Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was negative in a Ames test, MLA and in vitro CA.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-11-04 to 2016-11-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Name: Ethylene glycol dibenzoate
Product: EGDB
CAS No.: 94-49-5
Batch No.: 100000005282
Molecular Weight: 270.28 g/mol
Physical State: solid
Colour: white
Purity: 99.32%
Expiry Date: August 2018
Storage Conditions: room temperature, protected from light
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
Target gene:
His
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat S9 liver microsomal fraction
Test concentrations with justification for top dose:
Experiment I:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment II:
3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate
(without metabolic activation)
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
A. dest., Eurofins Lot No. 160915
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, Applichem Lot No. 0000772452
Positive controls:
yes
Positive control substance:
other:
Remarks:
Positive control without metabolic activation: sodium azide for tester strains TA 100, TA 1535; 4-nitro-o-phenylene-diamine for TA 98, TA 1537; methylmethanesulfonate for TA 102; with metabolic activation: 2-aminoanthracene for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
-For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


- Preincubation period: 60 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.



NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
experiment I and II
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Precipitation of the test item was observed in all tester strains used in experiment I and II (without metabolic activation) at concentrations of 1000 µg/plate and higher.

Toxic effects of the test item were noted in several tester strains evaluated in experiment I and II.

In experiment I toxic effects of the test item were observed in tester strain TA 1537 at concentrations of 1000 µg/plate and higher (without metabolic activation).

In experiment II toxic effects of the test item were noted in tester strains TA 98, TA 100, TA 1535 and TA 1537 at concentrations of 100 µg/plate and higher (without metabolic activation).

Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 of Salmonella typhimurium were exposed to Ethylene glycol dibenzoate at concentrations of 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I and experiment II (with mammalian metabolic activation)), and 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate (experiment II, without mammalian metabolic activation) according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II).

Ethylene glycol dibenzoate was tested up to the limit concentration of 5000 µg/plate in all tester strains used.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 April 2017 - 14 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- No correction factor required.
- Stability at higher temperatures: Yes, maximum temperature: 60°C, maximum duration: 1 hour
- Stability in DMSO: stable, solubility in DMSO: good
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: blood samples were collected by venepuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Suitability of cells: cells from healthy, adult, non smoking humans were used as recommended in international guidelines (e.g. OECD).
- Average Generation Time: first cytogenetic assay: 13.4 h (for test in absence of S9-mix) and 13.2 h (in presence of S9-mix); second cytogenetic assay: 14.2 h; cytogenetic assay 2A: 13.2 h
- Sex, age and number of blood donors: 27-31 years, sex unknown, at least 4 different donors
- Whole blood treated with heparin, added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, resp.), was used.


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
Dose range finding test (reported as part of experiment 1):
Without S9-mix, 3 hr exposure; 24 hr fixation: 15.7, 31.3, 62.5, 125 and 250 µg/mL
Without S9-mix, 24 hr exposure; 24 hr fixation: 15.7, 31.3, 62.5, 125 and 250 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 15.7, 31.3, 62.5, 125 and 250 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 15.7, 31.3, 62.5, 125 and 250 µg/mL
The highest tested concentration was determined by the solubility of the test substance in the culture medium.

First cytogenetic assay:
Without S9-mix, 3 hr exposure; 24 hr fixation: 62.5, 125 and 250 µg/mL
With S9-mix, 3 hr exposure; 24 hr fixation: 62.5, 125 and 250 µg/mL

Second cytogenetic assay:
Without S9-mix, 24 h exposure time, 24 h fixation time: 62.5, 125 and 250 µg/mL
Without S9-mix, 48 h exposure time, 48 h fixation time: 125, 250, 500 and 1000 µg/mL

Cytogenetic assay 2A:
Without S9-mix, 48 hr exposure; 48 hr fixation: 25, 125, 250, 300, 350, 400, 500 µg/mL
The test item precipitated in the culture medium at the highest dose levels (52 and 164 μg/ml).
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: as recommended in OECD test guideline 473.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
The stock solution of the test substance dissolved in DMSO was treated with ultrasonic waves until the test substance was completely dissolved. Test item concentrations were used within 1.5 hours after preparation. The final concentration of the solvent in the culture medium was 1.0% (v/v).

