500, 1581, 1000, 500, 158.1, 50 and 15.81 µg/plate.
In the Initial Mutation Test (using the plate incorporation method), there was no relevant increase of mutation factor with and without metabolic activation, there was no clear dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range. Indeed, this first assay was considered to be negative.
In the Confirmatory Mutation Test using the pre-incubation method, negative results were obtained with Salmonella typhimurium strains (TA98, TA100 and TA1537) and Escherichia coli WP2 uvrA strain with and without metabolic activation, and with TA1535 strain without metabolic activation. Positive effect of the test item was obtained in Salmonella typhimurium TA1535 bacterial strains with metabolic activation as the calculated mutation factor values were over the biologically relevant threshold value of 3 (at the two highest tested doses) and dose dependence was also observed.
The observed positive effect was confirmed in the Complementary Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 at 2500 µg/plate concentration with metabolic activation (the observed mutation factor value was 2.97). This borderline result demonstrated the slightly positive effect was reproducible and dose dependent.
In the Complementary Confirmatory Mutation Test, in Salmonella typhimurium TA98 bacterial strain without metabolic activation the highest revertant rate was observed at 158.1 µg/plate concentration. The observed mutation factor value was 2.48. Higher numbers of revertant colonies compared to the DMSO control were observed at several other concentrations in this strain. However, no dose-dependent relationship was observed. The number of revertant colonies was within the historical control range in each case. Furthermore, higher number of revertant colonies compared to the DMSO control were detected for untreated control (MF: 1.55) also in this strain without metabolic activation, indicating a higher than usual variability in this case. These results were also examined for reproducibility, but no increased revertant counts were detected in the first main test (using the same experimental method) in this strain. Thus, these values were considered as having no real biological relevance.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.
No precipitate was observed in the Initial Mutation Test, in the Confirmatory Mutation Tests and in the Complementary Confirmatory Mutation Test.
Inhibitory, cytotoxic effect of the test item (reduced background lawn development) was observed in the Confirmatory Mutation Test in all strains without metabolic activation at 5000 and/or 1581 and/or 500 µg/plate concentrations and a similar cytotoxic effect (reduced background lawn development/ reduced colony number) was seen in salmonella typhimurium TA1537 strains with metabolic activation at 5000 µg/plate concentration. The effect was excessive in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation at 5000, 1581 and 500 µg/plate concentrations, thus a complementary experiment was performed to ensure validity.
In the Complementary Confirmatory Mutation Test the background inhibition (reduced/slightly reduced background lawn development) was still observed in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation at 500 µg/plate concentrations.

 Summary Table of the Preliminary Concentration range Finding Test

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains
		TA 98		TA 100
		-S9	+S9	-S9	+S9
Untreated control	Mean	26.7	23.0	94.7	103.0
	MF	1.38	1.05	0.94	1.04
DMSO control	Mean	19.3	22.0	100.3	98.7
	MF	1.00	1.00	1.00	1.00
Distilled water control	Mean	--	--	96.7	--
	MF	--	--	0.96	--
5000		23.0	25.0	92.3	86.7
		1.19	1.14	0.92	0.88
2500	Mean	22.3	20.7	102.3	105.3
	MF	1.16	0.94	1.02	1.07
1000	Mean	24.7	29.3	98.7	107.0
	MF	1.28	1.33	0.98	1.08
316	Mean	25.7	28.0	98.7	113.7
	MF	1.33	1.27	0.98	1.15
100	Mean	21.0	36.0	97.3	110.0
	MF	1.09	1.64	0.97	1.11
31.6	Mean	26.0	34.0	101.0	118.3
	MF	1.34	1.55	1.01	1.20
10	Mean	22.0	28.3	109.7	117.0
	MF	1.14	1.29	1.09	1.19
NPD (4µg)	Mean	289.3	--	--	--
	MF	20.14	--	--	--
2AA (2µg)	Mean	--	2357.3	--	2325.3
	MF	--	107.15	--	23.57
SAZ (2µg)	Mean	--	--	1168.0	--
	MF	--	--	12.08	--

 

Summary Table of the Initial Mutation Test (Plate Incorporation Method)

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	20.0	25.0	109.7	119.7	13.0	8.7	6.3	9.3	32.7	33.0
	MF	1.05	0.99	0.98	1.04	1.39	0.74	0.76	1.00	1.01	0.93
DMSO control	Mean	19.0	25.3	112.3	114.7	9.3	11.7	8.3	9.3	32.3	35.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Distilled water control	Mean	--	--	104.3	--	11.3	--	--	--	35.0	--
	MF	--	--	0.93	--	1.21	--	--	--	1.08	--
5000	Mean	21.0	20.0	78.3	77.0	13.3	15.0	7.0	6.7	23.0	26.7
	MF	1.11	0.79	0.70	0.67	1.43	1.29	0.84	0.71	0.71	0.75
1581	Mean	21.0	27.7	101.0	100.0	10.0	22.3	9.3	5.3	28.7	22.0
	MF	1.11	1.09	0.90	0.87	1.07	1.91	1.12	0.57	0.89	0.62
500	Mean	24.7	26.0	96.3	102.3	9.0	20.3	5.3	6.7	25.3	24.0
	MF	1.30	1.03	0.86	0.89	0.96	1.74	0.64	0.71	0.78	0.67
158.1	Mean	24.3	28.0	91.3	100.7	11.7	17.0	10.7	9.0	22.3	30.0
	MF	1.28	1.11	0.81	0.88	1.25	1.46	1.28	0.96	0.69	0.84
50	Mean	20.3	29.7	83.0	94.7	11.3	11.3	11.7	8.3	24.0	26.7
	MF	1.07	1.17	0.4	0.83	1.21	0.97	1.40	0.89	0.74	0.75
15.81	Mean	24.0	28.3	86.3	95.3	10.7	14.3	7.7	13.0	23.7	25.0
	MF	1.26	1.12	0.77	0.83	1.14	1.23	0.92	1.39	0.73	0.70
NPD (4µg)	Mean	404.0	--	--	--	--	--	--	--	--	--
	MF	21.26	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2357.3	--	2417.3	--	222.7	--	201.3	--	--
	MF	--	93.05	--	21.08	--	19.09	--	21.57	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	233.7
	MF	--	--	--	--	--	--	--	--	--	6.55
SAZ (2µg)	Mean	--	--	1049.3	--	1178.7	--	--	--	--	--
	MF	--	--	10.06	--	104.00	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	494.7	--	--	--
	MF	--	--	--	--	--	--	59.36	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1066.7	--
	MF	--	--	--	--	--	--	--	--	30.48	--

 

Summary Table of the Confirmatory Mutation Test (Pre-Incorporation Method)

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	--	44.3	89.7	102.7	--	12.7	7.3	9.7	31.3	39.3
	MF	--	1.01	0.93	0.98	--	1.27	1.47	0.81	0.95	0.96
DMSO control	Mean	--	44.0	96.3	105.0	--	10.0	5.0	12.0	33.0	41.0
	MF	--	1.00	1.00	1.00	--	1.00	1.00	1.00	1.00	1.00
Distilled water control	Mean	--	--	96.7	--	--	--	--	--	39.0	--
	MF	--	--	1.00	--	--	--	--	--	1.18	--
5000	Mean	--	38.7	25.7	43.7	--	30.3	3.0	11.3	29.3	39.3
	MF	--	0.88	0.27	0.42	--	3.03	0.60	0.94	0.89	0.96
1581	Mean	--	37.3	65.7	85.3	--	37.7	3.3	9.7	32.7	38.3
	MF	--	0.85	0.68	0.81	00	3.77	0.67	0.81	0.99	0.93
500	Mean	--	40.7	91.3	115.3	--	21.3	6.3	11.0	31.0	46.7
	MF	--	0.92	0.95	1.10	--	2.13	1.27	0.92	0.94	1.14
158.1	Mean	--	36.3	101.7	106.7	--	16.7	10.7	9.7	31.0	41.7
	MF	--	0.93	1.06	1.02	--	1.67	2.13	0.81	0.94	1.02
50	Mean	--	42.0	110.3	113.3	--	12.7	9.7	12.3	31.0	35.3
	MF	--	0.95	1.15	1.08	--	1.27	1.93	1.03	0.94	0.86
15.81	Mean	--	49.0	108.3	123.7	--	15.0	13.0	10.7	29.0	41.0
	MF	--	1.11	1.12	1.18	--	1.50	2.60	0.89	0.88	1.00
5	Mean	--	41.0	139.0	100.3	--	11.0	6.0	11.3	29.3	33.0
	MF	--	0.93	1.44	0.96	--	1.10	1.20	0.94	0.89	0.80
NPD (4µg)	Mean	--	--	--	--	--	--	--	--	--	--
	MF	--	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2345.3	--	2457.3	--	252.7	--	213.7	--	--
	MF	--	53.30	--	23.40	--	25.27	--	17.81	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	199.0
	MF	--	--	--	--	--	--	--	--	--	4.85
SAZ (2µg)	Mean	--	--	1064.0	--	--	--	--	--	--	--
	MF	--	--	11.01	--	--	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	408.7	--	--	--
	MF	--	--	--	--	--	--	81.73	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1009.3	--
	MF	--	--	--	--	--	--	--	--	25.88	--

