6-[(C10-C13)-alkyl-(branched, unsaturated)-2,5-dioxopyrrolidin-1-yl]hexanoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-05-05 until 2011-07-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, well-performed and well-documented

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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:

first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

6-(3-tetrapropenyl-2,5-dioxopyrrolidin-1-yl)hexanoic acid

EC Number:

800-770-5

Cas Number:

1424148-99-1

Molecular formula:

C22H39NO4

IUPAC Name:

6-(3-tetrapropenyl-2,5-dioxopyrrolidin-1-yl)hexanoic acid

Details on test material:

Identity: (Tetrapropylensuccinimido)-capronsäure
Batch No.: ESD0009058
Molecular Weight: 379.54
Purity: >97.5%, dose calculation was adjusted to purity
Stability in Solvent: Not indicated by the sponsor
Storage: At room temperature
Expiration Date: March 29, 2013

On the day of the experiment (immediately before treatment), the test item was dissolved in DMSO (purity 99.9 %). The final concentration of DMSO in the culture medium was 0.5 % (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without S9 mix 1.9; 3.8; 7.5; 15.0; 22.5; 30.0 µg/mL
with S9 mix: 15.0; 30.0; 60.0; 120.0; 180.0; 240.0 µg/mL
Experiment II:
without S9 mix: 15.0; 30.0; 60.0; 120.0; 160.0; 200.0 µg/mL
with S9 mix: 30.0; 60.0; 120.0; 160.0; 180.0; 200.0 µg/mL
In the first experiment the cultures at the lowest concentration without metabolic activation was not continued since a minimum of only four analysable concentrations is required by the guidelines. The cultures at the highest concentration with metabolic activation were not continued due to exceedingly severe cytotoxic effects. In the second experiment the cultures at the two highest concentrations without metabolic activation and the highest concentration with metabolic activation were not continued due to exceedingly cytotoxic effects.

Vehicle / solvent:

DMSO
Justification for choice of solvent/vehicle: solubility properties

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


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): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected
- Effects of osmolality: Not increased
- Evaporation from medium: Not examined
- Water solubility: Not indicated by the sponsor
- Precipitation:
Pre-experiment: Precipitation occurred at 475.0 µg/mL and above in the presence and absence of metabolic activation after 4 hours treatment. Following continuous treatment for 24 hours without metabolic activation, precipitation was observed at the maximum concentration of 950 µg/mL.
Experiment I and II: No precipitation observed.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
In the range finding pre-experiment test item concentrations between 29.7 µg/mL and 3800 µg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.

Strong toxic effects were observed at 59.4 µg/mL and above in the absence of metabolic activation and at 237.5 µg/mL and above with metabolic activation (4 hours treatment). Following continuous treatment (24 hours) strong toxic effects occurred at 237.5 µg/mL and above.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 475.0 µg/mL and above in the presence and absence of metabolic activation after 4 hours treatment. Following continuous treatment for 24 hours without metabolic activation, precipitation was observed at the maximum concentration of 950 µg/mL.
In the pre-experiment the highest concentration was neutralised with 2 N sodium hydroxide. There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was placed into the lower range. Narrower spacing was used at high concentrations to cover the toxic range more closely.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects defined as a reduction of the relative cloning efficiency I and/or relative cell density to values below 50% in both parallel cultures were noted in the first experiment at 30.0 µg/mL without metabolic activation and at 180 µg/mL and above with metabolic activation. In the second experiment cytotoxic effects as described above occurred again at 180 µg/mL and above with metabolic activation. In the absence of metabolic activation cytotoxicity was noted at 120 µg/mL and above in culture I and at 200 µg/mL in culture II. The recommended toxic range of a relative cloning efficiency or a relative cell density of approximately 10-20% was covered by at least one of the parallel cultures with and without metabolic activation.