DURATION
- Preincubation period: 48 hr
- Exposure duration experiment 1: 3, 24 and 48 h (without S9)
- Exposure duration experiment 2: 24, 48 h (with S9)
- Fixation time: 24 h (for 3 or 24 hour exposure period) and 48 h (for 48 hour exposure period)

ENVIRONMENTAL CONDITIONS:
- Humidity: 38-91%
- Temperature: 34.4-37.1 °C

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 μg/ml medium)
STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper.

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 150 in each replicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity: for the 3 and 24 hours exposure time the test substance was not cytotoxic and was difficult to dissolve in aqueous solutions, the highest concentration analyzed was determined by the solubility in the culture medium.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
EVALUATION CRITERIA:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

ACCEPTABILITY CRITERIA:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analysed by the Fisher’s exact test (one-sided, p < 0.05).
Statistics:
See evaluation criteria above.
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
Key result
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
FIRST CYTOGENETIC ASSAY:
- The highest dose levels were determined by the solubility or had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.
- Both in the absence and presence of S9-mix, the substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
- Both in the absence and presence of S9-mix, Ethylene glycol dibenzoate did not induce a biologically relevant increase the number of polyploid cells and cells with endoreduplicated chromosomes. Although 1 endoreduplicated chromosome was observed in one of the duplicate cultures at the highest dose level in the presence of metabolic activation, which is outside the 95% control limits of the distribution of the historical negative control database, this was considered an isolated event and therefore considered not biologically relevant.

SECOND CYTOGENETIC ASSAY:
- The test item precipitated in the culture medium at the highest dose level and upwards (164 μg/ml).
- At the 48 h exposure time no appropriate dose levels could be selected for scoring of chromosome aberrations since the second dose level of 250 μg/ml appeared to be toxic (46%). Consequently, no intermediate dose level could be selected. Therefore, the experiment was repeated in cytogenetic assay 2A, using lower dose levels.

SECOND CYTOGENETIC ASSAY (2A):
- Dose levels were determined based on the mitotic index of the dose-range finding test and the first cytogenetic assay and the solubility of the test substance in the culture medium.
- The test substance did neither induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations nor did it induce a biologically relevant increase in the number of polyploid cells and cells with endoreduplicated chromosomes. Although at the 48 h exposure time 2 endoreduplicated chromosomes were observed in one of the duplicate cultures at the highest dose level, which is outside the 95% control limits of the distribution of the historical negative control database, this was considered an isolated event and therefore considered not biologically relevant.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: precipitation in the exposure medium was observed in the first experiment at a dose level of 250 µg/mL and in the second experiment at a dose level of 250 µg/mL (24 hr exposure time) and at 500 and 1000 µg/mL (48 hr exposure time). In cytogenetic assay 2A, the test substance precipitated at dose levels of 300 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest precipitating tested dose

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

Table 1 Historical data for the solvent control

 

3 hours exposure time

24 hours exposure time

48 hours exposure time

 

Gaps included

Gaps excluded

Gaps included

Gaps excluded

Gaps included

Gaps excluded

 

+ S9-mix

- S9-mix

+ S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

Mean number of aberrant cells per 100 cells

0.77

0.77

0.71

0.76

0.76

0.64

0.96

0.74

SD

1.00

1.09

1.02

1.12

1.05

1.02

1.23

1.12

n

284

282

284

282

278

278

270

270

Upper control limit
(95% control limits)

3.25

3.14

3.23

3.14

3.13

2.79

3.97

3.13

Lower control limit
(95% control limits)

-1.71

-1.60

-1.80

-1.61

-1.61

-1.51

-2.06

-1.66

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between January 2012 and November 2016.