 

Summary Table of the Complementary Confirmatory Mutation Test (pre-Incubation Method)

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains
		TA 98		TA 1535
		-S9	+S9	-S9	+S9
Untreated control	Mean	15.0	--	15.7	14.0
	MF	1.55	--	1.09	1.45
DMSO control	Mean	9.7	--	14.3	9.7
	MF	1.00	--	1.00	1.00
Distilled water control	Mean	--	--	12.7	--
	MF	--	--	0.88	--
5000	Mean	--	--	--	23.3
	MF	--	--	--	2.41
2500	Mean	--	--	--	28.7
	MF	--	--	--	2.97
1581	Mean	--	--	--	25.0
	MF	--	--	--	2.59
1000	Mean	--	--	--	25.0
	MF	--	--	--	2.59
500	Mean	17.3	--	14.3	17.0
	MF	1.79	--	1.00	1.76
158.1	Mean	24.0	--	11.7	14.7
	MF	2.48	--	0.81	1.52
50	Mean	20.3	--	14.3	10.7
	MF	2.10	--	1.00	1.10
15.81	Mean	20.0	--	14.0	8.3
	MF	2.07	--	0.98	0.86
5	Mean	16.3	--	12.7	--
	MF	1.69	--	0.88	--
1.581	Mean	19.7	--	13.3	--
	MF	2.03	--	0.93	--
NPD (4µg)	Mean	397.3	--	--	--
	MF	41.10	--	--	--
2AA (2µg)	Mean	--	--	--	199.7
	MF	--	--	--	20.66
SAZ (2µg)	Mean	--	--	1197.3	--
	MF	--	--	94.53	--

 

Conclusions:

The test item Tris(2-hydroxyethyl) isocyanurate triacrylate had slightly mutagenic activity in Salmonella typhimurium TA1535 bacterial strain with metabolic activation, no mutagenic activity was observed in Salmonella typhimurium TA98, TA100, TA1537 and Escherichia coli WP2 uvrA with and without metabolic activation and in Salmonella typhimurium TA1535 without metabolic activation under the test conditions used in this study.

Executive summary:

The test item Tris(2-hydroxyethyl) isocyanurate triacrylate was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/ß-naphthoflavone-induced rats.

 

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and a Complementary Mutation Test (Pre-Incubation Method).

 

Based on the results of a solubility tests, the test item was formulated in Dimethyl sulfoxide (DMSO). Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 µg/plate were examined in the Preliminary Concentration Range Finding Test.

Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 µg/plate and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 µg/plate.

Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation were 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate.

Examined concentrations in the Complementary Confirmatory Mutation Test in Salmonella typhimurium TA1535 strain with metabolic activation were 5000, 2500, 1581, 1000, 500, 158.1, 50 and 15.81 µg/plate.

 

In the Initial Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system.

 

In the Confirmatory Mutation Test in the case of Salmonella typhimurium TA1535 bacterial strain, positive effect was obtained using the pre-incubation method. In the Complementary Confirmatory Mutation Test a slightly positive effect was obtained in this bacterial strain using the pre-incubation method.

 

No precipitate was detected on the plates in the Preliminary Concentration Range Finding Test and in the main tests in all examined strains with and without metabolic activation.

 

Inhibitory, cytotoxic effect of the test item was observed in the Confirmatory Mutation Test in all strains without metabolic activation and a similar cytotoxic effect was seen in Salmonella typhimurium TA1537 strains with metabolic activation. The effect was excessive in Salmonella typhimurium TA98 and TA1535 strains without metabolic activation, thus a complementary experiment was performed to ensure validity. In the Complementary Confirmatory Mutation Test the background inhibition was still observed in these strains without metabolic activation.

 

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

 

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

In conclusion, the test item Tris(2-hydroxyethyl) isocyanurate triacrylate (Batch Number: F0766724VS) had slightly mutagenic activity in Salmonella typhimurium TA1535 bacterial strain with metabolic activation, no mutagenic activity was observed in Salmonella typhimurium TA98, TA100, TA1537 andEscherichia coliWP2uvrAwith and without metabolic activation and in Salmonella typhimurium TA1535 without metabolic activation under the test conditions used in this study.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well conducted and documented study according to OECD guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix Aroclor 1254 induced

Test concentrations with justification for top dose:

20, 100, 500, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Standard solvent

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, N-methly-N´-nitroso-N-nitroso-guanidine, 4-nitro-o-phenylenediamine9-aminoacridine chioride monohydrate and 9-aminocridine chloride monohydrate

Details on test system and experimental conditions:

METHOD OF APPLICATION: pre mixed suspension (test solution, S9mix or buffer, bacteria suspension, etc) were poured onto agar plates
DURATION
Preincubation:
- Preincubation period: 16h (37°C)
- Expression time (cells in growth medium): >10e8 bacteria / ml should reached
Exposure:
- Exposure duration: 48h (dark, 37°C)
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays): histidine


NUMBER OF REPLICATIONS: Triplicates


DETERMINATION OF CYTOTOXICITY
- Method: colonie count (cell growth)

Evaluation criteria:

- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Statistics:

no

Species / strain:

other: TA 1537, TA98 and TA100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight decrease for TA 98 above 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

weakly positive reactions

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
ambiguous with metabolic activation TA 100 TA1537 TA98 negative
negative without metabolic activation

According to the results of the present study. the test substance Trimethylolpropantriacrylat is weakly mutagenic
in the Ames test under the experimental conditions chosen here.

Executive summary:

The substance Trimethylolpropantriacrylat was tested for mutagenicity in the Ames test (standard plate test, according to OECD 471) both in the presence and in the absence of a metabolizing system obtained from rat liver (S-9 mix) using the

strains TA 1535, TA 100, TA 1537 and TA 98.

Tests without S-9 mix showed no increase in the number of his revertants. Tests with S-9 mix showed show also

no increase in the number of his revertants for TA 1537, TA 98 and TA 100 while TA 1535 reveals weakly positive reaction with no dose-dependency over a dose range of 500 µg - 5000 µg/plate.

According to cytotoxicity a slight decrease in the number of his revertants was observed only using TA 98 with S-9 mix from about

2500 µg/plate onward.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

other information

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test substance : - Source: NISSAN CHEMICAL INDUSTRIES, LTD.
- Lot No. 00915-1
- Purity: 99.0 %

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

E. coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation: S9mix from Phenobarbital and 5,6-benzoflavone induced rat liver microsomes.

Vehicle / solvent:

Water for injection

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

Positive controls:

yes

Positive control substance:

other: -S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (TA100, TA98, WP2 uvrA), Sodium azide (TA1535) and 9-Aminoacridine hydrochloride (TA1537) +S9 mix; 2 -Aminoanthracene (all strains)

Details on test system and experimental conditions:

Plates/test: 3
Number of replicates: 2
Positive and Negative control groups and treatment: yes,
Positive controls:
-S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (TA100, TA98, WP2 uvrA),
Sodium azide (TA1535) and 9-Aminoacridine hydrochloride (TA1537)
+S9 mix; 2 -Aminoanthracene (all strains)
Solvent: Water for injection
Metabolic activation: S9mix from Phenobarbital and 5,6-benzoflavone induced
rat liver microsomes.

