Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substancewas tested in two reverse mutation (Ames) assays; in the first, it was negative and in the second, it was positive only in S. typhimurium strain TA1535 in the presence of metabolic activation (no mutagenicity was observed in the otherS. typhimurium strains (TA1537, TA100 or TA98) or the Escherichia coli WP2uvrA with or without metabolic activation). Furthermore, the substance was negative in an in vitro gene mutation assay in mammalian cells and an in vivo mouse micronucleus test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 14 Nov, 1984 to 21 Dec, 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium: Histidine gene
Species / strain / cell type:
other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Aroclor-1254 induced rat liver S9-mix). This contained 0.10 µL of S9 supernatent (40.7 mg protein/mL) per 1 mL of S-9 mix.
Test concentrations with justification for top dose:
Preliminary test: 50, 166, 500, 1666 and 5000 µg/plate (for 2 strains TA 1538 and TA 100) without metabolic activation
Plate incorporation assay: 50, 166, 500, 1666 and 5000 µg/plate (for tester strains) with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (spectrophotometric grade)
- Lot no.: 744197
- Supplier: Mallinckrodt Inc, Paris, Kentucky, 40361
- Justification for choice of solvent/vehicle: Test substance was soluble in DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
TA 1535
Positive control substance:
other: sodium azide (10 µg/plate for TA 1535)
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
TA 1537
Positive control substance:
other: 9-aminoacridine (150 µg/plate for TA1537)
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
TA 98 and TA 1538
Positive control substance:
other: 2-nitrofluorene (5 µg/plate for TA 98)
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
TA 100
Positive control substance:
sodium azide
other: Sodium azide (10 µg/plate TA100)
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Positive control substance:
other: 2-aminoanthracene (5 µg/plate) for all tester strains
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar

DURATION:
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted (preliminary and plate incorporation tests).

COLONY COUNTING: The revertant colonies counting were counted on an electronic colony counter interfaced with an Apple computer for data acquisition

DETERMINATION OF CYTOTOXICITY
- Method: Reduction of the bacterial background lawn, increase in the size of the microcolonies and reduction of the revertant colonies.

Evaluation criteria:
In most tests with the Salmonella/Microsome Assay, results are either clearly positive or clearly negative.
A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay.
- If the solvent control is within the 95% confidence limits of the historical mean for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive.
- A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
All solvent and positive controls used in the evaluation of the test substance were within the acceptable limits of mean historical data.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Refer to attached document under 'Attached background material' for details on results.

Conclusions:
Under the test conditions, di-TMPTTA was not mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, with and without metabolic activation.
Executive summary:

A study was conducted to determine the mutagenic potential of di-TMPTTA according to a method equivalent or similar to OECD Guideline 471. In a preliminary study, the substance was tested up to concentrations of 5,000 µg/plate in Salmonella typhimuriurns trains TA1538 and TA100. No inhibition of bacterial lawn grown was observed at any dose. Based on these results, the substance was tested in strains TA1535, TA1537, TA1538, TA98 and TA100 both with and without metabolic activation with S9 mix at 50, 166, 500, 1,666 and 5,000 µg/plate. No mutagenicity was observed at any dose level. The test substance was therefore not considered to be mutagenic with and without metabolic activation under the conditions of this assay (Godek 1985).

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 26 April, 2012 to 10 May, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 33, 100, 333, 1000 and 3330 µg/plate
Experiment 2:
Without and with S9-mix: 33, 100, 333, 1000 and 3330 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Positive control substance:
2-nitrofluorene
Remarks:
without S Migrated to IUCLID6: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
sodium azide
Remarks:
without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
Key result
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at dose levels of 3330 and 5000 µg/plate


RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity was observed up to and including the top dose of 3330 µg/plate
Conclusions:
Under the study conditions, di-TMPTTA was mutagenic at up to 3330 μg/plate only in tester strain TA1535 of the Salmonella typhimurium in the presence of metabolic activation only. No mutagenicity was observed in the other Salmonella typhimurium tester strains (TA1537, TA100 or TA98) or the Escherichia coli tester strain WP2uvrA with or without metabolic activation.
Executive summary:

A study was conducted to evaluate the mutagenic activity of di-TMPTTA in areverse mutation assay according to OECD Guideline 471 and EU Method B.13/14. S. typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli tryptophan-requiring strain WP2uvrA were used. The test was performed in two independent experiments in the presence and absence of S9 mix. In a dose range-finder, the substance was tested up to concentrations of 5,000 μg/plate in the absence and presence of S9 mix in TA100 and WP2uvrA. Precipitation was observed on the plate at 3,330 and 5,000 μg/plate. The bacterial background lawn was not reduced at any concentrations and no biologically relevant decrease in the number of revertants was observed. Based on these results, the substance was tested in the first mutation assay at 33 to 3,330 μg/plate in the absence and presence of metabolic activation in S. typhimurium strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, the substance was tested at the same concentrations in the absence and presence of metabolic activation in S. typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2uvrA. Precipitation was observed on the plates at 3,330 μg/plate. The bacterial background lawn was not reduced at any concentration and no biologically relevant decrease in the number of revertants was observed. In the presence of S9 mix, the substance induced 14.5- and 8.5-fold increases in strain TA1535 in the first and second experiment, respectively. The increases observed were above the laboratory historical control data range, greater than three times the concurrent control, dose-related and observed in two independently repeated experiments. All other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The test substance at concentrations up to 3330 μg/plate was therefore mutagenic only in S. typhimurium strain TA1535 in the presence of metabolic activation. No mutagenicity was observed in the other S. typhimurium strains (TA1537, TA100 or TA98) or the Escherichia coli WP2uvrA with or without metabolic activation (Verspeek-Rip 2013).

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:
From7 January , 2014 to 31 March , 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
HPRT catalyzes the conversion of the nontoxic 6-TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6-TG. These cells are able to proliferate in the presence of 6-TG whereas the non-mutated cells die.
Metabolic activation:
with and without
Metabolic activation system:
S9 preparation form liver of male rats treated with three daily doses of a mixture of phenobarbitone and beta-naphthoflavone.
Test concentrations with justification for top dose:
Preliminary cytotoxicity test: 18.2; 36.41; 72.81; 145.63; 291.25; 582.5; 1,165; 2,330 and 4,660 µg/mL
Mutagenicity test - Experiment 14 h without S9: 0*; 0.25*; 0.5*; 1*; 1.5*; 2*; 2.5‡; 3; 4; 6; EMS 500* and 750* µg/mL 4 h with S9 (2%): 0*; 4*; 8*; 16*; 24*; 30*; 36*; 48; DMBA 1.0* and 2.0* µg/mL (* = dose levels plated out for mutant frequencyMutagenicity Test - Experiment 224 h without S9: 0*; 0.5*; 1*; 2*; 3*; 4*; 5; 6; 8; EMS 250* and 500* µg/mL 4 h with S9 (2%): 0*; 4*; 8*; 16*; 24*; 30*; 32*; 36*; 40*; DMBA 1.0* and 2.0* µg/mL
Vehicle / solvent:
Dimethyl sulphoxide (DMSO)
Untreated negative controls:
yes
Remarks:
Solvent treatment groups
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
500 µg/mL and 750 µg/mL as the positive control in the 4 h cultures without S9 and at 250 and 500 µg/mL for the 24 h cultures without S9.
Untreated negative controls:
yes
Remarks:
Solvent treatment groups
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
Remarks:
1.0 and 2.0 µg/mL as the positive control in cultures with S9.
Details on test system and experimental conditions:
Test Substance PreparationThe test substance was accurately weighed and dissolved in dimethyl sulphoxide (DMSO) and appropriate dilutions made. The molecular weight of the test substance was 466 and therefore the maximum recommended dose was the 10 mM concentration of 4660 µg/mL. The purity of the test substance was greater than 99% and therefore no purity correction was required. The test substance was formulated and dosed under safety light throughout the study. There was no significant change in pH when the test substance was dosed into media and the osmolality did not increase by more than 50 mOsm at the dose levels investigated.
Evaluation criteria:
The test substance was considered to be mutagenic if there was a reproducible dose-related increase in the mutation frequency where at least a threefold increase in the mutant frequency over the vehicle control value was observed. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency was not observed.
A test substance producing neither a dose-related increase of the mutant frequency nor a reproducible positive response at any of the test points was considered to be non-mutagenic in this system.
A single dose level that meets the minimum criterion for a positive response within a range of assayed concentrations was not sufficient to evaluate the test substance as a mutagen.
Key result
Species / strain:
mammalian cell line, other: V79 cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:

Preliminary cytotoxicity test
A dose range of 18.2; 36.41; 72.81; 145.63; 291.25; 582.5; 1,165; 2,330 and 4,660 µg/mL was used initially in the preliminary cytotoxicity test. The maximum dose tested was the maximum recommended dose level, the 10mM concentration. However, due to excessive toxicity, with no surviving dose levels in the 4h exposure in the absence of S9 and in the 24h exposure group, these exposures were repeated with a dose range of 0.125; 0.25; 0.5; 1; 2; 4; 8; 16 and 24 µg/mL.

A greasy/oily precipitate of the test substance was observed at the end of exposure at and above 2330 µg/mL in the 4 h exposure group in the presence of S9. In the 24 h exposure group cloudy precipitate was observed at 4 µg/mL at the end of the exposure period. No precipitate was observed at the end of the exposure period in the 4 h exposure group without S9. The cultures at and above 8 µg/mL were discarded in the 4h exposure group and in the 24 h exposure group as there were no viable cells remaining at the end of the exposure period. In the 4 h exposure group in the presence of S9, the dose levels at and above 145.63 µg/mL were not plated due to toxicity and no viable cells remaining at the end of the exposure period.

The results of the individual flask counts and their analysis are presented in Table 1. It can be seen that the test substance was markedly toxic and there was a dose related reduction in the plating efficiency (PE) demonstrated in all three exposure groups.

The maximum dose level selected for the main experiments was limited by toxicity and was 6 µg/mL and 48 µg/mL for the 4 h exposure groups of Experiment 1 in the absence and presence of S9 respectively. In Experiment 2, the maximum dose selected for the 24 h exposure group was 8 µg/mL and for the 4 h exposure group in the presence of S9 was 40 µg/mL.

Mutagenicity Test - Experiment 1

No precipitate of the test substance was seen at the end of the exposure period in either the absence or presence of S9.

 The Day 0 and Day 7 plating efficiencies for the without and with metabolic activation exposure groups are presented in Table 2 and Table 3. There was a marked reduction in plating efficiency demonstrated at Day 0 in both the presence and absence of S9. The 4 h exposure group in the absence of S9 demonstrated a dose related reduction in plating efficiency with decreases of 63% and 80% at 1.5 µg/mL and 2 µg/mL respectively. The dose level of 2.5 µg/mL achieved a 98% reduction in plating efficiency and although this dose level was plated, the data has been excluded as it was considered to be too toxic and beyond the acceptable minimum. In the presence of S9 a dose related reduction in plating efficiency at Day 0 was observed, with reductions of 46% and 66% at 24 µg/mL and 30 µg/mL respectively. The dose level of 36 µg/mL achieved a reduction in plating efficiency of 93% which although greater than the ideal maximum, was plated because it provided an intermediate dose level in the toxicity curve. The dose level of 48 µg/mL had no surviving cells remaining at the end of the exposure period and was discarded prior to plating. There was no marked reduction in the plating efficiencies at Day 7 in either the absence or presence of S9.

 The mutant frequency counts and mean mutant frequency per survivor values are presented in Table 2 and Table 3. There were no dose related increases in mutant frequency per survivor or any dose level which gave a threefold increase in the mutant frequency in either the absence or presence of S9. 

 It can be seen that the vehicle control values were all considered to be within an acceptable range, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

Mutagenicity Test - Experiment 2

No precipitate of the test substance was seen at the end of the exposure period in either the 24 h exposure group in the absence of S9 or in the 4 h exposure group in the presence of S9.

