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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 473 (in vitro cytogenicity in mammalian cells), Human peripheral blood lymphocytes, not clastogenic with and without metabolic activation

OECD 490 (in vitro gene mutation in mammalian cells), Mouse Lymphom TK cell lines, not mutagenic with and without metabolic activation

OECD 471 (in vitro gene mutation in bacteria), Ames test, not mutagenic with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
Blood was collected f rom male donors of age 24 and 30 years old for preliminary citotoxicity test and chromosomal aberation tests respectively, who were no smokers, not undergoing any drug treatment and not suspected of exposure to high levels of radation or hazardous chemicals or any viral infection
Cytokinesis block (if used):
colchicine at 0.2 microg/ml of final concentration in the culture media was used as the spindle inhibitor
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix:
The S9 homogeneate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 , 5 days prior to sacrifice. The S9 homogeneate was prepared in batches and stored in a deep freezer mantained at -68 to -86°. The S9 homogeneate was thawed immediately before use and mixed with the co-factor solution, containing 25 mg/ml NADP, 180 mg7ml Glucose-6-phosphate, 150 mM KCl, and S9 fraction (ratio 1:1:1:2 v/v)
Test concentrations with justification for top dose:
500, 1000 and 2000 mcrog/ml with and without metabolic activation for 3h exposure
500, 1000 and 2000 microg/ml without metabolic activation for 22 hours exposure
the test concentrations were based on the results of a preliminary citotoxicity test with the same conditions (with and without metabolic activation for 3h exposure, without metabolic activation for 22 hours exposure) at the dose 62.5, 125, 250, 500, 1000 and 2000 microg/ml that resulted at the highest dose level in a reduction in mitotic index by 45+/-5% of the solvent control.
Vehicle / solvent:
sterile water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments : three

METHOD OF TREATMENT/ EXPOSURE:
the target cells were exposed to the controls and three concetrations of the test itme as follows:
Experiment 1 and 3: the target cells in duplicate were exposed to the vechilce control positive control and test concentrations for 3h in the presence and for 22h in the absence of metabolic activation, respectively
Experiment 2: the target cells in duppkicate were exposed to the vehicle control and the test concentrations for 3h in the absence of metabolic activation

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3h with and without, 22h without metabolic activation
- Harvest time after the end of treatment (sampling/recovery times): each culture from the controls and treatmenet groups was harvested at approx 22h after beginning of treatment and processed separately for preparation of chromosomes.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colchicine at 0.2 microg/ml of final concentration in the culture media was added approximately 2h before harvest

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): cells were pelleted and re-suspended in a minimal amount of fresh fixative so as to give a milky suspension. Several drops of supsension were transferred on to clean glass slides, flame dried and dried on a slide warmer mainted at apprx. 40°C. Five slides per replicate were made.
The sldes were stained with freshly prepared 5% Giemsa stain in distilled water, rinsed with tap water , ari dreid, immersed in xylene and mounted with DPX.

- number of cells scored: 150 metaphases were scored from each replicate for chromosome aberrations.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): The number of chromosomes for each spread was counted , and those containing 44 to 48 chromosome were evaluated for aberration. The chromosome number was recorded for all cells analysed and the microscope coordinates were recorded for the aberrant cells. Aberrations were recorded as chromatid/chromosome gaps or breaks and exchanging figures.Since gaps are not considered as true aberrations, the results are presented as metaphases with aberration includinf gaps and excluding gaps.
- Determination of polyploidy:
- Determination of endoreplication:


METHODS FOR MEASUREMENT OF CYTOTOXICITY
-concurrent cytotoxicity was assesed for all treated and control cultures based on mitotic index


METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:
Evaluation criteria:
whne all the validaity criteria are fulfilled:
-A test chemicl is considered to be clearly positive if in any of the experimental conditions examined: 1) at least one of the test concentrations exhibits a statistically significant increase compared with concurrent vehicle control 2)the increase is dose-dependent when evaluated with an appropriate trend test 3)any of the results are outside the distribution of the historical vehicle control data
-A test chemicl is considered to be clearly negative if in all of the experimental conditions examined: 1) None of the test concentrations exhibits a statistically significant increase compared to the concurrent vehcile control 2)there is not concentration related increase when evaluated with an appropriate trend test 3)all results are within the distribution of the historical vehicle control data
-the results will be considered equivocal if they do no meet the criteria specified for a positive or a negative response
-An increase in the number of polyploidy cells may indicate that the test item has the potential to inhibit mitotic process and to induce numerical chromosome aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test item has the potential to inhiit cell cycle progression.
Statistics:
The statistical analysis was performed via SYSTAT statistical package version 12.0.Data were analysed for proportions of aberrant metaphases in each sample, excluding gaps as aberrations. Pooled data from each test concentrations and the positive control were compared with the vehicle control uing Fischer exact test. All analyses and comparison were evaluated at 5% (p<0.05) level
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Experiment 1: at the highest tested dose (2000 microg/ml) the mitothic inhibition was 41% compared to the vehicle control
The incidence of aberrations of the vehicle control was within the range of the in-houe historical control data
There was no statistically significant increase in the number of aberrant metaphases in any of the test concentrations when compared to the vehicle control. No incidences of polyploidy and endoreduplication were seen. The positive control cyclophosphamide monohydrate caused a statistically significant increase in the aberrant metaphases excluding gaps.

Experiment 2: at the highest tested dose (2000 microg/ml) the mitothic inhibition was 40% compared to the vehicle control
The incidence of aberrations of the vehicle control was within the range of the in-houe historical control data
There was no statistically significant increase in the number of aberrant metaphases in any of the test concentrations when compared to the vehicle control. No incidences of polyploidy and endoreduplication were seen.

Experiment 3: at the highest tested dose (2000 microg/ml) the mitothic inhibition was 45% compared to the vehicle control
The incidence of aberrations of the vehicle control was within the range of the in-houe historical control data
There was no statistically significant increase in the number of aberrant metaphases in any of the test concentrations when compared to the vehicle control. No incidences of polyploidy and endoreduplication were seen. The positive control ethylmethanesulphonate caused a statistically significant increase in the aberrant metaphases excluding gaps.

refer to the attached full study report for tables and details

Conclusions:
The substance was tested for in vitro citogenicity following OECD 473. Under the experimental conditions no evidence of induction of chromosome aberrations excluding gaps was obtained in any of the experiments at any test concentrations either in the presence or in the absence of metabolic activation.Similarly there were no incidences of polyploidy and endoreduplications.
It i concluded that the test item was not clastogenic in cultured human peripheral blodd lymphocytes up to the respective highest concentraion of 2000 microg/ml.
Executive summary:

The clastogenic potential of the test item Diethylene Glicole Diformate to induce chromosome aberration in mammalian cells was evaluted using cultured human peripheral blood lymphocytes.

Human lymphocytes in the blood cultre stimulated to divide by addition of PHA 48h prior to treatment, were exposed to the test item in the presence and absence of an exogenous metabolic activation sysmte (S9 fraction prepared from Aroclor 1254 induced rat liver). The study consisted in a preliminary cytotoxicity test and a chromosomal aberration assay. The latter consisted of three indipendent experiments: Experiment 1 and 2 in the presence and absence of metabolic activation sysem and 3h exposure, and Experiment 3 in the absence of metabolic activation with a 22h expoure.

The test item was soluble in sterile water at 200 mg/ml and was found to be stable in the Sterile water for 3h at room temperature and fortification levels of 300 microg/ml and 200000 microg/ml.

In a preliminary cytotoxicity test for the selection for the test concentrations for the chromosomal aberration assay, DEGDF exhibited the required level of cytotoxicity (reduction in the mitothic index by 45 +/- 5% of the concurrent vehicle control) at the highest tested concentration both in the presence and absence of metabolic activation with 3h expoure and also in the absence of metabolic activation with 22h exposure at and up to 2000 microg/ml. Based on these observation, in the chromosomal aberration assay, a top concentration of 2000 microg/ml was tested in the 3h exposure, in the presence and absence of metabolic activation and in the absence of metabolic activation with 22h exposure.

