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Toxicological information

Genetic toxicity: in vitro

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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:
2011-05-05 until 2011-07-18
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Rationale for reliability of 2: Guideline study, well-performed and well-documented, read-across Justification of read-across: the registration substance and the read-across supporting substance belong to homolog series of (Polypropylensuccinimido)-caproic acid.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
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. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
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
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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 106cells factor efficiency I density efficiency II 106cells 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 (Tetrapropenylsuccinimido)-caproic acid was investigated according to the OECD Guideline 471. No mutagenic property was found.
Executive summary:

The mutagenic potential of (Pentapropylensuccinimido)-caproic acid was assessed based on the data on the read-across substance (Tetrapentapropylensuccinimido)-caprioc acid.

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