The Day 0 and Day 7 cloning efficiencies for the without and with metabolic activation exposure groups are presented in Tables 4 and 5. It can be seen that, as in Experiment 1, the test substance was markedly toxic and there was a dose related reduction in plating efficiency in both exposure groups at Day 0. The 4 h exposure group in the presence of S9 was marginally less toxic than in Experiment 1 and an 88% reduction in plating efficiency was achieved at the maximum dose tested of 40 µg/mL at Day 0. In the 24 h exposure group at Day 0, there were reductions in plating efficiency of 53% and 66% at 3 µg/mL and 4 µg/mL respectively. The dose levels of 5 µg/mL, 6 µg/mL and 8 µg/mL were not plated as there were no viable cells remaining at the end of the exposure period. There was no marked reduction in the plating efficiencies at Day 7 in either exposure group. A Day 7 viability flask was lost from the ‘A’ replicate of the 1 µg/mL DMBA exposure due to a technical error. The calculations were therefore based on the remaining two flasks. 

The mutant frequency counts and mean mutant frequency per survivor per 106 cells values are presented in Tables 4 and 5. There were no increases in mutation frequency per survivor that gave a dose related or threefold increase over the vehicle control value in either exposure group. The mutant frequency of the ‘A’ and ‘B’ replicates of the 2 µg/mL dose level in the absence of S9 were markedly different with the ‘A’ replicate demonstrating a much higher mutant frequency. However, this was considered to be of no significance as it was not part of a dose related response and was considered to be normal biological variation. 

 It can be seen that the vehicle control values were all considered to be within an acceptable range, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

Conclusions:
Under the study conditions, Ditrimethylolpropane tetraacrylate was considered to be non-mutagenic to V79 cells at the HPRT locus.
Executive summary:

A study was conducted to assess the potential mutagenicity of the test substance on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line, according to OECD Guideline 476, in compliance with GLP. Chinese hamster (V79) cells were treated with the test substance at up to nine dose levels, in duplicate, together with the vehicle (dimethyl sulphoxide) and positive controls in the presence and absence of an S9 metabolic activation system. Three exposure condititons were used for the test, i.e. in Experiment 1, a 4 h exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration and a 4 h exposure in the absence of metabolic activation (S9). In Experiment 2, the 4 h exposure in the presence of S9 was repeated using the same S9 concentration and a revised dose range and a 24 h continuous exposure in the absence of S9 was performed. The dose range of the test substance was selected based on the results of a preliminary cytotoxicity test and were as follows:

 

Exposure Group

Final concentration oftest substance(µg/mL)

4 h without S9

0.25; 0.5; 1; 1.5; 2; 2.5; 3; 4; 6

4 h with S9 (2%)

4; 8; 16; 24; 30; 36; 48

24 h without S9

0.5; 1; 2; 3; 4; 5; 6; 8

4 h with S9 (2%)

4; 8; 16; 24; 30; 32; 36; 40

 

The vehicle (solvent) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system. The test substance demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The test substance was considered to be non-mutagenic to V79 cells at the HPRT locus under the study conditions (Morris 2014).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Based on the negative result in in vivo micronucleus test, the test substance was considered as non-genotoxic (non-clastogenic).

Link to relevant study records
Reference
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 30 Aug, 2012 to 14 Oct, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Hsd :ICR (CD-I)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, USA
- Date of receival: 30 August, 2012 (dose range finding) and 20 September, 2012 (definitive micronucleus study)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: DRF: 29.1-33.2 g (males) or 25.9-26.9 g (females); Definitive study: 30.7-37.0 g (males)
- Number of animals: Dose range finding: 20/sex, definitive study: 35/sex
- Assigned to test groups randomly: Yes, animals randomized using a computer program
- Housing: Animals were housed up to five/micro-barrier cage which were placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system
- Randomization: Animals were assigned to groups using a randomization procedure based on equalization of group mean body weights. At the time of randomization, the weight variation of animals will not exceed ±20% of the mean weight.
- Bedding: Heat treated hardwood chips were used
- Diet (e.g. ad libitum): Certified laboratory rodent chow (Harlen 2018C certified global rodent diet), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 5 to 11 d (mice were observed each day for signs of illness and other conditions of poor health.)