In the definitive chromosomal aberration assay, bllod cultures were exposed to the test item in duplicate at the concentration of 500, 1000 and 2000 microg/ml in Experiment 1 and 2 (3h exposure with and without metbaolic activation) and in Experiment 3 (22h exposure without metabolic activation).

In a similar way concurrent vehicle control and the positive controls (cyclophophamide monohydrate in the presence of metabolic activation and ehtylmethane sulfonate in the absecne of metabolic activation) were tested in duplicate cultures. In each case the cells in the c-metaphase were harvsted at approximately 22h after the start of treatment from the vehicle control, test item concentrationd and positive controls.

At the highest concentration tested, the reduction in mitothic index was 41, 40 and 45% in Experiments 1, 2 and 3 respectively, compared to the vehicle control.

A total of 300 metaphases from duplicate cultures from the controls and threee treatment levels were evaluated for chomosomal aberrations. The data from the treatment groups and the positive controls were statisically compared with the vehicle control. There were not statistically significant increase in the incidence of structually aberrant metaphases , either in the presence or in the absence of metabolic activation at any tested concentrations. Under identical conditions the respective positive controls produced statistically significant (p < 0.05) increases in aberrant metaphases confirming the sensitivity of the test item and the activity of the S9 mix.

The results of the concentration analyses were of the dose formulation samples were within the acceptable specification range and confirmed that the targeted top concentration level and the results support the validity of the study conclusion.

Under the experimental conditions DEGDf was not clastogenic in hman perypheral blood lymphocytes up to 2000 microg/ml.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
tk (thymidine kinase) locus of L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse Lymphoma L5178Y TK+/- -3.7.2C cell line with a population doubling time of 10 to 12 hours was used.
Source of frozen stock of Mouse Lymphoma L5178Y TK+/- -3.7.2C cell line: American Type Culture Collection (ATCC), P.O.Box: 1549, Manassas, VA 20108, USA
Stock at the test facility: as frozen permanents in liquid nitrogen

This cell line was screened for the absence of mycoplasma contamination at the test facility and certified free of mycoplasma contamination.
The modal chromosome number of this cell line is 40 and the mouse TK locus is located on the distal end of chromosome 11. Karyotype analysis was performed by an external laboratory to confirm two normal chromosome 11s.
Cells were grown in tubes on a tissue culture rotator and in microtiter plates, inside a humidified CO2 incubator at 37 ± 1°C with 5 ± 0.2 % CO2 in air and 95 – 96% humidity.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 mix
The S9 homogenate was prepared from male Wistar rats induced with a single intraperitoneal injection of 0.7 mL/rat ready to use Aroclor 1254 (500 mg/kg), 5 days prior to sacrifice. The S9 homogenate was prepared in batches and stored in a deep freezer maintained at -68 to -86 ºC. S9 mix was prepared by mixing the following, kept on an ice bath and used within one hour: S9 homogenate, Cofactor, PBS, basic medium
Test concentrations with justification for top dose:
16, 80, 400 and 2000 μg/mL in all the three experiments, experiments 1 and 2 (presence and absence of metabolic activation with 3-hour exposure) and in experiment 3 (absence of metabolic activation with 24-hour exposure).
In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the test item did not show evidence of significant growth inhibition (>90 % inhibition over the control) as relative total growth (RTG) in any of the tested concentrations either in the presence or in the absence of metabolic activation. The test item did not precipitate in the test medium and did not cause any appreciable change in the pH and osmolality of test medium.
Based on these observations, up to a maximum concentration of 2000 μg/mL was tested in all the three experiments of the gene mutation assay.

For the preliminary cytotoxicity test, the cell lines were exposed to 15.625, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL, along with the SW vehicle control.
Vehicle / solvent:
The test item is soluble in sterile water. Hence, sterile water (SW is Milli-Q water sterilized in an autoclave) was used as the vehicle control.
Positive controls:
yes
Positive control substance:
cyclophosphamide
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : Three