Table 2 Historical data for the positive controls

 

3 hours exposure time

24 hours exposure time

48 hours exposure time

 

Gaps included

Gaps excluded

Gaps included

Gaps excluded

Gaps included

Gaps excluded

 

+ S9-mix

- S9-mix

+ S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

Mean number of aberrant cells per 100 cells

32.63

29.33

31.98

28.86

30.90

30.00

35.95

34.96

SD

13.05

13.32

13.09

13.39

13.79

14.09

15.06

15.19

n

280

280

280

280

276

276

268

268

Upper control limit
(95% control limits)

57.86

53.16

57.12

52.57

58.23

57.87

66.11

65.15

Lower control limit
(95% control limits)

7.41

5.50

6.84

5.15

3.58

2.13

5.79

4.78

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between January 2012 and November 2016. 

Conclusions:
A chromosome aberration study with Ethyl glycol dibenzoate was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that Ethyl glycol dibenzoate is not clastogenic in human lymphocytes under the experimental conditions described in this report.
Executive summary:

A chromosome aberration study with Ethyl glycol dibenzoate was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that Ethyl glycol dibenzoate is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 March 2017 - 23 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
No correction factor for purity was applied.
Solubility in vehicle: good
Stability in vehicle: stable
Target gene:
- Thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines


MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
* Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
* Growth and exposure medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/ml trifluorothymidine
* Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not indicated
- Periodically 'cleansed' against high spontaneous background: not indicated
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
Since the test item was poorly soluble in exposure medium, the highest tested concentration was 500 μg/mL exposure medium.
Experiment 1: 2.5, 5, 10, 20, 30, 40, 50, 65, 80, 100, 150, 225, 350 and 500 μg/mL
Experiment 2: 2.5, 5, 10, 25, 50, 75, 100, 150, 225, 325, 425 and 500 μg/mL
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: the test item was checked for solubility in the vehicle based on visual assessment. The test item dissolved in the vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): below 1 x 10^6 cells/mL

DURATION
- Cleansing period: Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
- Exposure duration: 3 hours (experiment 1; with and without S9-mix); 24 hours (experiment 2; without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11/12 days

SELECTION AGENT: TFT

STAIN: 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
- test concentrations: 1
- positive control: 1
- solvent control: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: For determination of the cloning efficiency the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium. For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for cloning efficiency and
MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFTselection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the cloning efficiency and MF were scored with the naked eye or with the microscope.

NUMBER OF CELLS EVALUATED: 9.6 x 10^5 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
- A test item is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more than the mutation frequency in the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of the mutation frequency in the controls + 126.

ACCEPTABILITY CRITERIA:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small
colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility in medium: the test item precipitated in the exposure medium at concentrations of 250 μg/mL and above. The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item
concentration for the dose-range finding test was 500 μg/mL.

RANGE-FINDING/SCREENING STUDIES:
- After 3 hours of treatment, in the absence of S9-mix, the relative suspension growth was 18% at the test item concentration of 500 μg/mL compared to the relative suspension growth of the solvent control.
- After 3 hours of treatment, in the presence of S9-mix, no toxicity in the relative suspension growth was observed up to test item concentrations of 500 μg/mL compared to the solvent control.
- After 24 hours of treatment, the relative suspension growth was 7% at the test item concentration of 500 μg/mL compared to the relative suspension growth of the solvent control.

HISTORICAL CONTROL DATA: see table 1 and table 2

- In the second experiment (main study), the relative total growth of the highest test item was 33% compared to the total growth of the solvent controls at the highest precipitating dose level.
- In the second experiment (main study), increases above the 95% control limit were observed. Although these increases are above the historical control data range, no dose-related response is observed, the highest increase in the mutation frequency (172 per 10^6 survivors) is below the MF(controls) + 126 (270 per 10^6 survivors) and not more than a 1.2-fold increase was observed, therefore this increase is considered to be not biologically relevant.

ACCEPTABILITY:
- The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database
- Positive control chemicals both produced significant increases in the mutation frequency and the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
Remarks on result:
other: In both experiments

For detailed results, see attached illustration.