Evaluation criteria:

Criteria of evaluating results: Th e result was designated "mutagenic" when at
least two-fold increase over the control, and dose response-trend or
reproducibility was observed.

Statistics:

NA

Species / strain:

other: Salmonella typhimurium TA100, TA1535, TA98, TA1537, E. coli WP2 uvrA

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

Executive summary:

The genotoxicity of 1,3,5-TRIAZINE-2,4,6(1H,3H,5H)-TRIONE,1,3,5-TRIS(2HYDROXYETHYL) (S3) was assesed in a OECD TG 471 AMES test: Plates with Salmonella typhimurium TA100, TA1535, TA98, TA1537,E. coliWP2 uvrA were treated with 0, 156, 313, 625, 1,250, 2,500, 5,000 ug/plate.Tests without S-9 mix showed no increase in the number of revertants.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Data on target substance not available. Thus, read-across has been applied using data from the source substance TMPTA (S2).
See further read-acoss justification in attached document to section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results:
positive with metabolic activation
positive without metabolic activation

Trimethylolpropane triacrylate was considered to be mutagenic to L5178Y cells with and without metabolic activation.

Executive summary:

No data is available for Reaction mass of THEICTA and THEICDA (T).

A study was conducted to assess the mutagenic potential of trimethylolpropane triacrylate (TMPTA) on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The study was equivalent or similar to OECD guideline 476.

Three independent tests in the absence of exogenous metabolic activation and three independent tests in the presence of S9 mix were carried out using the concentrations based on results of the preliminary cytotoxicity trials.

 

In the absence of S-9 mix, significant increase in mutant frequency (3.6 times the background) was observed only at highest tested dose of 1.25 nL/mL in the first trial. This dose also showed high toxicity (19.1% relative growth). In the second trial, the same pattern of response was obtained. Significant increase in mutant frequency (4.2 times the background level) was observed only at the highest tested dose of 1.0 nL/mL (with 32.7% relative growth). Because the background frequency was unusually low in this trial, the assay was repeated again. In the third trial higher doses were tested and more extreme toxicities were obtained. Mutagenic activity was observed at the two highest doses (1.0and2.5nL/mL) and not at lower doses.

 

In the presence of S-9 activation, the results of first trial were inconclusive. In the second trial, no increase in mutant frequency was found for the 1.25 to 10.0 nL/mL dose range, in spite of the high toxicity at 10.0 nL/mL (14.9% relative growth). Because the background and positive control mutant frequency was unusually low in this trial, the assay was repeated again. The repeat assay showed a 6.4-fold increase in mutant frequency at 20 nL/mL. This dose was highly toxic (4.8% relative growth), whereas little toxicity was obtained with the other tested dose levels.

 

In conclusion, trimethylolpropane triacrylate (TMPTA) induced an increase in mutations at the TK locus in L5178Y mouse lymphoma cells in the dose range of 1.0 to 2.5 nL/mL without activation and at 20.0 nL/mL with microsomal activation. This mutagenic response was associated only with doses that were moderately to highly toxic.Trimethylolpropane triacrylate was considered to be mutagenic to L5178Y cells with and without metabolic activation.

Due to the very close structural similarity to TMPTA (S2), Reaction mass of THEICTA and THEICDA may have a mutagenic potential in mammalian cells as well.

See justification for read-across attached in section 13.

Reference 6

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 13, 1978 to January 24, 1979

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted similar or equivalent to OECD guideline 476

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: L5178Y mouse lymphoma cells TK (+/-) derived from the Fischer L5178Y line of Dr. Donald Clive.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Preliminary toxicity test:
0.004875 to 5 nL/mL (without S9); 0.004875 to 40 nL/mL (with S9).

Mutation test:
Test 1: 0.078 to 1.25 nL/mL without S9; 1.150 to 2.50 nL/mL with S9
Test 2: 0.150 to 1.00 nL/mL without S9; 1.250 to 10.00 nL/mL with S9
Test 3: 1.00 to 2.5 nL/mL without S9; 2.00 to 20 nL/mL with S9

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:

ethylmethanesulphonate

Remarks:

absence of S9 mix Migrated to IUCLID6: 0.5 µL/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

presence of S9 mix Migrated to IUCLID6: 0.3 µL/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: In suspension

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 48-72 h
- Selection time (if incubation with a selection agent): 10 d

NUMBER OF REPLICATIONS: Duplicate

Evaluation criteria:

A compound is considered mutagenic in this assay if:
- A dose-response relationship is observed in 3 of the 5 dose levels employed.
- The minimum increase at the low level of the dose-response curve is at least 2.5 times greater than the solvent and/or negative control values.
- The solvent and negative control data are within the normal range of the spontaneous background for the TK locus.

Statistics:

No data

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Preliminary cytotoxicity testing without activation indicated variable toxicity and complete lethality at 1.25 to 2.5 µL/mL. Three trials of the mutation assay are reported in which the inclusive applied dose ranges were 0.004875 to 5 µL/mL without activation, and 0.004875 to 40 µL/mL with activation. Dose levels showing excessive or insufficient toxicity to cell growth were eliminated from further testing
in order to select five doses for completion of the assay that would fall within the range of cytotoxicities where any mutagenic activity is normally observed.

MAIN STUDY:
Without metabolic activation - Significant increase in mutant frequency (3.6 times the background) was observed only at highest tested dose of 1.25 nL/mL in the first trial. This dose also showed high toxicity (19.1% relative growth). In the second trial, the same pattern of response was obtained. Significant increase in mutant frequency (4.2 times the background level) was observed only at the highest tested dose of 1.0 nL/mL (with 32.7% relative growth). Because the background frequency was unusually low in this trial, the assay was repeated again. In the third trial higher doses were tested and more extreme toxicities were obtained. Mutagenic activity was observed at the two highest doses (1.0 and 2.5 nL/mL) and not at lower doses.
 
With metabolic activation - The results of first trial were inconclusive. In the second trial, no increase in mutant frequency was found for the 1.25 to 10.0 nL/mL dose range, in spite of the high toxicity at 10.0 nL/mL (14.9% relative growth). Because the background and positive control mutant frequency was unusually low in this trial, the assay was repeated again. The repeat assay showed a 6.4-fold increase in mutant frequency at 20 nL/mL. This dose was highly toxic (4.8% relative growth), whereas little toxicity was obtained with the other tested dose levels.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

None

Conclusions:

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

Trimethylolpropane triacrylate was considered to be mutagenic to L5178Y cells with and without metabolic activation.

Executive summary:

A study was conducted to assess the mutagenic potential of trimethylolpropane triacrylate on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The study was equivalent or similar to OECD guideline 476.

Three independent tests in the absence of exogenous metabolic activation and three independent tests in the presence of S9 mix were carried out using the concentrations based on results of the preliminary cytotoxicity trials.

 

In the absence of S-9 mix, significant increase in mutant frequency (3.6 times the background) was observed only at highest tested dose of 1.25 nL/mL in the first trial. This dose also showed high toxicity (19.1% relative growth). In the second trial, the same pattern of response was obtained. Significant increase in mutant frequency (4.2 times the background level) was observed only at the highest tested dose of 1.0 nL/mL (with 32.7% relative growth). Because the background frequency was unusually low in this trial, the assay was repeated again. In the third trial higher doses were tested and more extreme toxicities were obtained. Mutagenic activity was observed at the two highest doses (1.0 and 2.5 nL/mL) and not at lower doses.

 

In the presence of S-9 activation, the results of first trial were inconclusive. In the second trial, no increase in mutant frequency was found for the 1.25 to 10.0 nL/mL dose range, in spite of the high toxicity at 10.0 nL/mL (14.9% relative growth). Because the background and positive control mutant frequency was unusually low in this trial, the assay was repeated again. The repeat assay showed a 6.4-fold increase in mutant frequency at 20 nL/mL. This dose was highly toxic (4.8% relative growth), whereas little toxicity was obtained with the other tested dose levels.

 

In conclusion, trimethylolpropane triacrylate induced an increase in mutations at the TK locus in L5178Y mouse lymphoma cells in the dose range of 1.0 to 2.5 nL/mL without activation and at 20.0 nL/mL with microsomal activation. This mutagenic response was associated only with doses that were moderately to highly toxic. Trimethylolpropane triacrylate was considered to be mutagenic to L5178Y cells with and without metabolic activation.

