Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No specific testing was performed on the reaction mass. However in all of the studies performed on separate components of the reaction mass (please see enlisting below) no evidence of genetic toxicity was seen. Therefore no evidence of genetic toxicity would be expected for the reaction mass.

DEGDB:

- Key study, reliabiltiy 1, OECD 471/ OECD 472 - negative with and without metabilic activation

- Supporting study, reliability 2 - negative

- Key study, reliability 1, OECD 473 - negative with and without metabolic activation

- Key study, reliability 1, OECD 476 - negative with and without metabolic activation

DPGDB:

- Key study, reliabiltiy 1, OECD 471/ OECD 472 - negative with and without metabolic activation

- Key study, reliability 1, OECD 473 - negative with and without metabolic activation

- Key study, reliability 1, OECD 476 - negative with and without metabolic activation

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:
23 July 1997 - 05 August 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD, EC and US EPA test guidelines and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, 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:
other: Japan Ministry of Health and Welfare (1989) Notification Yakushin 1 No 24: Guidelines for Toxicity Studies of Drugs, 4 I, Bacterial Reverse Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
First test - 5000, 1500, 500, 150, 50, 15, and 5 µg/plate plus a vehicle control.
Second test - 5000, 1500, 500, 150, and 50 µg/plate, plus a vehicle control.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was confirmed by the sponsor to be poorly soluble in water (solubility 51 ppm at 25°C). The solubility at 50 mg/mL was assessed in DMSO, ethanol, methanol, and acetone and the test substance was found to be fully soluble in each solvent tested. DMSO was selected as the solvent following confirmation by the sponsor. This solvent is the most commonly used solvent within the testing laboratory when water is not suitable.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
5 µg/plate for TA1535, 3 µg/plate for TA100, and 2 µg/plate for CM891 (In the absence of S9 mix)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537 (In the absence of S9 mix)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
1 µg/plate for TA98 (In the absence of S9 mix)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
In the presence of S9 mix. 2 µg/plate for TA1535; 10 µg/plate for CM891.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5µg/plate for TA1537, TA98, and TA100 (In the presence of S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation (second test only)


DURATION
- Preincubation period: 30 minutes (second test only)
- Exposure duration: 3 days


SELECTION AGENT (mutation assays): Histidine / tryptophan deficient agar


NUMBER OF REPLICATIONS: 3 replicate plates per concentration per strain


DETERMINATION OF CYTOTOXICITY
- Method: observation of the number of revertant colony counts and the presence or absence of the background bacterial lawn


Evaluation criteria:
The number of revertant colony counts for each test or positive control plate was determined and compared to the relevant concurrent vehicle control.
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:
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:
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: In the first test, at 5000 µg and 1500 µg/plate when the test substance solution was mixed with the top agar, a cloudy solution formed; slight cloudiness was observed at 500 µg/plate and at 150 µg/plate there was no observable cloudiness. In all cases the appearance was similar once the agar had set on the plates.
In the pre-incubation assay, a cloudy solution formed with a few small globules at 5000 µg/plate, a cloudy solution with no globules was observed at 1500 µg and 500 µg/plate; a slightly cloudy solution was observed at 150 µg/plate, and at 50 µg/plate no cloudiness was observed. In all cases, the appearance was similar following the pre-incubation period.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean revertant colony counts for the solvent controls were within the historical range.


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative
It was concluded that DEGDB showed no evidence of mutagenic activity in this bacterial system.
Executive summary:

A bacterial reverse mutation assay was performed to assess the potential of the test material DEGDB to cause gene mutation. The study was conducted according to OECD, EC, US EPA, and Japanese test guidelines and in compliance with GLP.

DEGDB was tested against four strains of Salmonella typhimurium and one strain of Escherichia coli, both in the presence and in the absence of metabolic activation (provided by S9 mix). Relevant positive controls were tested concurrently and provided positive results, demonstrating the sensitivity of the assay.