Table 1 Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

 

Mutation frequency per 106survivors

 

- S9-mix

+ S9-mix

 

3 hour treatment

24 hour treatment

3 hour treatment

Mean

86

81

87

SD

23

26

28

n

220

202

273

Upper control limit

(95% control limits)

135

135

145

Lower control limit

(95% control limits)

37

28

28

SD = Standard deviation

n = Number of observations

Table 2 Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay

 

Mutation frequency per 106survivors

 

- S9-mix

+ S9-mix

 

3 hour treatment

24 hour treatment

3 hour treatment

Mean

857

688

1710

SD

246

187

815

n

110

102

139

Upper control limit

(95% control limits)

1425

1124

4214

Lower control limit

(95% control limits)

289

253

-793

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between January 2013 and November 2016. 

Conclusions:
Based on the results of a in vitro mammalian cell gene mutation test, performed according to OECD guideline 490, Ethylene glycol dibenzoate is not mutagenic in the TK mutation test system under the experimental conditions described in this report.
Executive summary:

Based on the results of a in vitro mammalian cell gene mutation test, performed according to OECD guideline 490, Ethylene glycol dibenzoate is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

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

Genetic toxicity in vivo

Description of key information

The substance was negative in an in vivo CA.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2017 - 04September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
in vivo required for non-EU registration
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
3 November 2015
Type of assay:
other: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Specific details on test material used for the study:
Stability at higher temperatures: maximum temperature: 60°C for a maximum duration of 1 hour
Species:
mouse
Strain:
NMRI
Details on species / strain selection:
Species and strain are recommended by international guidelines (e.g. OECD, EC).
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: 6 weeks
- Weight at study initiation: 35.4 ± 2.0 g (within the range of 33-38 g)
- Assigned to test groups randomly: yes
- Fasting period before study: 3 - 4 hours prior to dosing until approximately 1 hour after administration of the test item.
- Housing: group housing in cages (maximum 5 animals per sex per cage) containing sterilized sawdust as bedding material.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: ≥5 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18.0-24.0 (actual: 21.5 - 22.4)
- Humidity (%): 40-70 (actual: 46-73)
- Air changes (per hr): ≥10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 31 July 2017 To: 25 August 2017
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: polyethylene glycol 400
- Justification for choice of vehicle: the test item was stable in PEG400 for at least 5 hours at room temperature under normal laboratory conditions and 8 days in the refrigerator over the concentration range 1 to 200 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Specific gravity of vehicle: 1.125 g/mL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: dosing solutions were prepared on the day of administration. No correction was made for the purity/composition of the test item. Before use the test item was crushed and ground in a mortar with pestle to improve the consistency. Test item concentrations were treated with ultra-sonic waves and heated up to a maximum temperature of 59.0°C until it had completely dissolved. Test item concentrations were dosed within 4 hours after preparation.

Duration of treatment / exposure:
Two treatments were performed, administered at a 24-hour interval.
Frequency of treatment:
Treatment was performed in 2 dosings with 2-3 hours in between.
Post exposure period:
The sampling time was 48 hours after first dosing.
Dose / conc.:
2 000 mg/kg bw (total dose)
No. of animals per sex per dose:
5 male animals per sampling time for the treatment group, positive control group and vehicle control group each (15 animals in total). Based on the results of the dose-range finding test that did not indicate differences in toxicity between sexes, the main test was performed with only male animals.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide dissolved in physiological saline

- Justification for choice of positive control: recommended in international guidelines (e.g. OECD, EC)
- Route of administration: oral gavage
- Doses / concentrations: single dose of 40 mg/kg bw
Tissues and cell types examined:
Polychromatic erythrocytes from the bone marrow were examined for micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: in the dose-range finding test performed with 3 male and 3 female animals, no toxicity was observed up to 2000 mg/kg bw. Therefore, the main test was performed as a limit test with a dose level of 2000 mg/kg bw (maximum recommended dose in accordance with regulatory guidelines).

TREATMENT AND SAMPLING TIMES: Two treatments were performed, administered at a 24-hour interval. The test item is administered as a split dose, i.e., two treatments on the same day separated by no more than 2-3 hours, to facilitate administering a large volume necessary due to limited solubility of the test item.
Bone marrow was sampled 48 hours after the first dosing.

DETAILS OF SLIDE PREPARATION: Femurs were flushed with approximately 2 ml of fetal calf serum. The cell suspension was collected and centrifuged at 216 g for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer (based on Giemsa).

