Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Key studies were available for the evaluation of bacterial reverse mutation, mammalian gene mutation and chromosomal aberration, demonstrating tthat there is no genotoxic potential.
Link to relevant study records
Reference
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:
March-May 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study has been performed according to GLP and state of the art and actual methods and is therefore considered reliable, relevant and adequate. The mutant frequencies were slightly increased in the test groups, however also the positive controls in the treatment regimes were always above the maximum of the historical control data. Therefore the study is considered to be influenced by higher response but still reliable with restrictions.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Principles of method if other than guideline:
The study did not apply the recent Global Evaluation Factor method to assign a positive response.
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase (TK) locus of L5178Y TK+/- mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK+/- mouse lymphoma cells were stored as frozen stocks in liquid nitrogen, the original cultures were obtained from American Type Culture Collection (ATCC), U.S.A. Prior to setting up the assay, the cells were cleansed of the spontaneous TK-/- cells by growing it in the selective medium (24-hour in the presence of Thymidine, Hypoxanthine, Methotrexate and Glycine (THMG) in DMEM A medium and 2 days in the presence of THG in DMEM A medium). Cells were screened for mycoplasma using the Mycoplasma detection kit (119K0675-1KT, Sigma) and no contamination was observed. For each experiment, one or more vials were thawed rapidly, the cells diluted in DMEM 10 and incubated in a humidified atmosphere of 5% (v/v) CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks. Cell characterization was performed upon receipt and once in 6 months.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
exogenous mammalian (S9) activation system
Test concentrations with justification for top dose:
Range finding study: 0.625, 1.25, 2.5 and 5.0 μL/mL along with concurrent solvent controls.
Main study: 0.019, 0.039, 0.078 and 0.156 μL/mL for 3-hour –S9; 0.625, 1.25, 2.5 and 5.0 μL/mL for 3-hour +S9 & 24-hour –S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (microwell plates)
DURATION
- Preincubation period: 3-24 hour
- Exposure duration: 3 or 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 14 days
SELECTION AGENT (mutation assays): TFT ( trifluorothymidine)
NUMBER OF REPLICATIONS: duplicate
DETERMINATION OF CYTOTOXICITY
- Method: relative survival growth percentage (relative cloning efficiency) ; relative total growth RTG; other: Viability scored by cell growth; mutation scored by colony count
Evaluation criteria:
The assay was considered valid as it met the following criteria:
1. The spontaneous mutant frequency of the negative/solvent control must be within 50 to 170 TFT-resistant mutants per 106 viable cells. The cloning efficiency of the solvent control group must be greater than 50%.
2. At least one concentration of each positive control exhibited mutant frequencies of >200 mutants per 106 cells. The colony size distribution for the MMS showed an increase in both small and large colonies.

A concentration-related or a reproducible increase in the mutant frequency was amongst the several criteria’s considered for determining a positive result. Biological relevance of the results was considered first and statistical methods may be used as an aid in evaluating the test results. However, statistical significance will not be the only determining factor for a positive response.
A test substance which does not meet the above criteria is considered non-mutagenic in this test system.
Colony sizing i.e. identifying the small and large colonies helps in predicting the clastogenic or mutagenic nature of the test substance, with small colony mutants indicating chromosomal aberrations and large colony mutants indicating point mutations or deletions.
Most studies give clear positive or negative results, however in certain cases the data set may preclude making definite judgment about the activity of the test substance wherein the results may remain equivocal irrespective of the number of times the experiment is repeated.
Positive results indicate the capacity of inducing gene mutations in the mouse lymphoma cells.
Statistics:
Statistical analysis was not performed.
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:
Range finding study
Four test concentrations of 0.625, 1.25, 2.5 and 5.0 μL/mL along with concurrent solvent controls were chosen for the range finding study. The maximum concentration (5 μL/mL) was achievable owing to the solubility of the test substance in DMSO. The stock solution (100x) of the test substance was diluted 1 in 100 in culture medium to achieve the desired final concentrations.At the time of treatment incubation, oil like emulsion of the test substance was seen in culture medium in doses 1.25 to 5.0 μL/mL in all treatment regimens and did not affect the pH or colour of the culture medium. In treatment regimen 3-hour, treatment of cultures with Proviplast 0142 resulted in cytotoxicity observed as Reduction in % RTG.
No contamination was observed in the viability plates.The cloning efficiencies in the solvent control cultures fell within the normal range.
For 3-hour treatment in the absence of S9 the %RTG ranged between 115.90 and 0.78; and for 3-hour treatment in the presence of S9 the %RTG ranged between 115.72 and 42.37. For 24-hour treatment in the absence of S9, the %RTG ranged between 110.39 and 51.02.In all three treatment regimens, treatment of cultures with Proviplast 0142 resulted in dose dependent reduction in % RTG in all concentrations (3-hour, +/-S9 and 24-hour, -S9).
Based on the % RTG results in the range finding studies, the following concentrations were selected for the main (mutation) study: 0.019, 0.039, 0.078 and 0.156 μL/mL for 3-hour –S9, 0.625, 1.25, 2.5 and 5.0 μL/mL for 3-hour +S9, 24-hour –S9.