 

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Data on target substance not available. Thus, read-across has been applied using data from the source substance THEICTA (S1), TMPTA (S2) and Tris(2-hydroxyethyl)isocyanurate (S3).
See further read-acoss justification in attached document to section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Species / strain:

lymphocytes:

Remarks:

primary cell culture of human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at all concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

other: Chinese hamster lung cells (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results: positive

Trimethylolpropane triacrylate (S2) induced chromosome aberrations in cultured human lymphocytes, both with and without metabolic activation.
Tris(2-hydroxyethyl) isocyanurate did not induce chromosome aberrations.

Executive summary:

Data on target substance not available. Thus, read-across has been applied using data from the source substance, TMPTA (S2) and Tris(2-hydroxyethyl)isocyanurate (S3).

Mutagenicity, in vitro, chromosome aberration mammalian cells: OECD 471 testing performed with TMPTA andtris(2-hydroxyethyl) isocyanurate was positive for TMPTA and negative for tris(2-hydroxyethyl) isocyanurate. Due to the very close structural similarity to TMPTA,Reaction mass of THEICTA and THEICDA (T) should be considered as having a potential for a clastogenic effects in mammalian cells as well.

See justification for read-across attached in section 13.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 13 to December 20, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to OECD Guideline 473, EU Method B.10, OPPTS 870.5375 and Japanese guidelines in compliance with the Principles of Good Laboratory Practice.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: primary cell cultures from human peripheral blood

Details on mammalian cell type (if applicable):

They have a stable karyotype with 46 chromosomes and an average cell cycle time of 12-14 h.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction consists of Aroclor 1254 treated rat liver microsomal fraction prepared and preserved in sterile tubes at -80°C. S9 mix was prepared at +4°C immediately before use and maintained at this temperature until added to the culture medium.

Test concentrations with justification for top dose:

Preliminary experiment:
23.1, 45.7, 92.6, 185, 370, 740, 1480 and 2960 μg/mL for the preliminary experiment both with and without S9 mix

Main experiment:
1) 0.78, 1.56, 3.13, 6.25, 12.5, 18.75, 25 and 37.5 μg/mL for the first experiment without S9 mix
2) 1.56, 3.13, 6.25, 12.5, 18.75, 25, 37.5 and 50 μg/mL for the first experiment with S9 mix
3) 3.13, 6.25, 9.38, 12.5, 18.75 and 28.13 μg/mL, for the second experiment without S9 mix
4) 9.38, 18.75, 28.13, 37.5, 50 and 75 μg/mL, for the second experiment with S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO), batch nos. K32136150327 and K32311850346 (Merck Clévenot, Chelles, France)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: 3 μg/mL, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: 12.5 or 25 μg/mL, with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: In RPMI medium containing fetal calf serum, penicillin, L-glutamine, Streptomycin and Phytohaemagglutinin


DURATION
- Incubation period of culture without test material: 48 h at 37°C
- Exposure duration: 3 h
- Expression time (cells in growth medium): 17 h
- Selection time (if incubation with a selection agent): 1.5 h
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h


SELECTION AGENT (mutation assays): Colcemid (10 μg/mL)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN Used: Giemsa


NUMBER OF REPLICATIONS: Duplicate cultures in both the experiments


NUMBER OF CELLS EVALUATED:
Cytotoxicity - 1000 cells per culture.
Chromosomal aberration - Analysis of 200 metaphases/concentration (with 44 to 46 chromosomes) was made, with 100 metaphases/culture, whenever possible. Only 50 metaphases/culture were analyzed when at least 10% of the cells with structural chromosome aberrations were observed.


DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index (number of cells in mitosis/1000 cells examined)

Evaluation criteria:

A reproducible, concentration-related, and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the concentrations and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings, if needed.

Statistics:

For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the results were compared using the χ2 test, in which p = 0.05 was used as the lowest level of significance.

Species / strain:

lymphocytes: Primary cell culture of human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at all conc. of the preliminary experiment and observed in both first and second experiments at conc. ≥ 12.5 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Additional information on chromosomal aberration:

With metabolic activation-
In the first experiment, a statistically significant increase in the frequency of cells with structural chromosomal aberrations was noted at 18.75 μg/mL (46% versus 1.5% for the vehicle control, p < 0.001).
In the second experiment, statistically significant and concentration-related increases in the frequency of cells with structural chromosomal aberrations were noted at concentrations ≥ 9.38 μg/mL (6, 10.7 and 30% at the concentrations of 9.38, p < 0.01 ; 12.5, p < 0.001 and 18.75 μg/mL, p < 0.001, respectively, versus 0.5% for the vehicle control).

Without metabolic activation-
In the first experiment, statistically significant and concentration-related increases in the frequency of cells with structural chromosomal aberrations were noted at concentrations of 37.5 and 50 μg/mL (21 and 53%, respectively versus 1% for the vehicle control, p < 0.001).
In the second experiment, statistically significant increases in the frequency of cells with structural chromosomal aberrations, without any clear evidence of a concentration-relationship, were noted at concentrations of 28.13 and 50 μg/mL (4.5%, p < 0.05 and 19%, p < 0.001, respectively, versus 1% for the vehicle control).



TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the culture medium, the concentration of 2963.2 μg/mL showed a slight to moderate precipitate. At this concentration, the pH was approximately 7.1 and the osmolality equal to 337 mOsm/kg H2O. However, this concentration was rejected following severe cytotoxicity.


RANGE-FINDING/SCREENING STUDIES:
The preliminary study was with a treatment volume of 27.5 μL/5.5 mL culture medium and the treatment-levels were 23.1, 45.7, 92.6, 185, 370,740, 1480 and 2960 μg/mL both with and without S9 mix. High toxicity was observed at all concentrations resulting in rejection of the experiment.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity occurred at 90% for the lowest concentration and 100% for the other concentrations in the preliminary study. In view of the severe toxiciobserved, the first experiment was repeated at lower concentrations ranging from 0.78 to 37.5 μg/mL. The concentrations in the second experiment ranged from 3.13 to 28.13 μg/mL. Cytotoxicity ranged from 45 to 100% in the first experiment and 26 to 83% in the second experiment at concentrations ≥ 12.5 μg/mL.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Validity criteria followed:

This study was considered valid since the following criteria were met:

• the frequency of cells with structural chromosome aberrations in the vehicle controls was consistent with CIT historical data,

• The frequency of cells with structural chromosome aberrations in the positive control tests was significantly higher than that of the vehicle controls and consistent with CIT historical data.

Conclusions:

Interpretation of results (migrated information):
positive

Trimethylolpropane triacrylate induced chromosome aberrations in cultured human lymphocytes, both with and without metabolic activation.

Executive summary:

An in vitro mammalian cell chromosomal aberration study was carried out with trimethylolpropane triacrylate in accordance with OECD TG 473, EU Method B.10, Japanese guidelines and EPA OPPTS method 870.5375.

A preliminary experiment was carried out with trimethylolpropane triacrylate 23.1, 45.7, 92.6, 185, 370, 740, 1480 and 2960 μg/mL both with and without S9 mix. In the main experiment, primary cell cultured human peripheral blood from two donors were incubated with Trimethylolpropane Triacrylate at the following concentrations:

1) 0.78, 1.56, 3.13, 6.25, 12.5, 18.75, 25 and 37.5 μg/mL without S9 mix (first experiment);

2) 1.56, 3.13, 6.25, 12.5, 18.75, 25, 37.5 and 50 μg/mL with S9 mix (first experiment);

3) 3.13, 6.25, 9.38, 12.5, 18.75 and 28.13 μg/mL without S9 mix (second experiment);

4) 9.38, 18.75, 28.13, 37.5, 50 and 75 μg/mL with S9 mix (second experiment).

Cytotoxicity was observed at all concentrations of the preliminary experiment. Hence this study was rejected. Cytotoxicity ranged from 45 to 100% in the first experiment and 26 to 83% in the second experiment at concentrations ≥ 12.5 μg/mL.

