Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

PGDB

OECD 471 (Ames test): Negative with and without metabolic activation

OECD 473 (In vitro mammalian cell chromosome aberration test): Negative with and without metabolic activation

OECD 476 (In vitro gene mutation study in mammalian cells: Mouse Lymphoma Assay) - Negative with and without metabolic activation

DPGDB

OECD 471 (Ames test): Negative with and without metabolic activation

OECD 473 (In vitro mammalian cell chromosome aberration test): Negative with and without metabolic activation

OECD 476 (In vitro gene mutation study in mammalian cells: Mouse Lymphoma Assay) - 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:
25 June to 1 October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to approved guidelines and in compliance with GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Copy of certificate of compliance from UK GLP Monitoring Authority included in report
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
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compound insoluble in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 30 minutes (second test only)
- Exposure duration: 72h
- Expression time (cells in growth medium): not applicable to this test
- Selection time (if incubation with a selection agent): selection agent not used
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable to this test

SELECTION AGENT (mutation assays): not applicable to this test
SPINDLE INHIBITOR (cytogenetic assays): not applicable to this test
STAIN (for cytogenetic assays): not applicable to this test

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: not applicable to this test

DETERMINATION OF CYTOTOXICITY
- Method: observed as a reduction in revertant colony numbers


OTHER EXAMINATIONS:
None
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.

If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Statistics:
The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.
Key result
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was seen in strain WP2 uvrA (pKM101) following exposure to PGDB at 5000 µg/plate in the first test. No signs of toxicity were observed with any other strains following exposure to PGDB in either of the tests.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
It was concluded that PGDB showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

No evidence of toxicity was obtained following exposure to PGDB. No precipitate was observed on any plates containing PGDB. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to PGDB at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

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.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
Qualifier:
according to guideline
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
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.
Key result
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
Key result
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:

Key data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

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 May to 1 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to approved guidelines and in compliance with GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP Monitoring Authority included in report
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:
In the absence of S9 mix, 3-hour treatment: 25, 90 and 100 µg/mL.
In the presence of S9 mix, 3-hour treatment: 500, 2000 and 2843 µg/mL.
In the absence of S9 mix, 21-hour continuous treatment: 125, 150 and 225 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: PGDB was soluble in dimethyl sulphoxide (DMSO) at 284.3 mg/mL (1M)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
mitomycin C - with S9 mix; cyclophoshamide - without S( mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: none
- Exposure duration: 3 hours and 21 hours
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colcemid®
STAIN (for cytogenetic assays):10% Giemsa, prepared in buffered water (pH 6.8)

NUMBER OF REPLICATIONS: not reported

NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
:
Evaluation criteria:
The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
The increases exceed the vehicle control range of this laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
Statistics:
One-tailed Fisher exact test (Fisher 1973) for comparing the number of aberrant metaphase cells in each test substance group with the vehicle control value.
Cochran-Armitage test for trend (Armitage, 1955) applied to the control and all test substance groups.

The data was analysed using the SAFEStat (SAS statistical applications for end users, version 1.1) Chromosome Aberrations application (version 1.1) which was developed in SAS (SAS INSTITUTE 2002).
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
As PGDB was negative for both short term treatments (3-hour treatment in the absence and presence of S9 mix), in accordance with current guidelines, cultures treated continuously for 21 hours in the absence of S9 mix underwent metaphase analysis.

TEST-SPECIFIC CONFOUNDING FACTORS
None observed

RANGE-FINDING/SCREENING STUDIES:
In the absence of S9 mix following 3-hour treatment, PGDB caused a reduction in the mitotic index to 68% of the vehicle control value at 1023.48 μg/mL. At higher tested concentrations overt toxicity was observed.
In the presence of S9 mix following 3-hour treatment, PGDB caused a reduction in the mitotic index to 54% of the vehicle control value at 2843 μg/mL.
In the absence of S9 mix following 21-hour continuous treatment, PGDB caused a reduction in the mitotic index to 80% of the vehicle control value at 132.64 μg/mL. At higher tested concentrations overt toxicity was observed.

COMPARISON WITH HISTORICAL CONTROL DATA:

All mean values for the vehicle control (DMSO), and all PGDB treatment concentrations were within the laboratory historical control range, when taken at the 99% confidence limit.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Main test -S9, 3 hr exposure - reduction in the mitotic index to 52% of the vehicle control value at 100 μg/mL. The concentrations selected for metaphase analysis were 25, 90 and 100 μg/mL.

Main test +S9, 3 hr exposure - caused a reduction in the mitotic index 78% of the vehicle control value at 2843 µg/mL. The concentrations selected for metaphase analysis were 500, 2000 and 2843 µg/mL.

Main test -S9, 21 hr exposure - PGDB caused a reduction in the mitotic index to 56% of the vehicle control value at 225 µg/mL. The concentrations selected for metaphase analysis were 125, 150 and 225 µg/mL.





TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:


TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TEST-SPECIFIC CONFOUNDING FACTORS
None

RANGE-FINDING/SCREENING STUDIES:
Toxicity:
In the absence of S9 mix following 3-hour treatment, PGDB caused a reduction in the mitotic index to 68% of the vehicle control value at 1023.48 µg/mL. At higher tested concentrations overt toxicity was observed.

In the presence of S9 mix following 3-hour treatment, PGDB caused a reduction in the mitotic index to 54% of the vehicle control value at 2843 µg/mL.

In the absence of S9 mix following 21-hour continuous treatment, PGDB caused a reduction in the mitotic index to 80% of the vehicle control value at 132.64 µg/mL. At higher tested concentrations overt toxicity was observed.


COMPARISON WITH HISTORICAL CONTROL DATA:

All mean values for the vehicle control (DMSO), and all PGDB treatment concentrations were within
the laboratory historical control range, when taken at the 99% confidence limit.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Main test: -S9, 3 hrs exposure - PGDB caused a reduction in the mitotic index to 52% of the vehicle control value at 100 µg/mL. The concentrations selected for metaphase analysis were 25, 90 and 100 µg/mL.

Main test: +S9, 3 ht exposure - PGDB caused a reduction in the mitotic index 78% of the vehicle control value at 2843 µg/mL. The concentrations selected for metaphase analysis were 500, 2000 and 2843 µg/mL.

Main test: -S9, 21 hrs exposure - PGDB caused a reduction in the mitotic index to 56% of the vehicle control value at 225 µg/mL. The concentrations selected for metaphase analysis were 125, 150 and 225 µg/mL.


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.

The positive control compound, Mitomycin C, caused statistically significant increases (p<0.001) in the proportion of aberrant cells.

The positive control compound, Cyclophosphamide, caused statistically significant increases (p< 0.001) in the proportion of aberrant cells.

Conclusions:
Interpretation of results: negative

The test substance PGDB has shown no evidence of causing an increase in the frequency of structural chromosome aberrations under the
experimental conditions described therefore PGDB showed no evidence of clastogenic activity in this in vitro mammalian cell chromosome aberration test.
Executive summary:

Toxicity was observed in the absence of S9 mix at concentrations above 1023.48 µg/mL (3 hr exposure) and above 132.64 µg/mL (21 hr exposure). Precipitate (visible by eye) was observed, when compared to the vehicle control at concentrations of 2843 μg/mL (10 mM). PGDB showed no evidence of causing an increase in the frequency of structural chromosome aberrations under the experimental conditions described.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
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
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:
Migrated to IUCLID6: Used in absence of S9
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: 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).
Key result
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
Key result
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:

Key data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

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: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 June 2014 to 7 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study carried out in compliance with an internationally recognised guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (tk) locus (TK+/-),
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The following media, obtained from a suitable supplier, were used:
R0 RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 µg/mL gentamicin.
R10p R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R30p R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.

R10p medium was used for cell culture unless otherwise specified.
R20p medium was used for the cloning efficiency plating. This was prepared by mixing equal volumes of R10p and R30p.
Selective medium consisted of R10p containing 4 µg/mL trifluorothymidine (TFT).

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, cell stocks are periodically checked for freedom from mycoplasma contamination.
- Periodically checked for karyotype stability: not reported.
- Periodically "cleansed" against high spontaneous background: yes - Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24-hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock.



Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Test concentrations with justification for top dose:
Preliminary toxicity test: 5.55, 11.11, 22.21, 44.42, 88.84, 177.69, 355.38, 710.75, 1421.5 and 2843 µg/mL
Mutation tests:
-S9 mix (3 hours) 25, 150, 175, 200, 225, 250, 275, 300, 1000, 2000, 2500 and 2843 µg/mL
+S9 mix (3 hours) 25, 150, 200, 250, 300, 500, 750, 1000, 1500, 2000, 2500 and 2843 µg/mL
-S9 mix (24 hours) 25, 300, 400, 450, 500, 550, 600, 650 and 700 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: soluble at maximum concentration tested with no fluctuations in osmolality or pH.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
in the absence of S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
in the presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 3 hours (with and without activation); 24 hours (without activation)
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): at least 7 days for viability plates and approximately 10 to 14 days for mutant plates


SELECTION AGENT (mutation assays): triflurothymidine (TFT)

NUMBER OF REPLICATIONS: 4 (vehicle control); 2 (test)

NUMBER OF CELLS EVALUATED: 2 x E5 cells/mL

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no

Evaluation criteria:
On completion of each main mutagenicity test, data were examined for cell growth parameters, cytotoxicity, plating efficiencies, spontaneous and positive control MF, and percent small colonies in positive control cultures.
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
None.

RANGE-FINDING/SCREENING STUDIES: Cells were exposed to the test substance for 3 hours in the absence and presence of S9 mix and for 24 hours in the absence of S9 mix. Precipitate (observed by eye at the end of treatment) was observed at concentrations of 1421.5 µg/mL and greater in both the absence and presence of S9 mix following a 3-hour exposure. Exposure to PGDB at concentrations from 5.55 to 2843 µg/mL in the absence and presence of S9 mix (3-hour exposure) resulted in relative suspension growth (RSG) values from 107 to 20% and from 118 to 1% respectively.