METHOD OF TREATMENT/ EXPOSURE:
For Experiments 1 and 2 (genotoxicity), 100 μL of vehicle (Sterile water) or stock/ dilution of Diethylene Glycol Diformate were transferred in to pre-labeled tubes containing the treatment stock culture (6x106 cells).
For Experiment 3, 200 μL of vehicle (Sterile water) or stock/ dilution of
Diethylene Glycol Diformate were transferred in to pre-labeled tubes containing the treatment stock culture (6x106 cells).
For the test in the presence of metabolic activation with 3-hour exposure (Experiment 1), 4 mL of S9 mix was added to each tube to achieve a concentration of 2 % (v/v) of S9.
For the test in the absence of metabolic activation with 3-hour exposure (Experiment 2), 4 mL of basic medium was added to each tube.
For the test in the absence of metabolic activation with 24-hour exposure (Experiment 3), 8 mL of complete medium was added to each tube.
The tube contents were gently mixed, loosely capped and kept for incubation on a tissue culture rotator inside a CO2 incubator for the respective exposure duration

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment 1 and 2 for 3h, Experiment 3 for 24h
- Harvest time after the end of treatment (sampling/recovery times):


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days

- Method used (mutant selection): A sample of the cell suspension of individual replicates from the phenotypic expression phase were suspended in the selective medium and plated at 2000 cells/well in to a microtitre plate for the estimation of mutant frequency.
The plates were incubated in the CO2 incubator for 12 days and then scored manually for the presence of colonies.

-Estimation of Cell Survival: After the phenotypic expression period, a sample of the cell suspension from each replicate culture was plated at 1.6 cells/well in a 96 well microtitre plate for the estimation of cell survival. The plates were incubated in the CO2
incubator for 12 days and then scored manually for the presence of colonies to estimate the Cloning Efficiency (CE) and to arrive at Relative Cloning Efficiency (RCE). From the RSG and RCE values, Relative Total Growth (RTG) was calculated.

-Scoring microtitre palted: The wells of the microtitre plates were scored under a microscope and the number of total wells (T) and the numbers of positive wells (P) were recorded. From this, the number of negative/empty wells (E) was derived as T – P.

- Small and Large Colony Mutants: Wells of the microtitre plates containing mutant colonies were observed using an inverted microscope and the colonies were sized based on a subjective scale.
Large colonies covered more than 25 % of the well and sometimes the entire well. In contrast, small colonies were visibly smaller, covering less than 25 % of the well’s diameter.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: RTG (relative total growth). which includes Relative Suspension Growth (RSG) during the 2 day expression period and the Relative Cloning Efficiency (RCE) obtained at the end of expression period in the preliminary cytotoxicity test and at the time of mutant selection in the gene mutation test.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mutant frequency (MF) is the cloning efficiency of the mutant colonies in selective medium (CEM) adjusted by the cloning efficiency in non-selective medium at the time of mutant selection (CEV).
i.e., MF = CEM / CEV
Induced Mutant Frequency = MF (treatment) – MF (control)


Evaluation criteria:
Providing that all acceptability criteria are fulfilled, the test item is considered able to induce mutation in this test system if in any of the experimental conditions examined, the increase in MF above the concurrent vehicle control exceeds the Global Evaluation Factor (GEF) of 126 x 10-6 and the increase is concentration related.
• Providing that all acceptability criteria are fulfilled, the test item is considered unable to induce mutation in this test system if in all the experimental conditions examined, there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF of 126 x 10-6.
• There is no requirement for verification of a clearly positive or negative response.
• In cases where the response is neither clearly positive nor clearly negative, a repeat experiment may be performed using modified experimental conditions (eg. concentration spacing, other metabolic conditions etc.).
• In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results. In such cases, the test item response should be concluded as equivocal.
Statistics:
The number of negative control values, are assumed to follow Poisson Distribution. Test of significance of difference between negative control values over different dose groups and the positive control will be carried out using a method of anlaysis suggested by M.R.Thomas (Cole and Arlett, 1984), based on χ2. All analysis and significance tests will be evaluated at a 5% level of significance (p ≤ 0.05).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Diethylene Glycol Diformate showed evidence of significant growth inhibition (80-90 % inhibition over the control) as relative total growth (RTG) at the highest tested concentration of 2000 μg/mL, in the presence and absence of metabolic activation and in the absence of metabolic activation with 24-hour exposure.
Based on these observations, a maximum concentration of 2000 μg/mL was tested in all the three experiments of the gene mutation assay.