Statistically significant and concentration-related increases in the frequency of cells with structural chromosomal aberrations were noted at concentrations ≥ 9.38 μg/mL with metabolic activation. Statistically significant increases in the frequency of cells with structural chromosomal aberrations were noted at concentrations at and above 28.13 μg/mL.

In conclusion trimethylolpropane triacrylate induced chromosome aberrations in cultured human lymphocytes both with and without metabolic activation.

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Source: NISSAN CHEMICAL INDUSTRIES, LTD.
- Lot No. 00915-1
- Purity: 99.0 %

Species / strain / cell type:

other: Chinese Hamster Lung cells (CHL/IU)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, with and without (short-term treatment), without (continuous treatment)

Test concentrations with justification for top dose:

0, 653, 1,306, 2,612 ug/mL for short-term treatment (6 h).
0, 653, 1,306, 2,612 ug/mL for -S9 continuous treatment (24 h).

Vehicle / solvent:

Physiological saline

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Plate/concentration: 2
no further information available

Evaluation criteria:

Criteria of evaluating results: The results were considered to be negative if the incidence was less than 4.9 %, equivocal if it was between 5.0 and 9.9 %, and positive if it was more than 10.0 %.

Statistics:

not specified

Key result

Species / strain:

other: Chinese Hamster Lung cells (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No increase in chromosomal aberrations was observed in the test with either the short-term treatment (-S9 and +S9) or continuous treatment (-S9).

Executive summary:

The in vitro genotoxicity of 1,3,5-TRIAZINE-2,4,6(1H,3H,5H)-TRIONE,1,3,5-TRIS(2HYDROXYETHYL) (S3) was assessed in a OECD TG 473 In vitro Mammalian Chromosome Aberration Test. Chinese Hamster Lung cells (CHL/IU) were treated with 0, 653, 1,306, 2,612 ug/mL for short-term treatment (6 h) and 0, 653, 1,306, 2,612 ug/mL for -S9 continuous treatment (24 h) with or without metabolic activation (S-9-mix). No increase in chromosomal aberrations was observed in the test with either the short-term treatment (-S9 and +S9) or continuous treatment (-S9).

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

 No data is available for Reaction mass of THEICTA and THEICDA (T).

For making a conclusive assessment of the gentoxic potential of TMPTA found in the in vitro studies using mammalian cells two in vivo studies an OECD 474 study and an OECD 489 study have been conducted.

Mutagenicity, in vivo, micronucleus formation, mice: OECD 474 as well as OECD 489 (Comet assay) testing performed with TMPTA were negative for genotoxic effectsin vivo.

Due to the very close structural similarity to TMPTA, Reaction mass of THEICTA and THEICDA may be considered without genotoxic potential in mammalian cellsin vivoas well.  

See justification for read-across attached in section 13.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Data on target substance not available. Thus, read-across has been applied using data from the source substance TMPTA (S2).
See further read-acoss justification in attached document to section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Only in males (piloerection at 875 mg/kg bw; 2 deaths and piloerection in surviving animal at 1750 mg/kg bw)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information): negative
TMPTA (S2) was considered to be non-genotoxic in micronucleus test in the mouse.
Due to the very close structural similarity to TMPTA (S2), the target substance Reaction mass of THEICTA and THEICDA (T) should be considered be non-genotoxic in the micronucleus test as well.

Executive summary:

No data is available for the target substance Reaction mass of THEICTA and THEICDA (T).

A study was performed to investigate the potential of the source substance Trimethylolpropane triacrylate (TMPTA)(S2) to induce micronuclei in bone marrow cells of mice. The study was performed according to guidelines (OECD 474, Commission Directive No. B12, OPPTS 870.5395 and Japanese guidelines) and in compliance with the Principles of Good Laboratory Practice. 

 

A preliminary toxicity test was performed to define the highest possible dose to be used for the cytogenetic study. In the main study, four groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice received a single oral administration of the test material at the following doses: 437.5, 875 and 1750 mg/kg bw (for males) or 500, 1000 and 2000 mg/kg bw (for females). Two groups of five males and five females received the vehicle (corn oil) acted as the control groups. One group of five males and five females received Cyclophosphamide (positive control) once by the oral route at a dose of 50 mg/kg bw. The animals were killed 24 (control, low, intermediate and high dose groups) or 48 h (control and high dose groups) after treatment. The animals of the positive control group were killed 24 h after treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE). 

 

In males, no clinical signs and no mortality attributed to the treatment were observed in controls and 437.5 mg/kg bw group. At 875 mg/kg bw, piloerection was noted and at 1750 mg/kg bw, two males were found dead 24 h following the treatment and piloerection was noted at the same time in the surviving males. In females, no clinical signs and no mortality attributed to the treatment were observed in animals given 0, 500, 1000 or 2000 mg/kg bw.

 

Mean values of MPE as well as the PE/NE ratios in the treated group were equivalent to those of the control group, for both harvest times. All frequencies of micronucleated cells (MPE) obtained in treated animals were clearly within or consistent with the historical vehicle control range. Cyclophosphamide induced a significant increase in the frequency of MPE, demonstrating the sensitivity of the test system under the experimental conditions of this study.

 

In conclusion TMPTA (S2) was considered to be non-mutagenic in the micronucleus test. 

 

Due to the very close structural similarity to TMPTA (S2), the target substance Reaction mass of THEICTA and THEICDA should be considered be non-mutagenic in the micronucleus test as well.

See justification for read-across attached in section 13.

Reference 2

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:

From December 08, 2005 to June 08, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study is according to OECD Guideline 474, EU Method B.12, OPPTS 870.5395 and Japanese guidelines in compliance with the Principles of Good Laboratory Practice.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

; requirements of the Japanese Government under the revised Chemical Substance Law (1986)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Swiss Ico: OF1 (IOPS Caw)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, 1'Arbresle, France
- Age at study initiation: 6 wk
- Weight at study initiation: 32.3 g for males (ranging from 28.4 to 35.2 g) and 23.9 g for females (ranging from 21.0 to 26.5 g).
- Assigned to test groups randomly: Yes
- Housing: Housed by groups in polycarbonate cages
- Diet (e.g. ad libitum): A04 C pelleted maintenance diet, ad libitum
- Water (e.g. ad libitum): FG Millipore membrane filtered water, ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C,
- Humidity (%): 30- 70 %
- Air changes (per h): 12 cycles/h
- Photoperiod (h dark / h light): 12 h/12 h


IN-LIFE DATES: From: 2005-12-13 to: 27-01-27

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Concentration of test material in vehicle: Preliminary tests - 175 and 200 mg/mL; Main test - 43.75, 50, 87.5, 100, 175 and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no. (if required): 015K0115

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Preliminary tests - The test item was dissolved in the vehicle in order to achieve the concentrations of 175 and 200 mg/mL and then homogenized using a magnetic stirrer. The resulting formulations were light yellow limpid solutions. Using a treatment volume of 10 mL/kg bw, the target doses were 1750 and 2000 mg/kg bw.

Main test - The test item was dissolved in the vehicle in order to achieve the concentrations of 43.75, 50, 87.5, 100, 175 and 200 mg/mL and then homogenized using a magnetic stirrer. Using a treatment volume of 10 mL/kg bw, the target doses were 437.5, 500, 875, 1000, 1750 and 2000 mg/kg bw.

The preparations were made immediately before use.

Duration of treatment / exposure:

Single treatment

Frequency of treatment:

Single treatment

Post exposure period:

24 h (control, low, intermediate and high dose groups); 48 h (for control and high dose groups)

Remarks:

Doses / Concentrations:
437.5, 875 and 1750 mg/kg bw (for males) or 500, 1000 and 2000 mg/kg bw (for females)
Basis:
nominal conc.

No. of animals per sex per dose:

5 animals/sex/dose (with exception of 8 animals/sex/high dose for 48 h sampling time)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 50 mg/kg bw

Tissues and cell types examined:

Bone marrow cells of mice

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Preliminary test was performed on groups of 6 animals (3 males and 3 females). The initial preliminary test indicated that males were the more sensitive sex. Additional preliminary testing was conducted on a group of 3 male mice to determine the high dose for this sex. Clinical signs and any mortality were recorded for a period of 48 h after each preliminary dosing.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 24 or 48 h following dosing

DETAILS OF SLIDE PREPARATION: The femurs of the animals were removed and the bone marrow was flushed out using fetal calf serum. After
centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded so that the scorer was unaware of the treatment group of the slide under evaluation ("blind" scoring).