3-hour treatment, +S9: The %RTG and mutant frequencies from the 3-hour treatment in the presence of S9 are given in Table 2. The mutant frequencies in the solvent control cultures fell within the normal range. The cloning efficiencies were 141.46 and 133.04% for solvent control cultures, replicate cultures 1 and 2, respectively. The positive control chemical, B(a)P induced a significant increase in mutant frequencies compared to concurrent control cultures. The assay was therefore considered valid.A dose related decrease in %RTG was observed. The mutant frequencies of all Proviplast 0142 treated cultures fell within or close to the historical control values and were not significantly different from the concurrent solvent control cultures.There was a dose related increase in mutant frequencies associated with Proviplast 0142 treatments. However, the proportion of small colonies, in all three treatment regimens associated with Proviplast 0142 treatments, were not significantly different from the concurrent solvent control cultures (Table 5).

3-hour treatment, -S9: The %RTG and mutant frequencies from the 3-hour treatment in the absence of S9 are given in Table 3. The mutant frequencies in the solvent control cultures fell within the normal range. The cloning efficiencies were 207.76 and 184.78% for solvent control cultures, replicate cultures 1 and 2, respectively. The positive control chemical, MMS induced a significant increase in mutant frequencies compared to concurrent control cultures. The assay was therefore considered valid.Treatment of cultures with Proviplast 0142 resulted in dose related decrease in %RTG. The mutant frequencies of all Proviplast 0142 treated cultures fell within or close to the historical control values and not significantly different from the concurrent solvent control cultures, except in concentrations 0.078 and 0.156 μL/mL. There was a dose related increase in mutant frequencies associated with Proviplast 0142 treatments. However, the proportion of small colonies, in the treatments associated with Proviplast 0142, were not significantly different from the concurrent solvent control cultures (Table 5)

24-hour treatment,-S9: The %RTG and mutant frequencies from the 3-hour treatment in the absence of S9 are given in Table 4. The mutant frequencies in the solvent control cultures fell within the normal range. The cloning efficiency of the solvent control cultures were 144.54 and 129.97%, in replicate cultures 1 and 2, respectively. The positive control chemical, MMS induced a significant increase in mutant frequencies compared to concurrent control cultures. The assay was therefore considered valid. Treatment of cultures with Proviplast 0142 resulted in dose dependent reduction in % RTG with the top dose of 5.0 μL/mL resulting in a RTG of 21.26 and 18.30% (replicate cultures 1 and 2), respectively. The mutant frequencies of all Proviplast 0142 treated cultures fell within or close to the historical control values and not significantly different from the concurrent solvent control cultures, except in concentrations 1.25 and 2.5 μL/mL. There was a dose related increase in mutant frequencies associated with Proviplast 0142 treatments. However, the proportion of small colonies, in the treatments associated with Proviplast 0142, were not significantly different from the concurrent solvent control cultures (Table 5).

In all three treatment regimes (3-hour, +/-S9 and 24-hour, -S9), treatment of cultures with Proviplast 0142, resulted in an acceptable dose related decrease in %RTG. The mutant frequencies of all Proviplast 0142 treated cultures fell within or close to the historical control values and were not significantly different from the concurrent solvent control cultures.
It is therefore concluded that Proviplast 0142 did not induce mutations in L5178Y TK+/- cells. The colony sizing data is reported for completeness.
Remarks on result:
other: strain/cell type: L5178Y cells TK+/-
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded, that under the conditions of the test, Proviplast 0142 did not induce mutations in L5178Y TK+/- cells when tested up to concentrations inducing acceptable levels of cytotoxicity.
Executive summary:

Proviplast 0142 was evaluated for its capacity to induce mutations at thymidine kinase (TK) locus in mouse lymphoma L5178Y TK+/- cell lines.Four test concentrations of 0.625, 1.25, 2.5 and 5.0 μL/mL along with concurrent solvent controls were chosen for the range finding study. The maximum concentration (5.0 μL/mL) was achievable owing to the solubility of the test substance in DMSO. At the time of treatment incubation, oil like emulsion of the test substance was seen in culture medium in doses 1.25 to 5.0 μL/mL in all treatment regimens and did not affect the pH or colour of the culture medium. Based on the results of the range finding study, the following concentrations were selected for the main (mutation) study: 0.019, 0.039, 0.078 and 0.156 μL/mL for 3-hour –S9; 0.625, 1.25, 2.5 and 5.0 μL/mL for 3-hour +S9 & 24-hour –S9. The mutant frequencies and cloning efficiencies in the solvent control cultures fell within the normal range. The positive control chemicals, MMS and B(a)P induced a significant increase in mutant frequencies compared to concurrent control cultures. In all three treatment regimens (3-hour, +/-S9 and 24-hour, -S9), treatment of cultures with Proviplast 0142, resulted in an acceptable dose related decrease in % relative total growth. The mutant frequencies of all Proviplast 0142 treated cultures fell within or close to the historical control values and were not significantly different from the concurrent solvent control cultures, except in concentrations 0.078 and 0.156 μL/mL (3-hour –S9) and 1.25 to 5.0 μL/mL (24-hour –S9).There was a dose related increase in mutant frequencies associated with Proviplast 0142 treatments, however the proportion of small colonies, in all three treatment regimens associated with Proviplast 0142 treatments, were not significantly different from the concurrent solvent control cultures.

