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

TEGDB:

- 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 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 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 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 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:
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:
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 to 05 August 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to appropriate International Test Guidelines
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
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:
other: US Environmental Protection Agency 1984 Method: HG Gene Muta S typhimurium The Salmonella typhimurium & Method HG-Gene Muta E. Coli The Escherichia coli reverse mutation assay
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, 4I, Bacterial Reverse Mutation Test
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
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
5, 15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test substance completely soluble in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthrascene
Remarks:
with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 3 days

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2 x 10E9 per mL

DETERMINATION OF CYTOTOXICITY
- Method: other: revertent colonies counted.
Evaluation criteria:
a). If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls with some evidence of a positive dose relationship in a specific bacterial strain reproduced with or without S9 mix it is considered to show evidence of mutagenic activity in this test system.
b). If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls at any dose level with any bacterial strain it is considered to show no evidence of mutagenic activity in this test system.
c). If the results obtained fail to satisfy the criteria for a clear positive or negative response given in paragraphs a and b additional testing may be performed in order to resolve the issue of the substance s mutagenic activity in this test system.
Statistics:
If no clear positive response can be obtained, the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al 1989 and will usually be analysis of variance followed by Dunnett s test.
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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2
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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported
- Effects of osmolality: Not reported
- Evaporation from medium: see below
- Other confounding effects: The observations of the test substance made indicate that there was a fine even dispersion of the test substance throughout the top agar during the incubation periods on the plates. A good dispersion was also achieved during the pre incubation period in the second test. The slight reduction in cloudiness during the incubation period was probably due to either evaporation of the test substance metabolism by the bacteria or S9 or diffusion into the minimal agar.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results: negative
It is concluded that TEGDB showed no evidence of mutagenic activity in this bacterial system.
Executive summary:

A study was performed to assess the mutagenetic potential of TEGDB. The study was conducted to GLP and according to EU Method B.13/14. and OECD 471/472 Test Guidelines as well as to EPA and Japan Ministry of Health Test Guidelines.

An in vitro (Ames) test was conducted using histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA1535 TA1537 TA98 and TA100) and a tryptophan dependent mutant of Escherichia coli (strain CM891), which were exposed to the test substance diluted in dimethyl sulphoxide (DMSO). Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254 induced rats S9 mix (metabolic activation). The first was a standard plate incorporation assay the second involved a pre incubation stage. Dose levels of up to 5 mg plate were tested in the mutation tests. Other dose levels used were a series of approximate half-log10 dilutions of the highest concentration.

No toxicity was observed in either mutation test and no evidence of mutagenic activity was seen at any dose level of TEGDB in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that TEGDB showed no evidence of mutagenic activity in this bacterial system. According to the criteria laid down in Council Directive 67/548/EEC (and subsequent adaptations), TEGDB is considered as not mutagenic and is not classified.

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:
02 July 1997 to 29 August 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to appropriate International Test Guidelines
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
other: Joint Directives of JEPA, JMHW and JMITI (31 October 1997) Kanpoan No 287, EISEI No. 127, Kikyoku No. 2 (31 October 1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Human
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
First Test: 6.25 12.5 25, 50, 100, 200, 400 and 800 µg/mL
Second Test: Without S9 mix: 50, 100, 150, 200, 300, 400, 500 and 600 µg/mL
With S9 mix: 50, 100, 200, 400, 500, 600, 700 and 800 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Found to be the most appropriate after assessment and comparisons with water, ethanol and acetone
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
mitomycin C
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 21 hours (3hrs plus 18 hours)
- Fixation time (start of exposure up to fixation or harvest of cells): 21 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: proportion of mitotic cells per 1000 cells in each culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures. From these results the dose level causing a decrease in mitotic index of approximately 50% of the solvent control value or if there was no decrease the maximum achievable concentration was used as the highest dose level for the metaphase analysis.
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:
lymphocytes: First Test
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
800 & 400 µg/mL
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: First Test
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
800 µg/mL
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: First Test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: Second Test
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
300 - 600 µg/mL
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: Second Test
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
800 µg/mL
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results: negative
Benzoflex S-358 has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.
Executive summary:

A study was performed to assess the ability of TEGDB to induce chromosomal aberrations in human lymphocytes cultured in vitro. The study was conducted to GLP and according to OECD 473 Test Guidelines as well as to EPA and Japan Ministry of Health Test Guidelines. Human lymphocytes in whole blood culture were stimulated to divide by addition of phytohaemagglutinin and exposed to the test substance both in the presence and absence of S9 mix derived from rat livers. Solvent and positive control cultures were also prepared. After the appropriate incubation time cell division was arrested using Colcemid, the cells harvested and slides prepared so that metaphase figures could be examined for chromosomal damage. In order to assess the toxicity of TEGDB to cultured human lymphocytes the mitotic index was calculated for all cultures treated with the test substance and the solvent control. In both the absence and presence of S9 mix TEGDB 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 test. All positive control compounds caused large statistically significant increases in the proportion of aberrant cells.

