p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-Jul-2011 to 29-Aug-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline

EC Number:

225-716-2

EC Name:

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline

Cas Number:

5026-74-4

Molecular formula:

C15H19NO4

IUPAC Name:

4-[(oxiran-2-yl)methoxy]-N,N-bis[(oxiran-2-yl)methyl]aniline

Test material form:

liquid: viscous

Details on test material:

- Substance type: organic
- Physical state: liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark

Method

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Source American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

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:

Dose range finding test 1:
Without and with S9-mix, 3 hours treatment: 33, 100, 333, 1000 and 2773 µg/mL
Dose range finding test 2:
Without S9-mix, 3 hours treatment: 0.01, 0.1, 1, 10 and 33 µg/mL
Mutation Experiment:
Without S9-mix, 3 hours treatment: 0.001, 0.01, 0.03, 0.1, 0.6, 1, 3.3 and 6.6 µg/mL
With S9-mix, 3 hours treatment: 0.3, 1, 3.3, 10, 33, 66, 100 and 125 µg/mL

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

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide.

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: 15 µg/mL

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Migrated to IUCLID6: 7.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Short-term treatment, With and without S9-mix: 3 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiment)

Evaluation criteria:

Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
Solvent control: 7.2
1000 µg/ml: 7.2
- Effects of osmolality: No
Solvent control: 0.417 mOsm/kg
1000 µg/ml: 0.582 mOsm/kg
- Precipitation: Precipitation in the exposure medium was observed at the dose level of 2773 µg/mL (= 0.01 M)

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 0.01 µg/mL in the absence of S9, 3 hours treatment; at dose levels of 100 µg/mL in the presence of S9, 3 hours treatment.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The relative total growth of the highest test substance concentration was reduced by 67 and 90% compared to the total growth of the solvent controls in the absence and presence of S9-mix, respectively.

Remarks on result:

other: strain/cell type: Test system L5178Y/TK+/-3.7.2C

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

P-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline was tested up to concentrations of 6.6 and 125 µg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Since the relative suspension growth (after 48 hours cell culture) was 13% at the test substance concentration of 6.6 μg/ml in the absence of S9-mix and 16% at the test substance concentration of 125 μg/ml in the presence of S9-mix, these dose levels were selected as highest dose level to be tested. After 11 days of culturing the CE_day2 appeared very low in the 2 highest dose groups. High toxicity was observed resulting in a relative total growth of 1 and 0. Consequently only 6 dose levels were analysed for expression of the TK mutation.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

The mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles.
It is concluded that p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline is mutagenic in the mouse lymphoma L5178Y test system.

Executive summary:

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

In the absence of S9-mix, p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline induced an up to 15-fold increase in the mutation frequency.The increases observed were above the GEF + MF_(controls)(203 per 10⁶survivors), more than three-fold, outside the historical control data range and in a dose dependent manner. Therefore, these increases are considered relevant and p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline is considered mutagenic in the absence of S9-mix.

 

In the presence of S9-mix, p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline induced an up to 33-fold increase in the mutation frequency.The increases observed were above the GEF + MF_(controls)(182 per 10⁶survivors), more than three-fold, outside the historical control data range and in a dose dependent manner. Therefore, these increases are considered relevant and p-(2,3 -epoxypropoxy)-N,N-bis(2,3 -epoxypropyl)aniline is considered mutagenic in the presence of S9 -mix.