Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are multiple in vitro studies that evaluated the genotoxicity potential of propyl gallate (target substance).

A number of bacterial reverse mutation assays (conducted similarly to OECD guideline 471) observed no genotoxic potential of propyl gallate in majority of the Salmonella typhimurium strains (Ishidate et al., 1984; Mortelmans et al., 1986; Tennant et al., 1987). However, one study by Fujita et al. (1988), as cited in EFSA (2014), did report a weak positive result of the Ames test in the oxidative damage-sensitive Salmonella typhimurium TA102 strain.

In vitro studies in mammalian cells have demonstrated conflicting outcomes regarding genotoxicity of propyl gallate; however, this discrepancy might be a result of the cell types used in these studies. It has been shown in rodent cell lines such as CHO and CHL, propyl gallate induced an increase in chromosomal aberrations, micronuclei formation and sister chromatid exchanges in the absence or presence of metabolic activation (Ishidate et al., 1984; Gulati et al., 1989; Tennant et al., 1987). Also, positive mutagenic responses from propyl gallate were observed in mouse lymphoma assay (MLA) (Tennant et al., 1987; McGregor et al., 1988).

Fowler et al. (2012) compared the genotoxic potential of propyl gallate in various cell types including rodent-based cell lines like CHO, V79, CHL and human primary lymphocytes as well as human liver cell line (HepG2). This study reported that rodent cell lines were more susceptible to cytotoxicity and micronuclei formation than human cells due to the p53-deficiency in rodent cell lines, which might lead to increased frequency of misleading positive results.

Indeed, several in vitro studies using human cells such as embryonic lung cells, fibroblasts, primary lymphocytes and liver cell line HepG2, propyl gallate did not induce genotoxic effects as determined from cytogenetic study, micronucleus assay and Comet assay.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
see box "Principles of method if other than guideline"
Principles of method if other than guideline:
Study conducted similar to OECD 471. However, only four Salmonella typhimurium strains were used.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name: Propyl gallate
- CAS No.: 121-79-9
- Supplier: Fluka Chemicals Co.
- Purity (%): >98
- Storage: at -70 °C
- Testing lab: SRI International, Menlo Park, California (K.M.)
Target gene:
Histidine locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Cells were obtained from Dr. Bruce Ames, University of California, Berkeley, USA.

Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat and hamster metabolic activation systems
Test concentrations with justification for top dose:
Test concentrations: 10, 33, 100, 333, 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol (95%)
Untreated negative controls:
yes
Remarks:
Potassium chloride
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535 and TA100; without S9
Untreated negative controls:
yes
Remarks:
Potassium choride
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
TA98; without S9
Untreated negative controls:
yes
Remarks:
Potassium chloride
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA97 and TA1537; without S9
Untreated negative controls:
yes
Remarks:
Potassium chloride
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
all strains; with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

EXPERIMENTAL PERFORMANCE
All chemicals were assayed for mutagenicity in the preincubation assay [Haworth et al., 1983]. To each of 13 x 100-mm test tubes maintained at 37 °C were added in the following order: 0.5 mL of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 mL of the overnight culture, and 0.05 mL of solvent or chemical dilution. The mixture was mixed and allowed to incubate without shaking at 37 °C for 20 min, at whichtime 2.5 mL (EGG) or 2.0 mL (CWR, SFU) of molten (45 °C) top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubes were mixed and poured onto 25 mL of minimal glucose bottom agar [Vogel and Bonner, 1956] in 15 x 100-mm plastic petri dishes and Fisher Scientific plates. When the top agar had solidified, the plates were inverted and incubated at 37 °C for 48 hr.

DURATION
- Preincubation period (Experiment II): 20 min at 37 °C
- Exposure duration: 48 h in the dark at 37 °C

NUMBER OF REPLICATIONS: 3 plates/strain/dose level per experiment. All experiments were repeated.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity can be detected by appearance of his negative pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn.
Evaluation criteria:
The criteria used for data evaluation were the same as those described previously [Haworth et al., 1983] and are summarized as follows: 1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; 2) non-mutagenic response: when no increase in the number of revertants was elicited by the chemical; 3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity. The initial determination of mutagenic, non-mutagenic, or equivocal was made by the testing laboratory; the final determination was made by the project officer (E.Z.). The chemicals were decoded by the chemical repository (Radian Corporation) only after the mutagenicity or non-mutagenicity of the chemicals had been determined.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
For detailed results please refer to table 1 in box "Any other information on results incl. tables"

Table 1: Propyl gallate (Lab: SRI, Solvent: ET95)