METHOD OF ANALYSIS:
For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Evaluation criteria:

- Statistically significant increase in the frequency of MPE when compared to the vehicle control group was required for a positive finding;
- Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained

Statistics:

- Normality and homogeneity of variances was tested using a Kolmogorov Smirnov test and a Bartlett test.
- Student t-test (2 groups) or a one-way analysis of variance (≥ 3 groups) followed by a Dunnett test (if necessary): If normality and homogeneity of variances were demonstrated.
- Mann/Whitney test (2 groups) or a Kruskall Wallis test (≥ 3 groups) followed by a Dunn test (if necessary).: If normality or homogeneity of variances was not demonstrated.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Only in males (piloerection at 875 mg/kg bw; 2 deaths and piloerection in surviving animal at 1750 mg/kg bw)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals:
- Neither mortality nor clinical signs were noted in the 3 treated females; Piloerection and ocular secretion or soiled urogenital area were noted in 2 surviving males.
- Confirmatory assay was performed using 3 males at 2000 mg/kg bw in order to confirm the previous findings. Two males were found dead 24 and 48 h following treatment. The surviving male showed piloerection.
- Based on these results, an additional test was performed using 3 males at 1750 mg/kg bw. Only piloerection was noted in males given 1750 mg/kg bw in this second preliminary test and no mortality was induced.
- For males, since toxic effects were observed, the choice of the top dose was based on the level of toxicity, such that a higher dose was expected to induce lethality. Therefore, 1750 mg/kg bw was selected as the top dose for the main test.
For females, since no toxic effects were observed, 2000 mg/kg bw was selected as the top dose for the main test.

RESULTS OF DEFINITIVE STUDY
- Mean values of MPE as well as the PE/NE ratios in the groups treated with the test item, were equivalent to those of the vehicle control group, for both harvest times.
- All frequencies of micronucleated cells (MPE) obtained in treated animals were clearly within or consistent with the historical range.

None

Conclusions:

Interpretation of results: negative
Trimethylolpropane triacrylate (TMPTA) was considered to be non-genotoxic in micronucleus test in the mouse.

Executive summary:

A study was performed to investigate the potential of Trimethylolpropane triacrylate (TMPTA) to induce micronuclei in bone marrow cells of mice. The study was performed according to guidelines (OECD 474, Commission Directive No. B12, OPPTS 870.5395 and Japanese guidelines) and in compliance with the Principles of Good Laboratory Practice. 

 

A preliminary toxicity test was performed to define the highest possible dose to be used for the cytogenetic study. In the main study, four groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice received a single oral administration of the test material at the following doses: 437.5, 875 and 1750 mg/kg bw (for males) or 500, 1000 and 2000 mg/kg bw (for females). Two groups of five males and five females received the vehicle (corn oil) acted as the control groups. One group of five males and five females received Cyclophosphamide (positive control) once by the oral route at a dose of 50 mg/kg bw. The animals were killed 24 (control, low, intermediate and high dose groups) or 48 h (control and high dose groups) after treatment. The animals of the positive control group were killed 24 h after treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE). 

In males, no clinical signs and no mortality attributed to the treatment were observed in controls and 437.5 mg/kg bw group. At 875 mg/kg bw, piloerection was noted and at 1750 mg/kg bw, two males were found dead 24 h following the treatment and piloerection was noted at the same time in the surviving males. In females, no clinical signs and no mortality attributed to the treatment were observed in animals given 0, 500, 1000 or 2000 mg/kg bw.

Mean values of MPE as well as the PE/NE ratios in the treated group were equivalent to those of the control group, for both harvest times. All frequencies of micronucleated cells (MPE) obtained in treated animals were clearly within or consistent with the historical vehicle control range. Cyclophosphamide induced a significant increase in the frequency of MPE, demonstrating the sensitivity of the test system under the experimental conditions of this study.

In conclusion trimethylolpropane triacrylate (TMPTA) was considered to be non-mutagenic in the micronucleus test. 

 

Reference 3

Endpoint:

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

Remarks:

Comet assay

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

November 2016 -September 2017

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)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) (2011). Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Propyldidynetrimethyl triacrylate (TMPTA), Cas. Number: 15625-89-5.
Batch number: F0994478VS.
- Expiration date of the lot/batch: 31 August 2017
- Purity: 80.2%
- Purity test date: NA

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 to 25°C, protected from light.
- Stability under test conditions and in solvent/vehicle:
The test article formulation in vehicle at 0.025 mg/mL is stable for 21 days when stored at 15 to 25°C, protected from light and for 195 hours at 2 to 8°C, protected from light. Formulations at 1 mg/ml and 200 mg/ml are stable for 34 days deep frozen <-70°C and 5 hours at room temperature
(ABL Study ABL15006). Formulations will be held at room temperature and used after mixing within five hours of preparation. The formulations will be assumed stable for this period unless specified otherwise by the Sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Treatment of test material prior to testing: Samples will be gently stirred (on a magnetic stirrer) at least 30 minutes prior to and continuously during dosing.

Due to the viscosity of the vehicle all formulations to be performed aseptically and not filtered.


FORM AS APPLIED IN THE TEST (if different from that of starting material)
Test substance in vehicle Polyethylene glycol 400 (PEG 400)

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

- Dose-finder study: Male and female mice.
- Main study: Female mice (only).

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Mouse CD-1, females.

- Age at study initiation: Approximately 8 to 10 weeks old.
- Weight at study initiation: Approximately 23 to 27 g on arrival.
- Assigned to test groups randomly: [no/yes, under following basis:]
- Fasting period before study:
- Housing: Cages conforming to the 'Code of practice for the housing and care of animals bred, supplied or used for scientific purposes' (Home Office, London, 2014).
- Diet (e.g. ad libitum): Ad libitum access to 5LF2 EU Rodent Diet 14% (manufactured by LabDiet St. Louis USA).
- Water (e.g. ad libitum): Ad libitum access to mains water via water bottles.

- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C.
- Humidity (%): 45 to 65%.
- Air changes (per hr): 15 to 20 air changes/hour.
- Photoperiod (hrs dark / hrs light): 12 hours nominal light/dark cycle.

IN-LIFE DATES: From To:
-Range-finding study: Medio November 2016 – Medio December 2016.
-Main study: April 2017 – September 2017

Route of administration:

intravenous

Vehicle:

- Vehicle(s)/solvent(s) used: [Polyethylene glycol 400 (PEG400)]
- Justification for choice of solvent/vehicle: The test article is insoluble in water, therefore formulations used on other toxicology studies are considered inappropriate.
- Concentration of test material in vehicle: 0.025 mg/mL, 1 mg/ml and 200 mg/ml.
- Lot/batch no. (if required): NA
- Purity: NA

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Route = Intravenous (I.V.)
Slow intravenous bolus injection directly into the femoral vein via the surgical cannula.

Maximum tolerated dose (MTD) based on Range-Finder data.
The dose route has been selected in order to maximise exposure of the target organs to the test article.

Duration of treatment / exposure:

Two times within 24 hours

Frequency of treatment:

Two administrations, at 0 and 23.5 hours, animals sampled at 24 hours.

Post exposure period:

0.5 hours

Dose / conc.:

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

Remarks:

Individual animal dose volumes will be based on the last recorded individual body weight

Dose / conc.:

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

Remarks:

Individual animal dose volumes will be based on the last recorded individual body weight

Dose / conc.:

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

Remarks:

Individual animal dose volumes will be based on the last recorded individual body weight

No. of animals per sex per dose:

- 6 female in each exposure group incl. a vehicle control group (PEG400).
- Three females for the positive control group (EMS).

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate (EMS) in water.

- Justification for choice of positive control(s): the oral route of exposure of EMS has previously been performed by the laboratory that have historical control data on this route of exposure.
- Route of administration: oral gavage on unfasted animals.
- Doses / concentrations: (Individual animal dose volumes will be based on the last recorded individual body weight).150 mg/kg, single oral administration at 21 hours and animals sampled at 24 hours.
- Dose volume = 10 mL/kg.

Tissues and cell types examined:

Range-Finder study: no tissue sampling.