It is concluded that TEGDB has 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: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 July 1997 to 26 August 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted to appropriate International Test Guidelines
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
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Preliminary Toxicity test: 5, 10, 25, 50, 100, 150, 200, 400, 800 µg/mL
Mutation Tests: Test 1: 25, 50, 100, 150, 200, 400 µg/mL (without S9 mix), Test 2: 50, 100, 150, 200, 300, 400 µg/mL (without S9 mix)
Test 1: 25, 50, 100, 150, 200, 400 µg/mL (with S9 mix), Test 2: 50, 100, 150, 200, 300, 400 µg/mL (with S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: most suitable after comparisons with water, ethanol and acetone.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 20-MethyIcholanthrene
Remarks:
with S9 mix
Details on test system and experimental conditions:
1-PRELIMINARY TEST

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: approximately 30 minutes whilst the S9 mix and the test compound solutions
- Exposure duration: 3 hours at 37°C
- Expression time (cells in growth medium): 48 hours. Suspension growth was monitored by sampling twice at approximately 24 hour intervals.
- Selection time (if incubation with a selection agent): 12 days

SELECTION AGENT (mutation assays): selective medium

NUMBER OF REPLICATIONS: 1 with S9 and 1 without S9 at each concentration.

2-MAIN TEST

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: approximately 30 minutes whilst the S9 mix and the test compound solutions
- Exposure duration: 3 hours.
- Expression time (cells in growth medium): 48 hours. Suspension growth was monitored by sampling twice at approximately 24 hour intervals.
- Selection time (if incubation with a selection agent): 12 days

SELECTION AGENT (mutation assays): selective medium

NUMBER OF REPLICATIONS: Two cultures both in the presence and absence of S 9 mix were used for each concentration of the test substance. At least four serial dilutions of the test compound were used when possible at concentrations expected to span the LC80 and LC0 range.

NUMBER OF CELLS EVALUATED: Viability was assessed by plating 200 cells in cloning medium into 90 mm bacteriological culture plates three plates were prepared for each culture. Mutant frequency was assessed by plating 10^6 cells in selective medium into 90 mm plates, three plates were again prepared for each culture.

DETERMINATION OF CYTOTOXICITY
- Method: viability, mutant frequency
Evaluation criteria:
The criteria for a positive response were:
. An increase of at least 100 in the mutant frequency in treated cultures relative to the concurrent control.
. The demonstration of a statistically significant increase in mutant frequency following treatment with the test substance.
. Evidence of a dose relationship over at least two consecutive dose levels in any increases in mutant frequency.
. Demonstration of reproducibility in any increase in mutant frequency.
. An increase in absolute colony numbers in the treated cultures.
. The RTG of cultures showing an increase in mutant frequency should not be less than 10%.
Statistics:
The statistical significance of the data was analysed by weighted analysis of variance following the methods described by (Arlett et al 1989).
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
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: N/A
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results: negative
It was concluded that TEGDB did not demonstrate mutagenic potential in this in vitro gene mutation assay.
Executive summary:

A study was performed to investigate TEGDB for its potential mutagenicity in the (in vitro) mouse lymphoma L5178Y cell mutation test. The study was conducted to GLP and according to the method OECD 476 as well as to EU & EPA Test Guidelines.

TEGDB was tested for mutagenic potential in an in vitro mammalian cell mutation assay; it is based on detection and quantitation of forward mutation in a subline of mouse lymphoma L5178Y cells from the heterozygous condition at the thymidine kinase locus TK+/- to the thymidine kinase deficient genotype TK-/-. Two independent tests in the absence of exogenous metabolic activation S-9 mix and two independent tests in the presence of S-9 mix were carried out. Toxicity was observed after treatment with TEGDB in all the tests both in the absence and the presence of S-9mix. 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 TEGDB 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), diethylene glycol dibenzoate (DEGDB) and triethylene glycol dibenzoate (TEGDB). No testing has been performed on the reaction mass itself but data are available for DPGDB, DEGDB and TEGDB. 

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, DEGDB and TEGDB 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, DEGDB and TEGDB 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

Chromosome aberration tests were performed to determine the potential of DPGDB, DEGDB and TEGDB to cause chromosome aberrations in Chinese Hamster Lung (CHL) cells cultured in vitro (DPDGB and DEGDB) or human lymphocytes (TEGDB).  In both the presence and the absence of metabolic activation (S9 mix), the 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, DEGDB and TEGDB 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, DEGDB or TEGDB 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, DEGDB and TEGDB 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.