No increases in the number of revertant colonies relative to the concurrent vehicle controls were observed for the test material in any of the strains tested. It was concluded that DEGDB showed no evidence of mutagenic activity in this bacterial system.

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:
23 July 1997- 5 August 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD, EC and US EPA test guidelines and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, 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 OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
This system employs mutant strains of Escherichia coli which are incapable of synthesising the amino acid tryptophan required for growth.
The strains used carry additional mutations which render them more sensitive to mutagens. The S. typhimurium strains have a defective cell coat which allows greater permeability of test substances into the cell. All the strains are deficient in normal DNA repair processes. In addition three of them possess a plasmid pKM101 which introduces an error prone repair process resulting in increased sensitivity to some mutagens.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
First test - 5000, 1500, 500, 150, 50, 15, and 5 µg/plate plus a vehicle control.
Second test - 5000, 1500, 500, 150, and 50 µg/plate, plus a vehicle control.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent/vehicle: The test material is poorly soluble in water. The solubility at 50 mg/mL was assessed in DMSO, ethanol, methanol, and acetone and the test substance was found to be fully soluble in each solvent tested. DMSO was selected as the solvent following confirmation by the sponsor. This solvent is the most commonly used solvent within the testing laboratory when water is not suitable.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
5 µg/plate for TA1535, 3 µg/plate for TA100, and 2 µg/plate for CM891 Migrated to IUCLID6: In the absence of S9 mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537 Migrated to IUCLID6: In the absence of S9 mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
1 µg/plate for TA98 Migrated to IUCLID6: In absence of S9 mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
In the presence of S9 mix. 2 µg/plate for TA1535; 10 µg/plate for CM891.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5µg/plate for TA1537, TA98, and TA100 Migrated to IUCLID6: In presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation (second test only)


DURATION
- Preincubation period: 30 minutes (second test only)
- Exposure duration: 3 days


SELECTION AGENT (mutation assays): Histidine / tryptophan deficient agar


NUMBER OF REPLICATIONS: 3 replicate plates per concentration per strain


DETERMINATION OF CYTOTOXICITY
- Method: observation of the number of revertant colony counts and the presence or absence of the background bacterial lawn
Evaluation criteria:
The number of revertant colony counts for each test or positive control plate was determined and compared to the relevant concurrent vehicle control.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: In the first test, at 5000 µg and 1500 µg/plate when the test substance solution was mixed with the top agar, a cloudy solution formed with globules apparent at the higher level.; slight cloudiness was observed at 500 µg/plate and at 150 µg/plate there was no observable cloudiness. In all cases the appearance was similar once the agar had set on the plates.
In the pre-incubation assay, a cloudy solution formed with a few small globules at 5000 µg/plate, a cloudy solution with no globules was observed at 1500 µg and 500 µg/plate; a slightly cloudy solution was observed at 150 µg/plate, and at 50 µg/plate no cloudiness was observed. In all cases, the appearance was similar following the pre-incubation period.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean revertant colony counts for the solvent controls were within the historical range.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative
It is concluded that DPGDB showed no evidence of mutagenic activity in this bacterial system.
Executive summary:

A bacterial reverse mutation assay was performed to assess the potential of the test material DPGDB to cause gene mutation. The study was conducted according to OECD, EC, US EPA and Japanese test guidelines and in compliance with GLP.

DPGDB was tested against four strains of Salmonella typhimurium and one strain of Escherichia coli, both in the presence and in the absence of metabolic activation (provided by S9 mix). Relevant positive controls were tested concurrently and provided positive results, demonstrating the sensitivity of the assay.