It is concluded, that under the conditions of the test, Proviplast 0142 did not induce mutations in L5178Y TK+/- cells when tested up to concentrations inducing acceptable levels of cytotoxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

For the determination of the bacterial reverse mutation (Salmonella typhimurium) potential of bis(2-butoxyethylene)adipate, OECD QSAR toolbox was used. For the prediction read-across among category members was applied and 5 nearest neighbours were taken. Log Kow was taken into account as descriptor. For the target compound, log Kow is 3.78 and the 5 neighbours were within the log Kow range of 2.18 -6.21. Based on the data of these compounds, it can be concluded that the investigated compound is negative for genetic toxicity.

For the determination of mammalian forward muation, a key thymidine kinase (TK) locus study in mouse lymphoma L5178Y TK+/- cell lines was performed (IIBAT, 2012g). Four test concentrations of 0.625, 1.25, 2.5 and 5.0 μL/mL along with concurrent solvent controls were chosen for the range finding study. The maximum concentration (5.0 μL/mL) was achievable owing to the solubility of the test substance in DMSO. At the time of treatment incubation, oil like emulsion of the test substance was seen in culture medium in doses 1.25 to 5.0 μL/mL in all treatment regimens and did not affect the pH or colour of the culture medium. Based on the results of the range finding study, the following concentrations were selected for the main (mutation) study: 0.019, 0.039, 0.078 and 0.156 μL/mL for 3-hour –S9; 0.625, 1.25, 2.5 and 5.0 μL/mL for 3-hour +S9 & 24-hour –S9. The mutant frequencies of treated cultures fell within or close to the historical control values and were not significantly different from the concurrent solvent control cultures, except in concentrations 0.078 and 0.156 μL/mL (3-hour –S9) and 1.25 to 5.0 μL/mL (24-hour –S9). There was a dose related increase in mutant frequencies, however the proportion of small colonies, in all treatment regimens were not significantly different from the concurrent solvent control cultures. It was concluded, that under the conditions of the test, Proviplast 0142 did not induce mutations in L5178Y TK+/- cells when tested up to concentrations inducing acceptable levels of cytotoxicity.

For the chromosome aberration evaluation, a key study in cultured human peripheral blood lymphocytes was performed (IIBAT, 2012h).  Cells were treated for 4-hour in presence and absence of metabolic activation (S9) and continuously for 36-hour in the absence of S9, and allowed for 32-hour expression in the case of 4-hour exposure. The pH of the post treatment medium was approximately 7.2. No visible contamination was observed.  Based on the %MI inhibition results in the range finding study, the following concentrations were selected for the main study: 4-hour, +S9/-S9:0.625, 1.25 and 2.5μL/mL; Continuous, -S9:0.156, 0.313 and 0.625μL/mL. Duplicate cultures were treated in the main study. The pH of the post treatment medium was approximately 7.2. No visible contamination was observed. The percentage aberrations in the solvent control cultures fell within the normal range (historical data). The positive control chemicals, MMC and CYP induced a significant increase in percentage aberrations compared to concurrent solvent control cultures. The assay were thereforeconsidered valid. In all three treatment regimens (4-hour, +/-S9 and continuous, -S9), treatment of cultures withProviplast 0142,resulted in an acceptable dose related inhibition in %MI.The percentage aberrations of all treated cultures fell within or close to the historical control values and were not significantly different from the concurrent solvent control cultures. It is concluded, that under the conditions of the test, Proviplast 0142 did not induce genotoxic response in human lymphocytes when tested up to concentrations inducing acceptable levels of cytotoxicity.


Justification for selection of genetic toxicity endpoint
Although 1 endpoint was selected above, all the 3 endpoints records are key studies for the different genetic toxicity endpoints: bacterial reverse mutation, chromosomal aberration and mammalian gene mutation.

Justification for classification or non-classification

Based on the results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC) and CLP regulation (EC No. 1272/2008 of 16 December 2008), Bis(2-butoxyethyl)adipate (Proviplast 0142/DBEA) does not have to be classified and has no obligatory labelling requirement for genotoxicity.