Main study:
- blood, sampled via cardiac puncture.
- Femur + bonemarrow.
- Liver.
- sternum incl. bonemarrow.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
- Three numbers of slides per animal
- Pre-treated in normal melting point agarose (NMA) according to Covance SOP.
- Cell suspension: 100 μL of cell suspension/agarose mix (in a ratio of 1:10) per slide. Cover-slipped and gelled on ice.

-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.
- Lysis conditions: On ice or at 2-8°C, protected from light.
- Lysis incubation for a minimum of one hour up to a maximum overnight incubation.
- Electrophoresis buffer: 300 mM NaOH, 1 mM EDTA, pH>13.
- Electrophoresis Voltage: 0.7 V/cm.
- Unwinding Time: Tissue specific time.
- Electrophoresis Time: Immediately following unwinding in the same buffer, time
- Neutralisation: Immediately after electrophoresis in 0.4 M Tris, pH 7.0 (3 x 5 minute washes). Once neutralised all slides are air dried and stored at room temperature. Period as per Covance SOP.
- Staining: 100 μL of 2 μg/mL ethidium bromide applied to slide immediately prior to scoring.

METHOD OF ANALYSIS:
-Analysed by microscopy, 150 cells per animal per tissue scored for Comet analysis. All sample/slides were given a random coded unknown to the slide analyst.
Controls (positive and negative) were cheked for quality and response before scoring of the slides
All animal per group were analysed. For Cytotoxicity assessment the number of observed hedgehogs were recorded.

-Comet Criteria: 1) Only clearly defined non overlapping cells are scored. 2) Hedgehogs are not scored. 3) Cells with unusual staining artefacts are not scored.

-Slide analysis was performed by an analyst trained in accordance with Covance Laboratories Standard Operating Procedures.

OTHER:

Evaluation criteria:

For valid data, the test article will be considered to induce DNA damage if:
1. A least one of the test doses exhibits a statistically significant increase in tail
intensity, in any tissue, compared with the concurrent vehicle control
2. The increase is dose related in any tissue
3. The increase exceeds the laboratory’s historical control data for that tissue.

The test article will be considered positive in this assay if all of the above criteria are
met.

The test article will be considered negative in this assay if none of the above criteria
are met and target tissue exposure has been confirmed.

Results which only partially satisfy the criteria will be dealt with on a case-by-case
basis. Biological relevance will be 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. Analysis of additional cells
from vehicle and / or treated animals or further experimental work may be deemed
necessary to aid evaluation of the data.

A positive response will be based on scientific judgement and will include analysis
of related, concurrent cytotoxicity information (such as hedgehog assessment,
histopathological changes and any clinical pathology results) and the historical
control data. Positive results at clearly cytotoxic dose levels will be interpreted with
caution and discussed fully in the report.

Decreases in tail intensity may also be indicative of biologically relevant events and
may require additional testing and evaluation. Interpretation of any decreases in tail
intensity will be discussed fully in the report.

Additional work will be discussed with the Sponsor and where appropriate
documented by protocol amendment.

Statistics:

Tail intensity data will be supplied for each slide. The median of the log-transformed tail intensities from each slide will be averaged to give an animal summary statistic. If the median value on a slide is zero, a small constant (0.0001) will be added before taking the logarithm and calculating the average for the animal. This animal average will be used in the statistical analysis.

Data will be analysed using one-way analysis of variance (ANOVA) with the fixed factor for treatment group. The positive control group will be excluded from this analysis. Levene’s test for equality of variances across the groups will also be performed. Where this shows evidence of heterogeneity (p≤0.01), the data will be rank-transformed prior to analysis using ANOVA on the ranks. Comparisons between each treated group and control will be made using
Dunnett’s test. The test will be one-sided looking for an increase in response with increasing dose. The backtransformed difference and p-value will be reported. In addition, a linear contrast will be used to test for an increasing dose response.

The positive control group will be compared to the control group using a two-sample t-test. Levene’s test for equality of variances between the groups will also be performed. Where this shows evidence of heterogeneity (p≤0.01), the data will be rank-transformed prior to analysis using a two-sample t-test on the ranks. The test will be one-sided looking for an increase in response with increasing dose. The backtransformed difference and p-value will be reported.

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 0, 10, 20 and 30 mg/kg/day
- Solubility: full soluble in PEG400
- Clinical signs of toxicity in test animals:

Initial Range-Finder Experiments (First trial):
In the Range-Finder experiments groups of two or three male and two or three female animals were dosed with vehicle or TMPTA at 10 (females only), 20 mg/kg/day (males and females) or 30 mg/kg/day (males only Initial administration of the vehicle in two male and two female animals was well tolerated with no clinical signs of toxicity and no notable effects on bodyweight.
At 10 mg/kg/day in females, no clinical signs were observed following the first administration. Immediately following the second dose, 2/3 animals showed signs of rapid breathing. At 0.5 hours post-dose one of the animals was found missing its protective tail cuff and was sent to necropsy. Of the two remaining animals 1/2 animals had piloerection at 0.5 hours post-dose but was back to a normal state by 1 hour post-dose. The other did not show any further clinical signs. All surviving animals lost bodyweight (between -1.1% to -8.1%).
At 20 mg/kg/day, following the first administration, clonic convulsions and gasping were noted in 1/3 males immediately post dose. However, the animal recovered by 0.5 hours post-dose. No clinical signs were observed in the females following the first dose. Following the second administration, no clinical signs were observed in the males. Clonic convulsions, gasping and shallow respiration were observed in 2/3 females immediately following the second dose and subsequently died. The remaining female survived to the end of the observation period. Of the surviving animals all lost some bodyweight (males lost between -3.2% and 5.8% and the female lost -6.0%).
At 30 mg/kg/day in males, no clinical signs were observed following the first dose. Immediately following the second dose twitching was observed in 3/3 animals and pale extremities in 1/3 animals. Pale extremities was observed in 3/3 animals at 0.5 hours post-dose but all animals were back to a normal state by 1 hour post-dose. All animals lost bodyweight (between -1.6% to -4.1%).
Clonic convulsions and twitching were considered to be dose limiting in the initial Range-Finder Experiments. As no significant sex differences were observed, the Main Experiment will be conducted in female animals only.
Following the initial Range-Finder Experiments, in females 10 mg/kg/day was considered an appropriate MTD for the study. Intermediate and low doses would be 5 and 2.5 mg/kg/day respectively. However these concentrations were not stable in formulation, hence the concentrations/doses from the repeated range-finder study were used, see below.

Repeated Range-Finder (Second trial):
At 10 mg/kg/day, there were no clinical signs of toxicity. All animals lost bodyweight (between -1.5% and -4.7%).
At 20 mg/kg/day, there were no clinical signs of toxicity following the first dose. Following the second dose, hunched posture was observed in 1/3 animals at 2 to 4-6 hours. All animals lost bodyweight (between -5.0% and -7.7%).
At 30 mg/kg/day, following the first dose 1/3 animals suffered a clonic convulsion immediately following the first dose. The animal was back to a normal state by 0.5 hours post-dose. Following the second dose, 3/3 animals suffered a clonic convulsion accompanied by gasping immediately following dosing. All animals were back to a normal state by 0.5 hours post-dose. At 4-6 hours post-dose, clinical signs of toxicity were observed in all animals including hunched posture (3/3), piloerection (1/3), lethargy (1/3), ataxia (1/3), laboured breathing (1/3) and hypothermia (1/3). All animals lost bodyweight (between -0.3% and 14.1%). Clinical signs including clonic convulsions and severe bodyweight loss were considered to be dose limiting in the repeated Range-Finder
Experiments in females. Following the repeated Range-Finder experiments, 20 mg/kg/day was considered an appropriate revised MTD for the study. Intermediate and low doses will be 10 and 5 mg/kg/day, respectively.

RESULTS OF DEFINITIVE STUDY
- No notable effect of treatment on body weights was observed.
- On macroscopic examination, no findings or changes were considered related to administration of TMPTA.
- Appropriateness of dose levels and route: Dose level selected on basis on the MTD of the range-finder study. The intravenous route of exposure was selected due to the irritation properties observed when administering TMPTA via the oral and dermal route of exposure.

