Amines, N-tallow alkyltrimethylenedi-, propoxylated

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-Propanol, 1,1'-[[3-[(3-aminopropyl)amino]propyl]imino]bis-, N-tallow alkyl derivs.

EC Number:

307-276-4

EC Name:

2-Propanol, 1,1'-[[3-[(3-aminopropyl)amino]propyl]imino]bis-, N-tallow alkyl derivs.

Cas Number:

97592-79-5

IUPAC Name:

97592-79-5

Constituent 2

Reference substance name:

2-Propanol, 1,1'4[3-[(3aminopropyl)amino]propyljimino] bis-, N-tallow alkyl derivatives

IUPAC Name:

2-Propanol, 1,1'4[3-[(3aminopropyl)amino]propyljimino] bis-, N-tallow alkyl derivatives

Details on test material:

- Name of test material (as cited in study report): POLYRAM SL
- Chemical Name: 2-Propanol, 1,1'-[[3-[(3aminopropyl)amino]propyl]imino] bis-, N-tallow alkyl derivatives
- Physical state: yellow viscous liquid
- Composition of test material, percentage of components: Fatty amine alkoxylated: 88.2%, Amines, tallow alkyl: 4.8 %, N-(tallow alkyl) triproplylenetetramine: 2.5%, Non amine: 2.5%, water content: 2.1%
- Purity test date: 12/01/2010
- Lot/batch No.: 96102715
- Expiration date of the lot/batch: 15/10/2011
- Storage condition of test material: at room temperature, protected from light

Method

Target gene:

Mammalian cell culture systems are used to detect mutations induced by chemical substances. This in vitro experiment is carried out to assess the potential of the test item to induce gene mutations by means of a Thymidine Kinase assay using the mouse lymphoma cell line L5178Y. The Thymidine Kinase (TK) system detects base pair mutations, frameshift mutations, small deletions as well as large, non-lethal deletions and rearrangements of the relevant chromosomes

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
see below under media in the section "Any other information on materials and methods incl. tables"
- Properly maintained: yes, see above
- Periodically checked for Mycoplasma contamination: yes/no
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 extract, male Wistar rats, induced with phenobarbital (80 mg/kg bw) and B-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.

Test concentrations with justification for top dose:

- short term pre-experiment (4 h exposure): 7, 15, 30 and 60 microg/mL with metabolic activation; 0.05, 0.2, 1.0, 4.0, 7.0 and 20.0 microg/mL
- experiment I (4 h duration) with metabolic activation: 3.0, 4.0, 6.0, 7.0, 7.75, 8.50, 9.25 and 10.0 microg/mL and without metabolic activation: 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 microg/mL
- experiment II (24 h duration) with metabolic activation: 2.0, 4.0, 5.5, 6.5, 7.25, 8.00, 8.75 and 9.50 microg/mL and without metabolic activation: 0.20, 0.35, 0.50, 0.65, 0.80, 0.90, 1.00 and 1.10 microg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none
- Justification for choice of solvent/vehicle: test item is dispersible in water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Benzo[a]pyrene (B[a]P), Ethylmethanesulfonate (EMS), Methylmethanesulfonate (MMS),

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: 4 h and 24 h (with and without activation)
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
see below in the section "Any other information on materials and methods incl. tables"

NUMBER OF REPLICATIONS:
not applicable

NUMBER OF CELLS EVALUATED:
number of cells seeded: 300'000, number of cells evaluated see result tables

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
Cytotoxicity is determined by measuring the colony-forming ability and the growth rate of cultures. The treated cultures are maintained 48-72 h to allow near optimal phenotypic expression of induced mutations.

Evaluation criteria:

There are several criteria for determining a positive result:
- clear and dose-related increase in the mutant frequency,
- biologically relevant response (at least a 2-fold increase of mutant frequencies related to the respective negative control values and higher than the historical range of negative controls) for at least one of the dose groups,
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low large/small colonies ratio (1.5 times the ratio of clastogenic controls MMS and/or B[a]P) is an indication for potential clastogenic effects and/or chromosomal aberrations.