 

TA100

TA1535

TA1537

TA98

Dose

Without hamster S9

With 10% hamster S9

With 10% rat S9

Without hamster S9

With 10% hamster S9

With 10% rat S9

Without hamster S9

With 10% hamster S9

With 10% rat S9

Without hamster S9

With 10% hamster S9

With 10% rat S9

µg/plate

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

0

159

7.5

134

1.9

127

6.7

41

2.1

30

2.8

41

1.8

10

1.9

8

2.1

10

2.8

28

1.5

40

4.3

38

3.8

100

115

6.4

141

6.3

123

7.3

26

4.1

30

1.2

30

1.7

12

0.0

6

0.9

12

1.8

20

2.7

35

1.0

24

2.1

333

92

4.4

120

18.9

113

2.3

23

2.7

24

6.9

27

0.9

12

1.2

9

2.5

10

2.5

18

4.2

35

3.6

30

3.5

1000

82

6.8

106

15.1

105

9.4

13

5.9

13

0.0

13

3.2

7

0.9

5

0.3

6

1.5

14

1.0

38

1.0

30

2.6

3333

63

8.4

81

4.8

82

4.8

4

0.3

4

2.0

4

0.6

11

0.3

7

1.0

8

0.3

11

2.5

28

4.4

15

7.9

10000

0s

 0.0

0s

0.0

0s

0.3

t

t

0s

0.0

8

 1.0

0s

0.0

0s

0.0

t

0s

0.0

1

1.0

POS

416

16.2

1785

44.9

2143

22.3

536

4.4

486

4.3

273

6.9

667

30.4

537

19.5

178

5.8

852

8.2

1738

38.5

560

28.4

POS = positive control

t = complete clearing of background lawn

s = slight clearing of background lawn

Conclusions:
Under the experimental conditions of the bacterial reverse mutation assay conducted similar to OECD test guideline 471, propyl gallate was tested negative and therefore can be considered as non-mutagenic.
Executive summary:

In a bacterial reverse mutation assay conducted similar to OECD test guideline 471, strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium were exposed to propyl gallate (>98 % purity) in 95% ethanol at concentrations of 10, 33, 100, 333 and 1000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains. Therefore, propyl gallate is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Principles of method if other than guideline:
See " Details on test system and conditions"
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Propyl gallate
- Source: Japan Food Additives Association, Tokyo
Target gene:
histidine locus
Species / strain / cell type:
other: S.typhimurium TA92, TA1535, TA100, TA1537, TA94 and TA98
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix)
Test concentrations with justification for top dose:
Six different concentrations (not specified in publication) were tested. 500 µg / plate was selected as the top dose as it was the highest non-cytotoxic dose used in the experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity and the test item is insoluble in water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
not specified
Details on test system and experimental conditions:
Reverse mutation assay was carried out according to the method of Ames, McCann & Yamasaki (1975).

METHOD OF APPLICATION: pre-incubation
- Pre-incubation: 20 min at 37 °C
- Incubation: 2 days at 37 °C

NUMBER OF REPLICATIONS: 2
Evaluation criteria:
The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays.
Statistics:
N.A.
Species / strain:
S. typhimurium, other: TA92
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium, other: TA94
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Propyl gallate is non-mutagenic in the bacterial reverse mutation test.
Executive summary:

In a bacterial reverse mutation assay (similar to OECD guideline 471), TA92, TA1535, TA100, TA1537, TA94 and TA98 of S. typhimurium were exposed to six concentrations (up to 0.5 mg per plate) of propyl gallate in the presence or absence of mammalian metabolic activation of the pre-incubation test. Propyl gallate did not induce any mutant colonies with and without metabolic activation. Based on the available data, propyl gallate is considered to be non-mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Study performed before adoption of currently valid test guideline (1997).
Deviations:
yes
Remarks:
only without metabolic activation
Principles of method if other than guideline:
See " Details on test system and conditions"
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Source: Mr H. Yoshino, Japan Food Additives Association, Tokyo
Target gene:
n.a.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Remarks:
V79 not specified
Details on mammalian cell type (if applicable):
CELLS USED
- Number of passages if applicable: maintained by 4-day passages
- Modal number of chromosomes: 25
- Doubling time: 15 h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
The cells were exposed "to each sample" at three different doses. 0.04 mg/mL was selected as the top dose. The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer.
- Maximum dose: 0.04 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiological saline
- Justification for choice of solvent/vehicle: The solvent was used because the sample was insoluble in water.
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
Remarks:
physiological saline
True negative controls:
no
Positive controls:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: not specified
- Cell density at seeding (if applicable): not specified

DURATION
- Exposure duration: The cells were exposed to each sample at three different doses for 24 and 48 hours. In the present study, no metabolic activation systems were applied.

