(carboxymethyl)dimethyl[3-[(1-oxoundecenyl)amino]propyl]ammonium hydroxide

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:

2009-10-21 to 2009-12-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

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:

C8-18 AAPB

IUPAC Name:

C8-18 AAPB

Method

Target gene:

Thymidine Kinase Locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: Clone 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment: 39.1; 78.1; 156.3; 312.5; 625; 1250; 2500; 5000 µg/mL

Experiment I:
without S9 mix (4 hours treatment): 2.4; 4.9; 9.8; 19.5; 39.0; 58.5; 78.0 µg/mL; evaluated: 2.4; 4.9; 9.8; 39 µg/mL
with S9 mix (4 hours treatment): 4.9; 9.8; 19.5; 39.0; 78.0; 117.0; 156.0 µg/mL; evaluated: 4.9; 9.8; 19.5; 39.0; 78.0 µg/mL

Experiment II:
without metabolic activation (24 hours treatment): 2.5; 5.0; 10.0; 20.0; 40.0; 50.0; 60.0 µg/mL; evaluated: 10; 20; 40; 50; 60 µg/mL
with metabolic activation (4 hours treatment): 10.0; 20.0; 40.0; 80.0; 100.0; 110.0; 120.0 µg/mL; evaluated: 40, 80, 100, 110, 120 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water (10 %)
- Justification for choice of solvent/vehicle: solubility properties

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation
Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT (Trifluorothymidine)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: approx. 4000 cells per well

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

Linear regression analysis (least squares) using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)

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: not effected
- Effects of osmolality: not increased
- Precipitation: No
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was chosen with regard to the solubility of the test item in deionised water. Test item concentrations between 39.1 and 5000 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation.
Strong toxic effects were observed at 78.1 µg/mL and above in the absence of metabolic activation (4 h treatment) and at 312.5 µg/mL and above in the presence of metabolic activation. Following continuous treatment (24 hours) toxic effects as described above occurred already at the lowest concentration of 39.1 µg/mL and above.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. No precipitation occurred up to the maximum concentration with and without metabolic activation at both treatment intervals.
The dose range of the main experiments was limited by toxicity of the test item. The individual concentrations were generally spaced by a factor of 2.0. A closer spacing was used in the upper concentration range to cover toxic effects more closely. A very narrow dose spacing was used in the second experiment with and without metabolic activation to cover the recommended cytotoxic range of approximately 10-20% relative total growth.

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment II the mutant frequency exceeded the range of the historical solvent control data at several test points without metabolic activation (both cultures) and at one test point with metabolic activation (culture I). However, the threshold described above was not reached at any test point of the second experiment and no dose dependent increase was indicated by statistical analysis.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative total growth of less than 50 % in both parallel cultures were observed in the absence of metabolic activation at 39 µg/mL in experiment I following 4 hour treatment and at 40 µg/mL and above in experiment II following 24 hours treatment. In the presence of metabolic activation toxic effects as described above occurred at 100 µg/mL and above in experiment II. No reproducible cytotoxic effects were noted in the first experiment with metabolic activation. The recommended toxic range of approximately 10-20 % RTG was covered in the second experiment with and without metabolic activation.
The isolated minor reduction of the relative total growth to 43.5 % in the first culture of experiment I with metabolic activation was not considered a real toxic effect since no comparable reduction was observed in the parallel culture under identical conditions.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Table