Any other information on results incl. tables

Summary Table

			relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL	mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
			%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12
Experiment I / 4 h treatment			culture I					culture II
Solvent control with DMSO		-	100.0	100.0	100.0	9.2	1.0	100.0	100.0	100.0	42.0	1.0
Positive control (EMS)	150.0	-	78.7	96.4	89.4	142.0	15.5	95.7	74.3	94.4	164.7	3.9
Test item	1.9	-	96.7	culture was not continued^#				98.0	culture was not continued^#
Test item	3.8	-	92.6	75.7	99.7	19.2	2.1	93.5	64.3	107.9	21.7	0.5
Test item	7.5	-	100.6	86.8	77.0	48.4	5.3	94.6	85.8	105.1	15.8	0.4
Test item	15.0	-	102.3	80.4	81.4	27.4	3.0	94.1	60.1	112.3	29.7	0.7
Test item	22.5	-	77.6	82.3	102.3	24.1	2.6	83.3	91.1	103.5	32.5	0.8
Test item	30.0	-	30.3	17.1	86.5	58.7	6.4	34.5	38.7	97.1	37.7	0.9
Solvent control with DMSO		+	100.0	100.0	100.0	17.1	1.0	100.0	100.0	100.0	14.3	1.0
Positive control (DMBA)	1.1	+	67.5	37.1	60.9	763.9	44.5	63.6	72.1	65.9	1012.5	70.9
Test item	15.0	+	93.5	96.4	101.3	11.1	0.6	90.8	169.6	97.4	12.3	0.9
Test item	30.0	+	90.0	115.5	97.2	10.9	0.6	97.9	153.9	64.9	27.3	1.9
Test item	60.0	+	99.7	86.9	108.5	11.1	0.6	89.6	196.1	70.1	26.3	1.8
Test item	120.0	+	93.3	112.3	96.3	8.9	0.5	113.2	157.0	104.3	24.1	1.7
Test item	180.0	+	0.0	17.5	41.1	0.0	0.0	0.0	30.5	61.3	26.1	1.8
Test item	240.0	+	0.0	culture was not continued^##				0.0	culture was not continued^##
Experiment II / 24 h treatment			culture I					culture II
Solvent control with DMSO		-	100.0	100.0	100.0	5.3	1.0	100.0	100.0	100.0	10.9	1.0
Positive control (EMS)	150.0	-	83.1	101.8	74.9	221.3	41.9	83.0	95.2	78.3	253.8	23.3
Test item	15.0	-	98.0	107.4	91.9	6.8	1.3	98.1	104.8	99.1	8.8	0.8
Test item	30.0	-	97.5	84.5	81.4	13.1	2.5	97.1	87.8	93.5	13.3	1.2
Test item	60.0	-	88.4	95.6	88.0	14.7	2.8	90.0	98.3	97.4	10.2	0.9
Test item	120.0	-	86.2	44.8	88.2	10.0	1.9	88.8	84.3	95.1	14.6	1.3
Test item	160.0	-	89.3	1.3	culture was not continued^##			85.9	89.7	96.8	19.2	1.8
Test item	200.0	-	0.0	6.6	culture was not continued^##			0.0	3.0	culture was not continued^##
Experiment II / 4 h treatment
Solvent control with DMSO		+	100.0	100.0	100.0	11.4	1.0	100.0	100.0	100.0	12.0	1.0
Positive control (DMBA)	1.1	+	59.0	133.4	48.4	1797.0	158.3	52.7	123.9	71.9	1345.2	112.1
Test item	30.0	+	96.8	114.6	92.4	15.4	1.4	97.4	129.3	109.7	16.8	1.4
Test item	60.0	+	116.5	111.5	83.6	5.6	0.5	107.2	118.6	108.9	17.9	1.5
Test item	120.0	+	95.8	125.9	88.1	7.6	0.7	93.4	105.8	87.2	10.1	0.8
Test item	160.0	+	15.8	58.2	83.9	16.1	1.4	51.2	106.9	97.2	15.7	1.3
Test item	180.0	+	8.0	36.4	82.7	18.3	1.6	24.7	108.3	95.5	22.9	1.9
Test item	200.0	+	0.0	culture was not continued^##				0.4	culture was not continued^##

^#        culture was not continued since a minimum of only four analysable concentrations is required

^##       culture not continued due to exceedingly strong toxic effects

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The mutagenic potential of (Tetrapropenyl succinimido)-caproic acid was investigated according to the OECD Guideline 471. No mutagenic property was found.

Executive summary:

The mutagenic potential of (Tetrapropenyl succinimido)-caproic acid was investigated according to the OECD Guideline 476. No mutagenic property was found.