-Validity of data: for the main experiment the data generated in this study confirm that:
The vehicle control data were comparable to laboratory historical control data for each tissue
1) The positive control induced responses that were comparable with the laboratory's historical control data and produced a marked increase in tail intensity compared to the concurrent vehicle control for each tissue
2) Adequate numbers of cells and doses were analysed
3) The high dose was considered to be the MTD
The assay data were therefore considered valid.

Text Table 3:TMPTA: Summary of Group Mean Data – Liver – Main Experiment 2

		Tail Intensity					Mean %Hedgehogs
Group / Dose Level (mg/kg/day)	Total No. Cells Scored	Mean	SEM	Back-Transformed Difference from Vehicle	P-value	Significance
6F / Vehicle (0)	900	0.48	0.08	-	-	-	0.52
7F /TMPTA(5)	900	0.31	0.12				0.21
8F /TMPTA(10)	900	0.24	0.05				0.54
9F /TMPTA(20)	900	0.59	0.17				0.21
10F / EMS (150)	450	17.60	1.78				3.16
Dose Response (Groups 6, 7, 8 & 9):to be added F                          Female SEM                   Standard error of means NS                       Not significant

 

Text Table 4:TMPTA: Summary of Group Mean Data – Bone Marrow – Main Experiment 2

		Tail Intensity					Mean %Hedgehogs
Group / Dose Level (mg/kg/day)	Total No. Cells Scored	Mean	SEM	Back-Transformed Difference from Vehicle	P-value	Significance
6F / Vehicle (0)	900	0.18	0.03	-	-	-	0.39
7F /TMPTA(5)	900	0.67	0.23				0.35
8F /TMPTA(10)	900	0.29	0.03				0.07
9F /TMPTA(20)	900	0.25	0.05				0.13
5F / EMS (150)	450	9.43	0.23				0.99
Dose Response (Groups 6, 7, 8 & 9):to be added F                          Female SEM                   Standard error of means NS                       Not significant Historical Control Ranges - Comet   MOUSE LIVER COMET HISTORICAL CONTROL RANGES Vehicle Control Data Tail Intensity (%) Hedgehogs (%) Number of Animals 78 72 Mean 1.28 4.07 Standard Deviation 1.37 3.37 Observed Range Minimum 0.04 0.00 Maximum 6.91 14.53 95% Reference Range Lower Limit 0.05 0.00 Upper Limit 5.30 11.30 Positive Control Data Tail Intensity (%) Hedgehogs (%) Number of Animals 64 59 Mean 21.89 9.55 Standard Deviation 18.06 7.00 Observed Range Minimum 2.99 0.00 Maximum 66.61 22.28 95% Reference Range Lower Limit 4.50 0.73 Upper Limit 63.81 21.32 Range compiled December 2016; generated from 13 studies dosed between January 2008 and June 2015   MOUSE BONE MARROW COMET HISTORICAL CONTROL RANGES Vehicle Control Data Tail Intensity (%) Hedgehogs (%) Number of Animals 5 5 Mean 0.44 7.38 Standard Deviation 0.18 2.30 Observed Range Minimum 0.24 3.37 Maximum 0.72 8.88 95% Reference Range Lower Limit NA NA Upper Limit NA NA Positive Control Data Tail Intensity (%) Hedgehogs (%) Number of Animals 3 3 Mean 11.45 10.06 Standard Deviation 2.44 0.69 Observed Range Minimum 8.68 9.55 Maximum 13.27 10.84 95% Reference Range Lower Limit NA NA Upper Limit NA NA Range compiled December 2016; generated from 1 study dosed in June 2015

Conclusions:

The toxicity of TMPTA on DNA damage in bonemarrow and liver in mice was evaluated according to OECD TG 489 (In Vivo Mammalian Alkaline Comet Assay, July 2016) under GLP. Female mice were intravenously exposed to TMPTA (5, 10, 20 mg/kg/bw) with PEG400 as vehicle. TMPTA, did not induce increases in tail intensity (DNA damage) in the liver or bone marrow as analysed by the comet assay when treated up to 20 mg/kg/day in females ( the maximum tolerated dose) under the experimental conditions employed.

Executive summary:

The toxicity of TMPTA on DNA damage in bonemarrow and liver in mice was evaluated according to OECD TG 489 (In Vivo Mammalian Alkaline Comet Assay, July 2016) under GLP.  In two dose-finder studies both male and female mice were intravenously exposed to TMPTA (0, 10, 20 ,30 mg/kg/bw) with PEG400 as vehicle. A MTD of 20 mg/kg/bw was considered appropriate based on clinical signs of mice in the 30 mg/kg exposure group e.g. hunched posture, piloerection, lethargy, ataxia, laboured breathing, hypothermia and loss of bodyweight.

In the main study there were no dose (5, 10, 20 mg/kg/bw) related increases in %hedgehogs in liver or bone marrow following treatment with TMPTA, thus demonstrating that treatment with TMPTA did not cause excessive DNA damage that could have interfered with comet analysis.

In the liver, female group mean tail intensity and tail moment values for all groups treated with TMPTA were comparable with the group mean vehicle control data. There were no marked increases in tail intensity between treated and the concurrent vehicle control group. Individual animal data at all dose levels were consistent with the vehicle control animals and fell within the laboratory’s historical control 95% reference range.

In the bone marrow, female group mean tail intensity and tail moment values for all groups treated with TMPTA were generally comparable with the group mean vehicle control data (the low dose of 5 mg/kg/day did exhibit a slightly elevated group mean tail intensity). There were no marked increases in tail intensity between treated groups at the intermediate or high doses (10 and 20 mg/kg/day) and the concurrent vehicle control group. Individual animal data at 10 and 20 mg/kg/day were consistent with the vehicle control animals. As there was no evidence of a dose response it is considered that the small increase in group mean tail intensity at 5 mg/kg/day is not biologically relevant.

Finally, it is concluded that TMPTA, did not induce biologically relevant increases in tail intensity (DNA damage) in the liver or bone marrow as analysed by the comet assay when treated up to 20 mg/kg/day in females (the maximum tolerated dose) under the experimental conditions employed

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

No data is available for Reaction mass of THEICTA and THEICDA (T).

Mutagenicity, in vitro, Salmonella typhimurium: 

OECD 471 testing performed with THEICTA as a mono-consituent substance was negative in all strains of Salmonella typhimuriumexcept strain TA 1535which showed positive response with metabolic activation.

OECD 471 testing for TMPTA andtris(2-hydroxyethyl) isocyanuratewere also showed mutagenic effects in this strain, however, only as a weak and non dose-related response.

Based on these data Reaction mass of THEICTA and THEICDA may be considered as having a mutagenic potential in bacteria.

Mutagenicity, in vitro, mutations mammalian cells:

OECD 476 testing performed with TMPTA was positive for mutagenic effects, however, only at cytotoxic exposure levels. Due to the very close structural similarity to TMPTA, Reaction mass of THEICTA and THEICDA may have a mutagenic potential in mammalian cells.

Mutagenicity, in vitro, chromosome aberration mammalian cells:

OECD 471 testing performed with TMPTA andtris(2-hydroxyethyl) isocyanurate was positive for TMPTA and negative fortris(2-hydroxyethyl) isocyanurate. Due to the very close structural similarity to TMPTA, Reaction mass of THEICTA and THEICDA should be considered as having a potential for a clastogenic effects in mammalian cellsin vitroas well.

Mutagenicity, in vivo, micronucleus formation, mice:OECD 474 as well as OECD 489 (Comet assay) testing performed with TMPTA were negative for genotoxic effectsin vivo. Due to the very close structural similarity to TMPTA, Reaction mass of THEICTA and THEICDA may be considered without genotoxic potential in mammalian cellsin vivoas well.  

Overall evaluation:

For THEICTA as mono-constituent substance, only data from Ames test is available, however, showing a positive response. Although a mutagenic and clastogenic potential of TMPTA could also be found in mammalian cells in vitro this could not be confirmed by in vivo testing OECD 474 and OECD 489. Based on the structural similarities between the two triacrylates read-across from the in vivo data on TMPTA is considered most relevant for assessment of the mutagenic potential in vivo of Reaction mass of THEICTA and THEICDA.

Thus, no classification applies for mutagenicity for Reaction mass of THEICTA and THEICDA.