No increases in the number of revertant colonies relative to the concurrent vehicle controls were observed for the test material in any of the strains tested. It was concluded that DPGD showed no evidence of mutagenic activity in this bacterial system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 January 1997 - 16 April 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with OECD, UK Department of Health, EC and EPA test guidelines, and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: UK Department of Health Report on Health and Social Subjects No. 35. Guidelines for the testing of chemicals for mutagenicity (1989).
Deviations:
no
Qualifier:
according to
Guideline:
other: US Environmental Protection Agency. Method: HG-Chrome - in vitro. In vitro cytogenetics (1982).
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
4.9, 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, and 625 µg/mL (Initial tests)
20, 40, 60, 80, 100, 120, 140, 160, 200 µg/mL (repeat of test without S9 mix).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility in four solvents (water, ethanol, acetone, and DMSO) was assessed, then the subsequent solutions assesed for precipitation or oily globules, etc. when added to culture medium. The data confirmed that DMSO was the most appropriate solvent for use in this study.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Used in the absence of S9 mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Used in the presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: 24 hours (cells in culture flasks, prior to dosing).
- Exposure duration: 18 hours (in the test with S9 mix, the medium containing S9 mix was removed and replaced by fresh medium after 3 hours). In the second test, cells were exposed for 42 hours, with harvests at 18 and 42 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 2 hours

SPINDLE INHIBITOR (cytogenetic assays): 0.1% Trypsin
STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: Duplicate assays in each test

NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Statistics:
The number of aberrant metaphase figures in each treatment group was compared with the solvent control value using Fisher's test, Fisher (1973).
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No significant reduction in mitotic indices was observed in the tests in the presence of S9 mix.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reductions in Mitotic indices were seen in each test with S9 mix; the concentrations selected for metaphase analysis were determined based on the extent of the reductions in mitotic index.
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative
It was concluded that DEGDB had shown no evidence of clastogenic activity in this in-vitro cytogenetic test system.
Executive summary:

A chromosome aberration test was performed to determine the potential of the test substance DEGDB to cause chromosome aberrations in Chinese Hamster Lung (CHL) cells cultured in vitro. The study was conducted according to OECD, EC, UK, and US EPA test guidelines, and in compliance with GLP. In both the presence and the absence of metabolic activation (S9 mix), DEGDB caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared with the solvent control in either of two tests. It was concluded that DEGDB had shown no evidence of clastogenic activity in this in-vitro cytogenetic test system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 January 1997 - 16 April 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD, UK Department of Health, EC and EPA test guidelines, and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: UK Department of Health Report on Health and Social Subjects No. 35. Guidelines for the testing ofchemicals for mutagenicity (1989).
Deviations:
no
Qualifier:
according to
Guideline:
other: US Environmental Protection Agency. Method HG-Chrome in vitro. In vitro mammalian cytogenetics (1982).
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix.
Test concentrations with justification for top dose:
Without S-9 mix; 19.5, 39.1, 50, 78.1, 100, 156.3 and 200 µg/mL
With S-9 mix; 39.1, 78.1, 156.3, 312.5, 400, 500 and 625 µg/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: Prior to commencing testing, the solubility of the test substance in solvents compatible with the test system was assessed. This consisted of a semi-quantitative determination of the solubility of DPGDB in water, dimethyl sulphoxide (DMSO), ethanol and acetone. The data confirmed that DMSO was the most appropriate solvent to use in this study.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Used in absence of S9
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Used in presence of S-9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: as impregnation on paper disk

DURATION
- Preincubation period: 24 hours (cells in culture flasks, prior to dosing)
- Exposure duration: 18 hours (in the test with S9 mix, the medium containing S9 mix was removed and replaced by fresh medium after 3 hours). In the second test, cells were exposed for 42 hours, with harvests at 18 and 42 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 2 hours.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: Duplicate assays in each test

NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Statistics:
The number of aberrant metaphase figures in each treatment group was compared with the solvent control value using Fisher's test, Fisher (1973).
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No significant reduction in mitotic indices was observed in the tests in the presence of S9 mix.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative
It was concluded that DPGDB had shown no evidence of clastogenic activity in this in-vitro cytogenetic test system.
Executive summary:

A chromosome aberration test was performed to determine the potential of the test substance DPGDB to cause chromosome aberrations in Chinese Hamster Lung (CHL) cells cultured in vitro. The study was conducted according to OECD, EC, UK, and US EPA test guidelines, and in compliance with GLP. In both the presence and the absence of metabolic activation (S9 mix), DPGDB caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared with the solvent control in either of two tests. It was concluded that DPGDB had shown no evidence of clastogenic activity in this in-vitro cytogenetic test system.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: other: Cell mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 February 1997 - 07 May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with OECD, EPA, and EC guidelines, and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: US Environmental Protection Agency: HG-Gene Muta-Somatic Cells: Detection of gene mutations in somatic cells in culture, 1983.
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Directive 87/302/EEC, 1988.
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
Preliminary toxicity test - 9.8, 19.6, 39.1, 78.1, 156.3, 312.5, 625, 1250 µg/mL.
Mutation tests - 50, 100, 150, 200, 250, 275, 300, 325, 350 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility was determined in water, DMSO, Acetone, and Ethanol; the test substance was found to be miscible in Ethanol, Acetone and DMSO, but imiscible with water. Solutions of the test material were then added to culture medium at 1% and observations made. All solvents gave precipitation on dosing, and above approximately 625µg/mL globular droplets were observed. On consultation with the sponsor, DMSO was chosen as the vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
10 µg/mL, used in the absence of S-9 mix.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 20-Methylcholanthrene
Remarks:
2.5 µg/mL, used in the persence of S-9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days (48 hours' expression time followed by 12 days' incubation).

NUMBER OF REPLICATIONS: 3 plates were prepared for each culture

NUMBER OF CELLS EVALUATED: 200 Assessed for viability; 10^6 assessed for Mutant Frequency

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (growth in treated groups relative to growth in untreated control groups)
Evaluation criteria:
Relative Total Growth (RTG) over the experimental period and the Mutant Frequency per 10^6 survivors (MF) were calculated.
Statistics:
The statistical significance of the data was analysed by weighted analysis of variance following the methods described by Arlett et aI (1989).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative
It was concluded that DEGDB did not demonstrate mutagenic potential in this in vitro gene mutation assay.
Executive summary:

A mammalian cell mutation assay was conducted to assess the potential of the test material DEGDB to cause mutation within mouse lymphoma L5178Y cells. The study was conducted according to OECD, EPA, and EC test guidelines, and in compliance with GLP.

Toxicity was observed after treatment with DEGDB in all the tests both in the absence and the presence of metabolic activation (S-9 mix). No biologically significant increases in mutant frequency were observed in any of the tests either in the absence or the presence of S-9 mix. It was concluded that DEGDB did not demonstrate mutagenic potential in this in vitro mammalian cell mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: other: Cell mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 February 1997 - 7 May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD, EPA and EC test guidelines and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: US Environmental Protection Agency, Method; HG-Gene Muta-Somatic Cells: Detection of gene mutations in somatic cells in culture, 1983
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Directive 87/302/EEC, 1988.
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Preliminary toxicity test - 4.9, 9.8, 19.6, 39.1, 78.2, 156.3, 312.5, 625 µg/mL
Mutation tests - 25, 50, 75, 100, 150, 200, 225 and 250 µg/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent/vehicle: Solubility was determined in water, DMSO, acetone, and ethanol; the test substance was found to be miscible in ethanol, acetone and DMSO, but imiscible with water. Solutions of the test material were then added to culture medium at 1% and observations made. All solvents gave precipitation on dosing, and above approximately 625 µg/mL globular droplets were observed. On consultation with the sponsor, DMSO was chosen as the vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
10 µg/mL, used in absence of S-9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 20-Methylcholanthrene
Remarks:
2.5 µg/mL, used in presence of S-9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days (48 hours' expression time followed by 12 days' incubation).

