Reaction mass of bis(oxiran-2-ylmethyl) terephthalate and tris(oxiran-2-ylmethyl) benzene-1,2,4-tricarboxylate

Genetic toxicity: in vitro

Administrative data

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:

2013-12-03 to 2014-01-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,2,4-tris[(oxiran-2-yl)methyl] benzene-1,2,4-tricarboxylate; 1,4-bis[(oxiran-2-yl)methyl] benzene-1,4-dicarboxylate

EC Number:

940-592-6

Molecular formula:

-

IUPAC Name:

1,2,4-tris[(oxiran-2-yl)methyl] benzene-1,2,4-tricarboxylate; 1,4-bis[(oxiran-2-yl)methyl] benzene-1,4-dicarboxylate

Details on test material:

Physical State: solid/granulate
Storage Conditions: at room temperature
Molecular Weight (Base Form): 278 g/mol
Purity: 100%

Method

Target gene:

His

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver microsomal fraction

Test concentrations with justification for top dose:

Experiment I:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(TA 98 with and without metabolic activation and TA 100 with metabolic activation)
0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate (TA 100 without metabolic activation, TA 1535, TA 1537 and TA 102)

Experiment II:
1.58, 5.00, 15.8, 50.0, 158, 500 and 1500 µg/plate
(TA 98 with and without metabolic activation
TA 100, TA 1535, TA1537 with metabolic activation)
0.158, 0.50, 1.58, 5.00, 15.8, 50.0 and 158 µg/plate
(TA 100 and TA 1535 without metabolic activation)
0.50, 1.58, 5.00, 15.8, 50.0, 158 and 500 µg/plate
(TA 1537 without metabolic activation)
5.00, 15.8, 50.0, 158, 500, 1500 and 3000 µg/plate
(TA 102)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Remarks:

A. dest., BSL Lot No. 131122

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, AppliChem Lot No. 3T003218

Positive controls:

yes

Positive control substance:

other:

Remarks:

Positive control without metabolic activation: sodium azide for tester strains TA100, TA1535; 4-nitro-o-phenylene-diamine for TA98, TA1537; methylmethanesulfonate for TA102. Positive control with metabolic activation: 2-aminoanthracene for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 60 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Toxic effects of the test item were noted in all tester strains evaluated:

In experiment I toxic effects of the test item were observed in tester strain TA 98 at concentrations of 316 µg/plate and higher (withoutmetabolic activation) and at concentrations of 1000 µg/plate and higher (withmetabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 31.6 µg/plate and higher (withoutmetabolic activation) and at concentrations of 316 µg/plate (withmetabolic activation). In tester strain TA 1535 toxic effects of the test item were observed at concentrations of 100 µg/plate and higher (withoutmetabolic activation) and at concentrations of 316 µg/plate and higher (withmetabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 100 µg/plate and higher (withoutmetabolic activation) and at concentrations of 1000 µg/plate and higher (withmetabolic activation). In tester strain TA 102 toxic effects of the test item were observed at a concentration of 2500 µg/plate (withoutmetabolic activation).

In experiment II toxic effects of the test item were noted in tester strains TA 98 and TA 1537 at concentrations of 158 µg/plate and higher (withoutmetabolic activation) and at concentrations of 500 µg/plate and higher (withmetabolic activation). In tester strains TA 100 and TA 1535 toxic effects of the test item were noted at concentrations of 50 µg/plate and higher (withoutmetabolic activation) and at concentrations of 500 µg/plate and higher (withmetabolic activation). Biologically relevant increases of revertant colony numbers were observed as follows:

Table4: Overview ofConcentrations Showing Mutagenic Effects

	Experiment I		Experiment II
Tester Strain	- S9	+ S9	- S9	+ S9
TA 98	10.0 to 316 µg/plate	-	15.8 to 158 µg/plate	-
TA 100	10.0 µg/plate	-	-	-
TA 1535	10.0 to 31.6 µg/plate	10.0 to 316 µg/plate	15.8 to 50.0 µg/plate	158 to 500 µg/plate
TA 1537	-	-	-	-
TA 102	≥ 1000 µg/plate	≥ 1000 µg/plate	≥ 1500 µg/plate	≥ 1500µg/plate

- S9:    without metabolic activation

+ S9:   with metabolic activation

The threshold value of 2.0 was exceeded and a maximum mutation factor of 8.8 was reached in tester strain TA 98 in experiment II at a concentration of 158 µg/plate (withoutmetabolic activation). Also the threshold value of 3.0 was exceeded and a maximum mutation factor of 9.2 was reached in tester strain TA 1535 in experiment I at a concentration of 100 µg/plate (withmetabolic activation). Moreover, a dose-response relationship was found in these tester strains up to the bacteriotoxic concentrations.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test material caused gene mutations by base pair changes and frameshifts in the genomes of the tester strains TA 102, TA 100, TA 1535 and TA 98, respectively.
Therefore, the test material is considered to be mutagenic in this bacterial reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to the test material at different concentrations (see above), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I and II).

The test material was tested up to the limit concentration of 5000 µg/plate depending on the particular tester strain.

The positive controls induced the appropriate responses in the corresponding strains. There was evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.