The test item, Diethylene Glycol Diformate, did not cause a significant increase in the frequencies of mutants compared to vehicle control both in the presence and absence of metabolic activation at the tested concentrations. However, under similar conditions the positive controls (Methyl methanesulfonate and Cyclophosphamide monohydrate) induced a significant increase in the mutant frequency as compared with the vehicle control.

In the presence of metabolic activation, at the highest concentration tested, the reduction in the cell growth as RTG was 78 % compared to the vehicle control.
Similarly, in the absence of metabolic activation, with the 3 and 24-hour exposure period, at the highest concentration tested, the reduction in the cell growth as RTG was 79 and 85 % compared to the respective vehicle controls.

Since, Diethylene Glycol Diformate did not show any positive mutagenic response, colonies were scored using the criteria of small and large colonies only on the negative and positive controls.

refer to the attached full study report for tables and details

Conclusions:
The substance was tested for gene mutation in mammalian cells following OECD 490 (Mouse Lymphoma cell lines L5178T TK+/- -3.7.2C). Under the experimental conditions no evidence for the induction of gene mutation was observed in any of the
concentrations of the test item either in the presence or in the absence of metabolic activation. Taken together, the results of the three independent experiments suggest that the test item does not have the potential to cause gene mutation either in the
presence or in the absence of metabolic activation. It is concluded that the test item, Diethylene Glycol Diformate does not have the potential to induce gene mutation in Mouse Lymphoma L5178Y TK+/- -3.7.2C cells at the tested concentrations.
Executive summary:

The genotoxic potential of the test item, Diethylene Glycol Diformate, to induce gene mutation in mammalian cells was evaluated using cultured Mouse Lymphoma L5178Y TK+/- -3.7.2C cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver). The study consisted of a preliminary cytotoxicity test and a gene mutation

assay. The gene mutation assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 24-hour exposure.

The test item formed a solution in Sterile Water (SW) at 200 mg/mL and was found stable in Milli-Q water for 3 hours at room temperature at the concentrations of 500 and 200000 μg/mL.

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the test item did not show evidence of significant growth inhibition (>90 % inhibition over the control) as relative total growth (RTG) in any of the tested concentrations either in the presence or in the absence of metabolic activation. The test item did not precipitate in the test medium and did not cause any appreciable change in the pH and osmolality of test medium.

Based on these observations, up to a maximum concentration of 2000 μg/mL was tested in all the three experiments of the gene mutation assay.

In the cell gene mutation assay, mouse lymphoma cells were exposed to the test item in duplicate at the concentrations of 16, 80, 400 and 2000 μg/mL in all the three experiments, experiments 1 and 2 (presence and absence of metabolic activation with 3-hour exposure) and in experiment 3 (absence of metabolic activation with 24-hour exposure). Concurrent vehicle and positive controls Cyclophosphamide monohydrate in the presence of metabolic activation and Methyl methanesulfonate in the absence of metabolic activation were also tested in duplicate.

The reduction in cell growth as relative suspension growth (RSG) was 64, 65 and 72 % in Experiments 1, 2 and 3, respectively, compared to the vehicle control.

The reduction in cell growth as relative total growth (RTG) was 78, 79 and 85 % in Experiments 1, 2 and 3, respectively, compared to the vehicle control.

There was no evidence of induction of gene mutation in any of these experiments either in the presence or absence of metabolic activation. In each of these experiments, the respective positive controls produced a statistically significant increase in the frequencies of mutants, under identical conditions. The study indicated that the test item, Diethylene Glycol Diformate was not mutagenic under the conditions of testing employed

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
lymphocytes: human peripherla bllod
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 homogenate
Test concentrations with justification for top dose:
50, 158, 500, 1581 and 5000 μg/plate for the initial and confirmatory experiment
The top dose was seleted based on a preliminary toxicity test resutls, where the testt item did not exhibit toxicity to the tester strain at any of the tested doses; therefore the test item was tested up to the highest OECD 471-recommended dose of 5000 μg/plate in the mutation assay.
Vehicle / solvent:
sterile water (Milli-Q water autoclaved before use.)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate for both initial and confirmatory experiment
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in mediu: palte incorporation for initial experiment; per-incubation for confirmatory experiment