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3°F
- Humidity: 50 ± 20%
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
- Lot/batch no. (if required): MKBD6671
- CAS no.: 8001-30-7
- Expiration date: 24 september, 2013
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance dose formulations were prepared fresh for each phase of the study prior to use in dose administration. All formulations for the dose range finding (DRF) assay (0.1, 1, 10, 100, and 200 mg/mL) and all formulations for the definitive micronucleus assay (50, 100, and 200 mg/mL) were prepared as follows:
An appropriate amount of the test substance was combined with an appropriate volume, 80% of the target volume, of the vehicle. Each formulation was vortexed for 2 min and stirred for 10 min using a magnetic stir bar/plate. Remaining volume of the vehicle was added to reach the final targeted volume and each formulation was stirred for an additional 10 min. All formulations appeared as yellow solutions. All formulations were stirred using a magnetic stir bar/plate continuously prior to and during dose administration.

Positive control dose formulation:
An aqueous dosing formulation of cyclophosphamide (CP) at a concentration of 5 mg/mL was prepared fresh on the day of dose administration. An appropriate amount of CP was dissolved in an appropriate volume of sterile water for injection (B. Braun Medical, CAS number: 7732-18-5, lot number: J1L003 expiration date: September 2013). The formulation was vortexed for 2 min. The accuracy of preparation and stability of the CP formulation was demonstrated by acceptable results that met the criteria for a valid test.
Duration of treatment / exposure:
24 or 48 h
Frequency of treatment:
Single administration
Remarks:
Doses / Concentrations:
1, 10, 100, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
(for dose range-finding study)
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
(for definitive micronucleus study)
No. of animals per sex per dose:
-Range-finding study: 5 male and 5 female mice at 2000 mg/kg and two male mice each exposed to the test substance at 1, 10, 100 or 1000 mg/kg bw
-Main study: Five male mice/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate (CP)
- CAS number: 6055-19-2
- Lot number: SLBC0666V
- Expiration date: 31 March 2015, was obtained from Sigma-Aldrich.
Tissues and cell types examined:
Femurs were removed for marrow extraction and the prepared slides were examined for polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs) and total erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose selection was based on a preliminary dose range-finding test conducted on 5 male and 5 female mice at 2000 mg/kg bw and two male mice each to the test substance at 1, 10, 100 or 1000 mg/kg bw. No mortality and/or toxic signs were recorded after 2 d of exposure.

TREATMENT AND SAMPLING TIMES: 5 mice per treatment were euthanized by CO2 asphyxiation verified by toe pinch reflex at 24 or 48 h after exposure. Immediately following euthanasia. The femurs were exposed, cut just above the knee and the bone marrow was aspirated into a syringe containing fetal bovine serum.The bone marrow cells were transferred to a labeled centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at about 100 x g for about five minutes and the supernatant was drawn off, leaving a small amount of
serum with the remaining cell pellet

DETAILS OF SLIDE PREPARATION: Bone marrow cells extracted, preparations spread on slides and air-dried. The slides were fixed in methanol. One set of slides was stained with a nucleic acid-specific stain acridine orange and was used in microscopic evaluation. Second set was packaged for storage until finalization for the report. s

METHOD OF ANALYSIS: Slides were scanned to determine the frequency of micronuclei in 2,000 polychromatic erythrocytes (PCEs) per animal. In addition, at least 1,000 total erythrocytes (PCEs + NCEs) were scored per animal to determine the proportion of PCEs as an index of bone marrow cytotoxicity. PCE proportions <20% of vehicle control value were considered excessively cytotoxic and the animal data were excluded from evaluation.
Evaluation criteria:
Criteria for a Valid Test
The micronucleated polychromatic erythrocytes (mnPCE) frequency of the vehicle controls must be within the historical vehicle control range and the positive control must induce a significant increase (p≤0.05) in mnPCE frequency as compared to concurrent vehicle control.

Evaluation of Test Results
Once the criteria for a valid assay were met, the results were evaluated as follows:
- The test substance is considered to be positive if it induces a significant increase in mnPCE frequency (p≤0.05) at any dose level or sampling time compared to the concurrent vehicle control.

- The test substance is considered to be negative if no significant increase in mnPCE frequency is observed (p> 0.05) compared to the concurrent vehicle control.