NUMBER OF REPLICATIONS: 3 plates were prepared for each culture

NUMBER OF CELLS EVALUATED: 200 Assessed for viability; 10^6 assessed for Mutant Frequency

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (growth in treated groups relative to growth in untreated control groups)
Evaluation criteria:
Relative Total Growth (RTG) over the experimental period and the Mutant Frequency per 10^6 survivors (MF) were calculated.
Statistics:
The statistical significance of the data was analysed by weighted analysis of variance following the methods described by Arlett et aI (1989).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative
It was concluded that DPGDB did not demonstrate mutagenic potential in this in vitro gene mutation assay.
Executive summary:

A mammalian cell mutation assay was conducted to assess the potential of the test material DPGDB to cause mutation within mouse lymphoma L5178Y cells. The study was conducted according to OECD, EPA, and EC test guidelines, and in compliance with GLP.

Toxicity was observed after treatment with DPGDB in all the tests both in the absence and the presence of metabolic activation (S-9 mix). No biologically significant increases in mutant frequency were observed in any of the tests either in the absence or the presence of S-9 mix. It was concluded that DPGDB did not demonstrate mutagenic potential in this in vitro mammalian cell mutation assay.

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

Genetic toxicity in vivo

Description of key information

There are no in vivo studies for genetic toxicity avaialable.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

This substance is a reaction mass of dipropylene glycol dibenzoate (DPGDB) and diethylene glycol dibenzoate (DEGDB). No testing has been performed on the reaction mass itself but data are available for DPGDB and DEGDB. 

The results of the gene mutation in bacteria, chromosomal aberration test and gene mutation in mammalian cells studies indicate that the components of the reaction mass are not classified for genetic toxicity; therefore the reaction mass itself is also not classified for genetic toxicity.

All main studies were performed according to international test guidelines and in compliance with GLP. An earlier, supporting study on DEGDB was not in compliance with GLP. The results are presented below by the test type.

 

Gene mutation in bacteria

DPGDB and DEGDB were separately tested against four strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and one strain of Escherichia coli (CM891), both in the presence and in the absence of metabolic activation (provided by S9 mix). Relevant positive controls were tested concurrently and provided positive results, demonstrating the sensitivity of the assay. No increases in the number of revertant colonies relative to the concurrent vehicle controls were observed for any of the test materials in any of the strains tested. 

A supporting study which was largely consistent in design with the test guideline was performed on DEGDB. Salmonella typhimurium TA 1538 and Saccharomyces cerevisiae were tested in addition to the strains of Salmonella typhimurium listed above in the main tests. No evidence of mutagenic activity was observed.

 

It was concluded that DPGDB and DEGDB showed no evidence of mutagenic activity in this bacterial system. The reaction mass would therefore also show no sign of mutagenic activity.

Chromosomal aberration – in vitro test

Chromosomal aberration tests were performed to determine the potential of DPGDB and DEGDB to cause chromosome aberrations in Chinese Hamster Lung (CHL) cells cultured in vitro.  In both the presence and the absence of metabolic activation (S9 mix), the separately tested substances caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared with the solvent control in either of two tests performed in each study.

 

It was concluded that DPGDB and DEGDB showed no evidence of clastogenic activity in this in-vitro cytogenetic test system. The reaction mass would also show no clastogenic activity.

 

 

Gene mutation in mammalian cells

Mammalian cell mutation assays (mouse lymphoma L5178Y cells) were conducted and toxicity was observed after treatment with DPGDB or DEGDB in all the tests both in the absence and the presence of metabolic activation (S-9 mix). No biologically significant increases in mutant frequency were observed in any of the tests either in the absence or the presence of S-9 mix.

 

It was concluded that DPGDB and DEGDB did not demonstrate mutagenic potential in this in vitro mammalian cell mutation assay.


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

On the basis of the findings in the reported different in-vitro investigations, and according to the criteria laid down in EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, the components of the reaction mass is not considered to be mutagenic and therefore the reaction mass itself is also not classified for mutagenicity.