NUMBER OF REPLICATIONS: not specified

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Chromosome preparations were made as follows. Colcemid (final concn 0.2 ug/mL) was added to the culture 2 hours before cell harvesting. The cells were then trypsinised and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a no-cover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): A hundred well-spread metaphases were observed under the microscope (x 600 with a no-cover objective lens).
Evaluation criteria:
The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
V79 not specified
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Additional information on results:
Propyl gallate induced chromosomal abberations at doses lower than 10 mM. The incidence of cells with structural chromosomal aberrations after 24-hour exposure was 69%
Remarks on result:
other: D20, 6.72; TR, 0.25. Positive only at this dose, 0.04 mg/mL.
Conclusions:
In conclusion, propyl gallate is considered to be mutagenic according to the results of a chromosome aberration test in this study.
Executive summary:

In a mammalian cell cytogenetic assay (chromosome aberration test), primary lung fibroblast cultures were exposed to propyl gallate in physiological saline at three different doses for 24 and 48 hours in the absence of mammalian metabolic activation. Propyl gallate was tested up to 0.04 mg/mL and induced chromosomal aberrations. The incidence of cells with structural chromosomal aberrations after 24-hour exposure was 69%. Based on the results, propyl gallate is considered to be mutagenic.

This study is classified as partly acceptable. This study partly satisfies the requirements for test guideline OECD 473 for in vitro cytogenetic mutagenicity data.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
no guideline followed
Principles of method if other than guideline:
In a study from Litton Bionetics (1974), as cited by CIR (2007) and EFSA (2014), three different assays, a host-mediated assay, a cytogenetic assay and a dominant lethal assay, were used to evaluate the mutagenicity of Propyl gallate. In particular, in the in vitro cytogenetic study, human embryonic lung cultures (WI-38) were used. Doses tested were 0.5, 5 and 50 μg/mL. The cells were examined at 24, 48 and 72 hours. One hundred metaphases or anaphases per concentrations were examined.
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
no data
Target gene:
NA
Species / strain / cell type:
other: Human embryonic lung cultures (WI-38)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Test concentrations with justification for top dose:
0.5, 5 and 50 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: no data
Untreated negative controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
- Cells were examined at 24, 48 and 72 hours.
- One hundred metaphases or anaphases per concentration were examined.
Rationale for test conditions:
no data
Evaluation criteria:
Chromosomal damage was scored.
Statistics:
no data
Species / strain:
other: Human embryounic lung cultures (WI-38)
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
The low and high doses caused 2% and 3% chromosomal aberrations, respectively, compared to 1% in the negative control; these variations were not considered as significant. Anaphase preparations were examined in this test. There was no demonstration of a clastogenic effect with 0, 3 and 2% cells with aberrations at 0.5, 5 and 50 μg/mL respectively, vs. 2% in the negative control.

According to the EFSA (2014) report, this study applied a limited experimental protocol, which was based on the examination of only 100 metaphases or 100 anaphases per concentration.

Conclusions:
Based on an in vitro cytogenetic study from Litton Bionetics (1974), as cited by EFSA (2014) and CIR (2007), Propyl gallate is considered to be non-mutagenic.
Executive summary:

In an in vitro cytogenetic study from Litton Bionetics (1974), as cited by EFSA (2014) and CIR (2007), Propyl gallate was added to human embryonic lung cultures in anaphase at doses of 0.5, 5 and 50 µg/mL. The cells were examined at 24, 48 and 72 hours. One hundred metaphases or anaphases per concentrations were examined. Chromosomal damage was scored. The low and high doses caused 2% and 3% chromosomal aberrations, respectively, compared to 1% in the negative control; these variations were not considered as significant. Anaphase preparations were examined in this test. There was no demonstration of a clastogenic effect with 0, 3 and 2% cells with aberrations at 0.5, 5 and 50 μg/mL, respectively vs. 2% in the negative control. Based on this study, Propyl gallate is considered to be non-mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Type of assay:
in vitro mammalian cell micronucleus test
Conclusions:
Propyl gallate was shown to have potential to induce micronuclei formation in rodent cell lines (V79, CHO and CHL) and in one human cell line (TK6) but not in primary human lymphocytes and human HepG2 cell line. The authors of the study concluded that rodent cell lines were consistently more susceptible to cytotoxicity and micronuclei induction than the p53-competent human cells.
Executive summary:

In a study from Fowler et al. (2012), as cited by EFSA (2014), Propyl gallate was tested in an in vitro micronucleus assay without metabolic activation in a 3-hour treatment plus a 21-hour recovery period protocol, in which the response of rodent (V79, CHO and CHL) and human cells (peripheral lymphocytes, TK6 and HepG2) were compared. In this study Propyl gallate induced high levels of toxicity in all cell types and a significant increase of micronuclei in V79, CHO, CHL and TK6 cells. Conversely, primary human lymphocytes and HepG2 cell were negative for micronucleus induction despite similar levels of cytotoxicity observed when compared to the other cells.

Fowler et al. concluded in this study that the rodent cell lines (V79, CHO and CHL) were consistently more susceptible to cytotoxicity and micronuclei induction than p53-competent cells (e.g. human peripheral blood lymphocytes, TK6 and HepG2), and are therefore more susceptible to giving misleading positive results.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test:
Testing of mutagenicity using Ames' tester strains S. typhimurium TA97 and TA102
- Short description of test conditions:
The mutation test was carried out using the preincubation procedure described by Ames et al. The test chemicals were preincubated with S9 mix or phosphate buffer (pH 7.4) for 20 min
GLP compliance:
not specified
Conclusions:
In the EFSA scientific opinion on the re-evaluation of propyl gallate, 2014 the results of the publication of Fujita, 1988 were cited. It was reported, that weak mutagenic acitivity was reported for propyl gallate in Salmonella typhimurium strains TA102 in the presence and absence of metabolic activation in the preincubation method at the top dose of 100 µg/plate. It was negative in strain TA97 with and without metabolic acitivation.
Executive summary:

In the EFSA scientific opinion on the re-evaluation of propyl gallate, 2014 the results of the publication of Fujita, 1988 were cited. It was reported, that weak mutagenic acitivity was reported for propyl gallate in Salmonella typhimurium strains TA102 in the presence and absence of metabolic activation in the preincubation method at the top dose of 100 µg/plate. It was negative in strain TA97 with and without metabolic activation. As no further information on the methodology were presented this information will only be assessed as supporting information in a weight-of-evidence approach.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
- Principle of test: A chromosome aberration assay with chinese hamster ovary cells were conducted in vitro. 27 chemicals were tested including propyl gallate, each with and without metabolic activation.
- Short description of test conditions: Cells were treated for about 10 hours with the test material.
- Parameters analysed / observed: The chromosome number was recorded for each cell and chromosome or chromatid type aberrations were classified into categories.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Purity: not specified

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: National Toxicology Program Chemical Repository (Radian Corporation, Austin, TX), supplier: Tennessee Eastman
- Expiration date of the lot/batch: Not reported
- Purity test date: Not reported

OTHER SPECIFICS:
- All test substances were handled under yellow light; the identity of each chemical was not released until all testing was completed and the data evaluated.
Target gene:
N.A.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Litton Bionetics (Kensington, MD)
- Number of passages if applicable: up to 15 passages
Cells were stored in liquid nitrogen and was tested yearly for mycoplasma. Results from all test for mycoplasma contamination were positive

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy’s 5A medium (modified) supplemented with L-glutamine (2 mM), antibiotics, and 10% fetal bovine serum (FBS). All stock and experimental cultures were maintained at 37 °C in an atmosphere of 5% CO2 in air and 95% relative humidity.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no
Metabolic activation:
with and without
Metabolic activation system:
Liver fraction (S9) from Aroclor 1254-induced male Sprague Dawley rats
Test concentrations with justification for top dose:
Without S9 (trial 1/3): 0, 16.0, 30.0, 50.0 µg/mL
Without S9 (trial 2/3): 0, 5.0, 16.0, 30.0 µg/mL
Without S9 (trial 3/3): 0, 1.0, 2.5, 5.0, 16.0 µg/mL
With S9 (trial 1/1): 0, 300.0, 400.0, 500.0 µg/mL

The doses used for the chromosome aberration study were chosen based on the toxicity of the test chemical observed in the sister-chromatid exchange study (see IUCLID section 7.6.1 Gulati (1989), SCE).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DSMO was the alternative choice to serum-free culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1.2 x 10E6 cells seeded per 75 cm² flask 24 h prior to treatment

DURATION
- Preincubation period: 24 hours
- Exposure duration: 10 hours
- Harvest time: 12-13.5 h
- Fixation time (start of exposure up to fixation or harvest of cells): without metabolic activation: 24 hours; with metabolic activation: 26 hours

SELECTION AGENT (mutation assays): Colcemid added 2-4 hours prior to cell harvest by mitotic shake-off

NUMBER OF REPLICATIONS: Positive results in initial tests were confirmed by additional tests. If both -S9 and +S9 studies gave a positive response and required confirmation, they were done sequentially (-S9 first). If the -S9 repeat was positive, the repeat +S9 study was not always performed.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 6% Giemsa for 5-10 minutes.