Mice were observed for clinical signs of toxicity prior to and following each dose administration and at least once daily thereafter. Body weights were recorded at least once on each day of dosing.
Statistics:
The frequency of mnPCEs and the proportion of PCEs to total erythrocytes were determined for each animal and treatment group. Statistical significance (p≤0.05) was determined using the binomial distribution (Kastenbaum-Bowman tables).
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Piloerection was noted in one of the mice at 500 and 1000 mg/kg bw and in all mice at 2000 mg/kg bw following dose administration.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DRF study:
No mortality was observed in any of the treatment groups. All mice at 1, 10, and 100 mg/kg bw appeared normal during the study period. Piloerection was noted in male mice at 1000 and 2000 mg/kg bw, while this clinical sign was noted transiently in female mice. No appreciable reductions to the mean group body weights were observed.

Definitive Study:
- Clinical Signs
No mortality was observed in any of the treatment groups. All mice in the control groups, and in the test substance groups at 500 mg/kg bw, appeared normal. Piloerection was noted at 1000 and 2000 mg/kg bw.
- Bone Marrow Evaluation
No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the test substance groups relative to the respective vehicle control groups were observed, suggesting that the erythropoiesis was not inhibited.
- No statistically significant increase in the incidence of mnPCE in test substance groups relative to the respective vehicle control groups was observed at 24 or 48 h.
CP, the positive control, induced a statistically significant increase in the incidence of mnPCEs (p≤ 0.05, Kastenbaum-Bowman Tables). The number of mnPCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol.

- Frequency of micronuclei in polychromatic erythrocytes and PCE:NCE ratio: See table 1

Results: Refer to the attached background material for details on results.

Conclusions:
Under the conditions of the mouse micronucleus assay, di-TMPTTA was negative (not clastogenic).
Executive summary:

An in vivo bone marrow micronucleus test was performed with di-TMPTTA according to OECD Guideline 474, in compliance with GLP. The study was conducted in two phases: a dose range-finding assay and a definitive micronucleus assay. In both phases of the study, test and/or control mice were administered at a dose volume of 10 mL/kg bw by a single oral gavage. The dose range-finding assay was performed exposing two males/group at 1, 10, 100, 1,000 mg/kg bw, while five male and five females were exposed to 2,000 mg/kg bw. No mortality was observed in any of the treatment groups. All mice at 1, 10 and 100 mg/kg bw appeared normal during the study period. Piloerection was noted in males at 1,000 and 2,000 mg/kg bw, while this clinical sign was noted transiently in females. No appreciable reductions in mean group body weights were observed. In the definitive micronucleus assay, mice were dosed either with the control (vehicle or positive) or with the test substance at 500, 1,000 or 2,000 mg/kg bw. No mortality was observed in any of the treatment groups. All mice in the control groups and in the treated groups at 500 mg/kg bw appeared normal. Piloerection was noted at 1,000 and 2,000 mg/kg bw. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the treated groups relative to the respective vehicle control groups were observed, suggesting that the test substance did not inhibit erythropoiesis. No statistically significant increase in the incidence of micronucleated polychromatic erythrocyte cells in treated groups relative to the respective vehicle control groups was observed at 24 or 48 h after dose administration. Under the conditions of the mouse micronucleus assay, the test substance was negative (not clastogenic) (Kulkarni, 2013).

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

Additional information

In vitro

A study was conducted to determine the mutagenic potential of the test substance according to a method equivalent or similar to OECD Guideline 471. In a preliminary study, the substance was tested up to concentrations of 5,000 µg/plate inSalmonella typhimuriumstrains TA1538 and TA100. No inhibition of bacterial lawn grown was observed at any dose. Based on these results, the substance was tested in strains TA1535, TA1537, TA1538, TA98 and TA100 both with and without metabolic activation with S9 mix at 50, 166, 500, 1,666 and 5,000 µg/plate. No mutagenicity was observed at any dose level. The test substance was therefore not considered to be mutagenic under the conditions of this assay.

