Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, N-(hydroxyethyl) derivs.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

05-Sep-2011 to 28-Nov-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

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:

Ethanol, 2,2'-[[3-[(2-hydroxyethyl)amino]propyl]imino]bis-, N-C12-18-alkyl derivs.

EC Number:

291-269-5

EC Name:

Ethanol, 2,2'-[[3-[(2-hydroxyethyl)amino]propyl]imino]bis-, N-C12-18-alkyl derivs.

Cas Number:

90367-21-8

Molecular formula:

(C8-18H17-37)-N-[C2H4O]3H-(CH2)3-N-[(C2H4O)3]2

Constituent 2

Reference substance name:

Tris (2-hydroxyethyl) Cocoalkyl diaminopropane

IUPAC Name:

Tris (2-hydroxyethyl) Cocoalkyl diaminopropane

Test material form:

other: clear amber liquid

Details on test material:

- Name of test material (as cited in study report): Tris(hydroxyethyl) C12-18 alkyl diaminopropane
- Substance type: Clear amber liquid
- Physical state: Liquid
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark under nitrogen

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):

- 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 2500 µg/mL
Without S9-mix, 24 hours treatment: 33, 100, 333, 1000 and 2500 µg/ml
Dose range finding test 2:
Without S9-mix, 3 and 24 hours treatment: 0.01, 0.1, 1, 3, 10 and 33 µg/mL

Experiment 1:
Without S9-mix, 3 hours treatment: 0.01, 1, 1.5, 2, 3, 3.5, 4 and 4.5 µg/mL
With S9-mix, 3 hours treatment: 0.01, 0.1, 1, 5, 10, 15, 20 and 25 µg/mL

Experiment 2
Without S9-mix, 24 hours treatment: 0.01, 0.03, 0.1, 0.3, 0.6, 1, 1.5 and 2 µg/mL
With S9-mix, 3 hours treatment: 0.1, 10, 15, 17.5, 25, 27.5, 32.5 and 35 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was stable and soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

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
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 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 experiments)

Evaluation criteria:

DATA EVALUATION
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.

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.

Statistics:

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.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

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: 7.4
2500 µg/ml: 7.6
- Effects of osmolality: No
Solvent control: 0.368 mOsm/kg
2500 µg/ml: 0.326 mOsm/kg

- Precipitation: No precipitation was observed up to and including the top dose of 2500 µg/mL

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 3 µg/mL in the absence of S9, 3 hours treatment; at dose levels of 33 µg/mL in the presence of S9, 3 hours treatment; at dose levels of 1 µg/mL in the absence of S9, 24 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:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 51 and 80% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period, respectively.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 89 and 60% compared to the total growth of the solvent controls after the 3 hours treatment period in the first and second experiment, respectively.

Remarks on result:

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

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles.
Tris(hydroxyethyl) C12-18 alkyl diaminopropane is not 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, Tris(hydroxyethyl) C12-18 alkyl diaminopropane did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

 

In the presence of S9-mix, Tris(hydroxyethyl) C12-18 alkyl diaminopropane did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.