			relative	mutant		relative	mutant
	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6cells	threshold	growth	10^6cells	threshold
Column	1	2	3	4	5	6	7	8
Experiment I / 4 h treatment			culture I			culture II
Solv. control with water		-	100.0	147	273	100.0	141	267
Pos. control with MMS	19.5	-	32.4	424	273	23.7	403	267
Test item	2.4	-	131.7	122	273	67.5	209	267
Test item	4.9	-	122.7	134	273	129.3	139	267
Test item	9.8	-	103.9	137	273	126.0	104	267
Test item	19.5	-	43.7	275	273	96.9	96	267
Test item	39.0	-	31.1	187	273	39.3	181	267
Test item	58.5	-	culture was not continued^#			culture was not continued^#
Test item	78.0	-	culture was not continued^#			culture was not continued^#
Solv. control with water		+	100.0	130	256	100.0	141	267
Pos. control with CPA	3.0	+	61.5	232	256	90.0	250	267
Pos. control with CPA	4.5	+	44.8	320	256	54.6	348	267
Test item	4.9	+	66.2	164	256	137.6	99	267
Test item	9.8	+	104.3	140	256	134.1	146	267
Test item	19.5	+	57.7	202	256	125.6	134	267
Test item	39.0	+	111.4	105	256	147.5	83	267
Test item	78.0	+	43.5	149	256	108.2	154	267
Test item	117.0	+	culture was not continued^#			culture was not continued^#
Test item	156.0	+	culture was not continued^#			culture was not continued^#
Experiment II / 24 h treatment			culture I			culture II
Solv. control with water		-	100.0	158	284	100.0	153	279
Pos. control with MMS	13.0	-	19.2	569	284	25.0	573	279
Test item	2.5	-	culture was not continued^##			culture was not continued^##
Test item	5.0	-	culture was not continued^##			culture was not continued^##
Test item	10.0	-	65.9	217	284	78.7	264	279
Test item	20.0	-	44.3	208	284	71.1	230	279
Test item	40.0	-	33.1	173	284	45.8	208	279
Test item	50.0	-	38.5	182	284	29.8	214	279
Test item	60.0	-	20.7	209	284	16.2	241	279
Experiment II / 4 h treatment			culture I			culture II
Solv. control with water		+	100.0	164	290	100.0	145	271
Pos. control with CPA	3.0	+	52.2	355	290	25.0	312	271
Pos. control with CPA	4.5	+	23.6	385	290	23.3	407	271
Test item	10.0	+	culture was not continued^##			culture was not continued^##
Test item	20.0	+	culture was not continued^##			culture was not continued^##
Test item	40.0	+	75.5	171	290	102.9	189	271
Test item	80.0	+	40.2	210	290	61.6	159	271
Test item	100.0	+	47.1	133	290	49.0	149	271
Test item	110.0	+	32.6	183	290	42.0	167	271
Test item	120.0	+	22.0	203	290	29.0	201	271

Threshold = number of mutant colonies per 10⁶cells of each solvent control plus 126

#      culture was not continued due to exceedingly severe cytotoxic effects
##    culture was not continued since a minimum of only four analysable concentrations is required

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of C8 -18 AAPB to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation.

The main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix:                     2.4; 4.9; 9.8; 19.5; and 39.0 µg/mL
with S9 mix:                         4.9; 9.8; 19.5; 39.0; and 78.0 µg/mL

Experiment II:

without S9 mix:                                10; 20; 40; 50; and 60 µg/mL

with S9 mix:                               40; 80; 100; 110; and 120 µg/mL

Relevant cytotoxic effects indicated by a relative total growth of less than 50 % in both parallel cultures were observed in the absence of metabolic activation at 39 µg/mL in experiment I following 4 hour treatment and at 40 µg/mL and above in experiment II following 24 hours treatment. In the presence of metabolic activation toxic effects as described above occurred at 100 µg/mL and above in experiment II. No reproducible cytotoxic effects were noted in the first experiment with metabolic activation. The recommended toxic range of approximately 10-20 % RTG was covered in the second experiment with and without metabolic activation.

The isolated minor reduction of the relative total growth to 43.5 % in the first culture of experiment I with metabolic activation was not considered a real toxic effect since no comparable reduction was observed in the parallel culture under identical conditions.

No substantial and reproducible dose dependent increase of the mutation frequency was observed with and without metabolic activation. The mutation frequency did not reproducibly reach or exceed the threshold of 126 above the mutation frequency of the corresponding solvent control in any of the experimental parts. An isolated increase exceeding the threshold was noted in the first culture of experiment I without metabolic activation at 19.5 µg/mL. However, this increase was judged as irrelevant fluctuation since it was not reproduced in the parallel culture under identical experimental conditions. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance. In experiment II the mutant frequency exceeded the range of the historical solvent control data at several test points without metabolic activation (both cultures) and at one test point with metabolic activation (culture I). However, the threshold described above was not reached at any test point of the second experiment and no dose dependent increase was indicated by statistical analysis.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT^â11statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in all experimental groups.

In this study the range of the solvent controls was from 130 up to 164 mutant colonies per 10⁶cells; the range of the groups treated with the test item was from 83 up to 275 mutant colonies per 10⁶cells. The solvent controls remained within the range of the historical data.

Methylmethanesulfonate (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and cyclophosphamide (3.0 µg/mL and 4.5 µg/mL in both main experiments) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at at least one of the concentrations of the controls.

There was no concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guidelines Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, February 1998, adopted July 21, 1997, Guideline No. 476 "In vitro Mammalian Cell Gene Mutation Test“ and Commission Regulation (EC) No. 440/2008 B.17: ”Mutagenicity –In vitro Mammalian Cell Gene Mutation Test“, dated May 30, 2008 for in vitro mutagenicity (mammalian forward gene mutation) data.