Following a continuous exposure for 24 hours, precipitation (assessed by eye at the end of treatment) was observed at concentrations of 710.75 µg/mL and greater. Exposure to concentrations from 5.55 to 2843 µg/mL resulted in RSG values from 109 to 0%.

Concentrations used in the main test were based upon these data.

ADDITIONAL INFORFORMATION ON CYTOXICITY: As there was some evidence of toxicity in the preliminary toxicity test, the maximum concentration tested in the 3-hour exposure in both the absence and presence of S9 mix was 2843 µg/mL, and in the 24-hour exposure in the absence of S9 mix was 700 µg/mL.
Remarks on result:
other: strain/cell type: heterozygous at the thymidine kinase locus, TK +/-.
Remarks:
Migrated from field 'Test system'.

The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Main mutation test – 3-hour treatment in the absence of S9 mix

Mean mutation frequency - 118 (control ); 77 to 93 (test solution); 1299 (positive control)

Main mutation test – 3-hour treatment in the presence of S9 mix

Mean mutation frequency - 82 (control ); 82 to 130 (test solution); 567 (positive control)

Main mutation test – 24-hour treatment in the absence of S9 mix

Mean mutation frequency - 71 (control ); 56 to 86 (test solution); 1386 (positive control)

Conclusions:
Interpretation of results: negative with and without metabolic activation

It was concluded that the test substance did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.
Executive summary:

Toxicity was observed in the preliminary test. Precipitate (visible by eye) was observed, when compared to the vehicle control in all tests at the higher test concentrations. PGDB showed no evidence of causing an increase in the frequency of mutations under the experimental conditions described.

Endpoint:
in vitro gene mutation study in mammalian cells
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.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
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
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).
Key result
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:

Key data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

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

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Key data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

Bacterial Reverse Mutilation Assay (AMES)

In a key OECD Guideline 471 bacterial reverse mutation assay (Huntingdon Life Sciences, 2014h; Klimisch score = 1), the mutagenic potential of the test material (PGDB) was evaluated using S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli strain WP2 uvr A pKM 101 in the absence and presence of metabolic activation (±S9). No evidence of toxicity was observed following exposure to PGDB and no precipitate was observed on any plates containing PGDB in the preliminary test. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to PGDB at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix. It was concluded that PGDB showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

 

In a key OECD Guideline 471 read across bacterial reverse mutation assay (Huntingdon Life Sciences, 1998k; Klimisch score = 1), 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). 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 DPGDB showed no evidence of mutagenic activity in this bacterial system.

 

Chromosomal Aberration Assay

In a key OECD Guideline 473 in vitro cytogenicity / chromosome aberration study in mammalian cells (Huntingdon Life Sciences, 2014h; Klimisch score = 1), the mutagenic potential of the test material (PGDB) was evaluated in human lymphocytes in the absence and presence of metabolic activation (±S9). Toxicity was observed in the absence of S9 mix at concentrations above 1023.48 µg/mL (3 hr exposure) and above 132.64 µg/mL (21 hr exposure). Precipitate (visible by eye) was observed, when compared to the vehicle control at concentrations of 2843 μg/mL (10 mM). PGDB showed no evidence of causing an increase in the frequency of structural chromosome aberrations under the experimental conditions described and was therefore not considered to exhibit any clastogenic activity in this in vitro mammalian cell chromosome aberration test.

 

In a key OECD Guideline 473 read across in vitro cytogenicity / chromosome aberration study in mammalian cells (Huntingdon Life Sciences, 1998l; Klimisch score = 1) the potential of DPGDB to cause chromosome aberrations was evaluated in Chinese Hamster Lung (CHL) cells cultured in vitro. 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.

 

Gene mutation in mammalian cells

In a key OECD Guideline 476 in vitro gene mutation study in mammalian cells (Huntingdon Life Sciences, 2014i; Klimisch score = 1), the mutagenic potential of the test material (PGDB) was evaluated in mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (±S9). Toxicity was observed in the preliminary test. Precipitate (visible by eye) was observed, when compared to the vehicle control in all tests at the higher test concentrations. PGDB showed no evidence of causing an increase in the frequency of mutations under the experimental conditions described and it was concluded that the test substance did not demonstrate mutagenic potential in this in vitro cell mutation assay.

 

In a key OECD Guideline 476 in vitro gene mutation study in mammalian cells (Huntingdon Life Sciences, 1998m; Klimisch score = 1), the mutagenic potential of the test material (DPGDB) was evaluated in mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (±S9). 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.

Justification for classification or non-classification

Based on available in vitro data, propylene glycol dibenzoate (PGDB) is not considered to be mutagenic or clastogenic and is therefore, not classified for mutagenicity under the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.