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes at 37°C


METHODS FOR MEASUREMENT OF CYTOTOXICITY
intensity of the bacterial background lawn and mean number of revertant colony counts compared to the SW control plates.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Revertant colonies of all the tester strains for the controls and each test concentration were counted manually.
- OTHER:
Evaluation criteria:
To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of
the metabolic activation system. The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above
or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Diethylene Glycol DiFormate was found to be stable in SW for 5 hours at room temperature at fortification levels of 500 μg/mL and 50000 μg/mL. The results of concentration analysis of Diethylene Glycol DiFormate dose formulations from the initial mutation assay indicated that the mean of percentage agreement with claimed concentration were 103 and 101% (with an RSD of 0.4 and 1.0 %, respectively) of their respective claimed concentrations of 500 and 50000 μg/mL, confirming that the concentration of the test item
was within the acceptable limits. No test item was detected in the vehicle control.

The results of concentration analysis of Diethylene Glycol DiFormate dose formulations from the confirmatory mutation assay indicated that the mean of percentage agreement with claimed concentration were 99.3 and 105% (with an RSD of 0.2 and 0.4 %, respectively) of their respective claimed concentrations of 500 and 50000 μg/mL, confirming that the concentration of the test item was within the acceptable limits. No test item was detected in the vehicle control.

The analysis results of the first set of samples from the initial and confirmatory mutation assays were meeting the acceptance limits, back up samples were disposed. These results from the initial and confirmatory mutation assays indicate that the top dose of 5000 μg/plate level was achieved and support the validity of the study conclusion

Solubility Test
The test item was insoluble in sterile water and soluble in SW at 50 mg/mL, on sonication.

refer to the attached fulls study report for tables and details

Conclusions:
The substance was tested for gene mutation in bacteria following OECD 471. Under the experimental conditions the substance was not mutagenic up to the highest OECD 471 recommended dose of 5000 μg/plate.
Executive summary:

The test item, Diethylene Glycol DiFormate was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in three phases, a preliminary toxicity test, an initial mutation assay and a confirmatory mutation assay. The bacterial tester strains were exposed to the test item in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

The test item was soluble in sterile water (SW) at 50 mg/mL and was found stable in SW for 24 hours at room temperature at the fortification levels of 500 μg/mL and 50000 μg/mL.

In a preliminary toxicity test for the selection of test doses for the mutation assay, the test item did not precipitate on the basal agar plates at any of the tested doses. The test item did not exhibit toxicity to the tester strain at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colony counts was comparable to the SW control plates. Based on these observations, Diethylene Glycol Di Formate was tested up to the highest OECD 471-recommended dose of 5000 μg/plate in the mutation assay.

The test item was exposed in triplicate at 50, 158, 500, 1581 and 5000 μg/plate test doses using the direct plate incorporation mode of exposure in the initial mutation assay and using the pre-incubation mode of exposure in the confirmatory assay. The vehicle control (SW) and the appropriate positive controls were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains. The results of the study from both the initial and confirmatory mutation assay showed that, Diethylene Glycol Di Formate did not show any positive mutagenic increase at any of the tested doses either in the presence or in the absence of metabolic activation. Under identical test conditions, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.

The results of the concentration analysis of the dose formulation samples of both the initial and confirmatory mutation assays confirmed that the top dose level of 5000 μg/plate was achieved in both assays and the results support the validity of the study conclusion.

The study indicated that the test item, Diethylene Glycol DiFormate was not mutagenic in this Bacterial Reverse Mutation Assay up to the highest OECD-471 recommended tested dose of 5000 μg/plate, under the conditions of testing employed.

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

Genetic toxicity in vivo

Description of key information

not required

Additional information

Justification for classification or non-classification

Classification for mutagenicity under Regulation 1272/2008 is warranted for substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans. The classification in Category 2 is based on:

— positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

From the in-vitro battery of tests performed on Diethylene Glycol Diformate the substance did not show any potential to be clastogenic nor mutagenic in both mammalian and bacteria cells. Therefore, based on the above criteria DiethyleneGlycol Diformate is not classified for mutagenicity under Regulation 1272/2008