METHOD OF ANALYSIS:
At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%).
The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

OTHER: Animals were checked for mortality at least twice a day. The toxic signs were recorded at least once a day. The animals were weighed just prior to dosing.
Evaluation criteria:
A test item is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control;
b) The increase is dose related when evaluated with a trend test;
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control;
b) There is no concentration-related increase when evaluated with a trend test;
c) All results are within the 95% control limits of the negative historical control data range.

ACCEPTABILITY CRITERIA:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. The positive control data will be analyzed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).
Statistics:
The positive control data were analyzed by the Students t test (one-sided, p < 0.05) to determined statistical significance.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: three males and three females were treated with 2000 mg/kg bw
- Clinical signs of toxicity in test animals: no
- Rationale for exposure: according to guidelines (e.g. OECD, EC)
- Other: observation period after dosing was 3 days

RESULTS OF DEFINITIVE STUDY (see table 1)
- Induction of micronuclei: no increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of Ethylene glycol dibenzoate treated animals compared to the vehicle treated animals.
- Ratio of PCE/NCE: no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group
- Appropriateness of dose levels and route: since no toxicity was observed in the dose-range finding test and no differences between the sexes were observed, it was justifiied to use 2000 mg/kg bw as only dose level.
- Statistical analysis: since a statistical effect was only observed in the treatment group treated with the positive control, statistical analysis was applied to this treatment group only.
- All animals tested showed no treatment related clinical signs of toxicity or mortality.

ACCEPTABILITY OF ASSAY:
- The concurrent vehicle control data were within the 95% control limits of the distribution of the historical negative control database (see table 1-3).
- The positive control induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes (p<0.001). The number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database (i. e. 29.8 +/- 7.9)

Table 1 Mean Number of Micronucleated Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic Erythrocytes

Group

Treatment

Dose

(mg/kg body weight)

Number of micronucleated polychromatic erythrocytes

Ratio polychromatic/ normochromatic erythrocytes

 

 

 

(mean ± S.D.)(a,b)

(mean ± S.D.)(a,c)

 

 

 

 

 

 

 

 

 

A

Vehicle Control

0

1.0

±

0.7

1.04

±

0.07

B

Test Item

2000

1.0

±

1.0

0.99

±

0.11

C

CP

20

29.8

±

7.9(d)

0.80

±

0.22

Vehicle control = PEG400

CP = Cyclophosphamide.

(a)Five animals per treatment group. ; (b)At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.; (c)The ratio was determined from at least the first 1000 erythrocytes counted.; (d)Significantly different from corresponding control group (Students t test, Welch t test, P < 0.001).

Table 2 Historical data for the vehicle controls (males)

Mean number of micronucleated polychromatic erythrocytes per 2000 cells

1.34

SD

1.10

n

230

Upper control limit

(95% control limits)

3.76

Lower control limit

(95% control limits)

-1.09

SD = Standard deviation; n = Number of observations

Distribution historical negative control data from experiments performed between January 2012 and October 2017.

Table 3 Historical data for the positive controls (males)

Mean number of micronucleated polychromatic erythrocytes per 2000 cells

24.80

SD

9.45

n

224

Upper control limit

(95% control limits)

47.18

Lower control limit

(95% control limits)

2.42

SD = Standard deviation; n = Number of observations

Distribution historical negative control data from experiments performed between January 2012 and October 2017.

Conclusions:
In a bone marrow micronucleus test with male mice, performed according to OECD guideline 474 and GLP principles, it is concluded that Ethylene glycol dibenzoate is not clastogenic or aneugenic up to and including a dose level of 2000 mg/kg bw.
Executive summary:

In a bone marrow micronucleus test with male mice, performed according to OECD guideline 474 and GLP principles, it is concluded that Ethylene glycol dibenzoate is not clastogenic or aneugenic up to and including a dose level of 2000 mg/kg bw.

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

Additional information

The in vivo testing was required for registration outside the EU.

Justification for classification or non-classification

The test substance is not classified based on the negative outcome of the available studies.