A second study was conducted to evaluate the mutagenic activity of the test substance in a reverse mutation assay according to OECD Guideline 471 and EU Method B.13/14.S. typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia coli tryptophan-requiring strain WP2uvrA were used. The test was performed in two independent experiments in the presence and absence of S9 mix. In a dose range-finder, the substance was tested up to concentrations of 5,000 μg/plate in the absence and presence of S9 mix in TA100 and WP2uvrA. Precipitation was observed on the plate at 3,330 and 5,000 μg/plate. The bacterial background lawn was not reduced at any concentrations and no biologically relevant decrease in the number of revertants was observed. Based on these results, the substance was tested in the first mutation assay at 33 to 3,330 μg/plate in the absence and presence of metabolic activation inS. typhimuriumstrains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, the substance was tested at the same concentrations in the absence and presence of metabolic activation inS. typhimuriumstrains TA1535, TA1537, TA98, TA100 andE. coliWP2uvrA. Precipitation was observed on the plates at 3,330 μg/plate. The bacterial background lawn was not reduced at any concentration and no biologically relevant decrease in the number of revertants was observed. In the presence of S9 mix, the substance induced 14.5- and 8.5-fold increases in strain TA1535 in the first and second experiment, respectively. The increases observed were above the laboratory historical control data range, greater than three times the concurrent control, dose-related and observed in two independently repeated experiments. All other bacterial strains showed negative responses over the entire dose range, i. e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The test substance at concentrations up to 3330 μg/plate was therefore mutagenic only inS. typhimuriumstrain TA1535 in the presence of metabolic activation. No mutagenicity was observed in the otherS. typhimuriumstrains (TA1537, TA100 or TA98) or theEscherichia coliWP2uvrA with or without metabolic activation.

A further study was conducted to assess the potential mutagenicity of the test substance on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line according to OECD Guideline 476. Chinese hamster (V79) cells were treated with the test substance at up to nine dose levels, in duplicate, together with the vehicle (dimethyl sulphoxide) and positive controls in the presence and absence of an S9 metabolic activation system. Three exposure condititons were used for the test, i.e. in Experiment 1, a 4 h exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration and a 4 h exposure in the absence of metabolic activation (S9). In Experiment 2, the 4 h exposure in the presence of S9 was repeated using the same S9 concentration and a revised dose range and a 24 h continuous exposure in the absence of S9 was performed. The dose range of the test substance was selected based on the results of a preliminary cytotoxicity test and were as follows:

Exposure Group

 Final concentration of test substance(µg/mL)

4 h without S9

0.25; 0.5; 1; 1.5; 2; 2.5; 3; 4; 6

4 h with S9 (2%)

4; 8; 16; 24; 30; 36; 48

24 h without S9

0.5; 1; 2; 3; 4; 5; 6; 8

4 h with S9 (2%)

4; 8; 16; 24; 30; 32; 36; 40

The vehicle (solvent) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system. The test substance demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The test substance was considered to be non-mutagenic to V79 cells at the HPRT locus under the study conditions.

Anin vitrocytogenicity study in mammalian cells was not conducted sincein vivotesting was available (mouse micronucleus study).

In vivo

Anin vivobone marrow micronucleus test was performed with the test substance according to OECD Guideline 474. The study was conducted in two phases: a dose range-finding assay and a definitive micronucleus assay. In both phases of the study, test and/or control mice were administered at a dose volume of 10 mL/kg bw by a single oral gavage. The dose range-finding assay was performed exposing two males/group at 1, 10, 100, 1,000 mg/kg bw, while five male and five females were exposed to 2,000 mg/kg bw. No mortality was observed in any of the treatment groups. All mice at 1, 10 and 100 mg/kg bw appeared normal during the study period. Piloerection was noted in males at 1,000 and 2,000 mg/kg bw, while this clinical sign was noted only transiently in females. No appreciable reductions in mean group body weights were observed. In the definitive micronucleus assay, mice were dosed either with the control (vehicle or positive) or with the test substance at 500, 1,000 or 2,000 mg/kg bw. No mortality was observed in any of the treatment groups. All mice in the control groups and in the treated groups at 500 mg/kg bw appeared normal. Piloerection was noted at 1,000 and 2,000 mg/kg bw. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the treated groups relative to the respective vehicle control groups were observed, suggesting that the test substance did not inhibit erythropoiesis. No statistically significant increase in the incidence of micronucleated polychromatic erythrocyte cells in treated groups relative to the respective vehicle control groups was observed at 24 or 48 h after dose administration. Under the conditions of the mouse micronucleus assay, the test substance was negative (not clastogenic).

Justification for classification or non-classification

Based on the results of in vitro and in vivo testing conducted with the test substance, the substance is not considered to require classification for genotoxicity according to CLP (EC 1272/2008) criteria.