Statistics:

According to the OECD guidelines, the biological relevance 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:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none reported
- Effects of osmolality: none reported
- Evaporation from medium: unlikely given the low vapour pressure of the substance
- Water solubility: test item reported to be dispersible in water
- Precipitation: Precipitation of the test item was noted only in the pre-experiments at 30 and 60 µg/mL with and at 7 and 20 µg/mL without metabolic activation
- Other confounding effects: none reported

RANGE-FINDING/SCREENING STUDIES:
yes, see results below

COMPARISON WITH HISTORICAL CONTROL DATA: yes, but historical control data not reported in detail

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

- Table 1: Pre-Experiment for Toxicity, with metabolic activation

Test Group	Concen-tration [µg/mL]	Number of Cells seeded	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	Relative Suspension Growth (RSG) [%]
NC1	0	300000	323000	925000	1410000	100.00
NC2		300000	360000	980000	1450000
1	7	300000	201000	322000	1280000	51.88
2	15	300000	11200	0	0	n.d.
3 (P)	30	300000	26700	0	0	n.d.
4 (P)	60	300000	26300	0	0	n.d.

NC: Negative control

(P): Precipitation

RSG: [(value TSG / value TSG of corresponding controls) x 100]

n.d.: no data

- Table 2: Pre-Experiment for Toxicity, without metabolic activation

Test Group	Concen-tration [µg/mL]	Number of Cells seeded	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	Relative Suspension Growth (RSG) [%]
NC1	0	300000	281000	803000	1610000	100.00
NC2		300000	287000	845000	1620000
1	0.05	300000	251000	742000	1570000	99.06
2	0.20	300000	313000	902000	1550000	95.34
3	1.00	300000	288000	676000	1570000	78.65
4	4.00	300000	57100	47600	179000	20.07
5 (P)	7.00	300000	9710	8300	5800	3.82
6 (P)	20.00	300000	121000	48800	31000	1.64

NC: Negative control

(P): Precipitation

RSG: [(value TSG / value TSG of corresponding controls) x 100]

- for the other tabulated resuts please see the attached pdf-document

Applicant's summary and conclusion

Conclusions:

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

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 2-Propanol, 1,1'-[[3-[(3-aminopropyl)amino]propyl]imino]bis-, N-tallow alkyl derivs. is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

Executive summary:

In the present study (Bioservice Scientific Laboratories 2010) 2-Propanol, 1,1'-[[3-[(3-aminopropyl)amino]propyl]imino]bis-, N-tallow alkyl derivs. was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations was based on data from the pre-experiments. In experiment I 10.0 .tg/mL (with metabolic activation) and 3.0 µg/mL (without metabolic activation) were selected as the highest concentrations. In experiment II 9.5 µg/mL (with metabolic activation) and 1.1 .tg/mL (without metabolic activation) were selected as the highest concentrations.

Experiment I and II with metabolic activation and experiment I without metabolic activation were performed as 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

The test item was investigated at the following concentrations:

Experiment I

with metabolic activation:

3.0, 4.0, 6.0, 7.0, 7.75, 8.50, 9.25 and 10.0 gg/mL

and without metabolic activation:

0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 p.g/mL

Experiment II

with metabolic activation:

2.0, 4.0, 5.5, 6.5, 7.25, 8.00, 8.75 and 9.50 .tg/mL

and without metabolic activation:

0.20, 0.35, 0.50, 0.65, 0.80, 0.90, 1.00 and 1.10 vg/mL

Precipitation of the test item was noted only in the pre-experiments.

Growth inhibition was observed in experiment I and II with and without metabolic activation.

In experiment I with metabolic activation the relative total growth (RTG) was 16.52% for the highest concentration (10.0 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 3.0 µg/mL with a RTG of 11.10%. In experiment II with metabolic activation the relative total growth (RTG) was 17.47% for the highest concentration (9.5 lig/mL) evaluated. The highest concentration evaluated without metabolic activation was 1.1 pg/mL with a RTG of 15.57%.

In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation). EMS, MMS and B[a]P were used as positive controls and showed distinct and

biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.