NUMBER OF CELLS EVALUATED: 100 cells scored for each dose in early studies and 200 cells per dose in later studies. All slides except high-dose positive controls were coded. Only metaphase cells in which the chromosome number was between 19 and 23 were scored.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Only metaphase cells in which the chromosome number was between 19 and 23 were scored.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
The chromosome number was recorded for each cell and chromosome or chromatid type aberrations were classified into three categories: simple (breaks, fragments, double minutes), complex (interchanges, rearrangements), and other (pulverized, more than ten aberrations/cell).
Statistics:
The percentage of cells with aberrations was analyzed. Both the dose-response curve and individual dose points were statistically analyzed. A statistically significant (P < 0.003) trend test or a significantly elevated dose point (P < 0.05) was sufficient to indicate a chemical effect.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
At doses of 5 -50 µg/mL propyl gallate produced a strong, dose-related increase in chromosomal aberrations, but only in the absence of S9 and with extended harvest times (harvest at 23 hours).
For individual results see Table 1 in box "Any other information on results incl. tables".

Table 1: Chromosome aberration propyl gallate

Trial Dose (µg/mL) Cells Percent cells with aberrations
Total Simple Complex
Without S9 - Trial 1 0.0 200 1.0 1.0 0.0
16.0 200 20.0 13.0 12.0
30.0 200 50.0* 46.0 11.0
50.0 64 89.0* 84.0 33.0
Without S9 - Trial 2 0.0 200 1.0 1.0 0.0
5.0 200 8.0* 5.0 3.0
16.0 200 43.0* 37.0 8.0
30.0 45 49.0* 40.0 18.0
Without S9 - Trial 3 0.0 200 3.0 2.0 1.0
1.0 200 2.0 2.0 1.0
2.5 200 0.0 0.0 0.0
5.0 200 1.0 0.0 1.0
16.0 200 55.0 49.0 12.0
With S9 - Trial 1 0.0 200 1.0 1.0 0.0
300.0 200 1.0 1.0 0.0
400.0 200 4.0 2.0 2.0
500.0 200 3.0 1.0 2.0

* statistical significant

Conclusions:
In this study, propyl gallate induced chromosome aberrations in absence of metabolic activation.
Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration) conducted similar to OECD guideline 473, CHO cells cultured in vitro were exposed to propyl gallate at concentrations of 0, 1.0, 2.0, 5.0, 16.0, 30.0 and 50.0 in dimethyl sulfoxide in absence and at concentrations of 0, 300.0, 400.0 and 500.0 µg/mL in dimethyl sulfoxide in presence of mammalian metabolic activation. Positive controls induced the appropriate response. At doses of 5 -50 µg/mL propyl gallate produced a strong, dose-related increase in chromosomal aberrations, but only in the absence of S9 and with extended harvest times (harvest at 23 hours). Based on the results, propyl gallate is considered to be mutagenic in the absence of mammalian metabolic activation.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: A sister chromatid exchange (SCE) test with Chinese hamster ovary cells were conducted in vitro. 27 chemicals were tested, each with and without metabolic activation.
- Short description of test conditions: The test was conducted both with and without metabolic activation. Cells were treated with test or control substances for 2 hours to allow interaction with cells before the addition of bromodeoxyuridine. After that, incubation was continued for 24 hours.
- Parameters analysed / observed: Fifty second-division metaphase cells were scored per dose for the incidence of SCE. The number of chromosomes in each cell was also recorded.
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Specific details on test material used for the study:
- Purity: not specified

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
National Toxicology Program Chemical Repository (Radian Corporation, Austin, TX), supplier: Tennessee Eastman
- Expiration date of the lot/batch:
Not reported
- Purity test date:
Not reported

OTHER SPECIFICS:
- All test substances were handled under yellow light; the identity of each chemical was not released until all testing was completed and the data evaluated.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Litton Bionetics (Kensington, MD)
- Number of passages if applicable: up to 15 passages
Cells were stored in liquid nitrogen and was tested yearly for mycoplasma. Results from all test for mycoplasma contamination were positive

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy’s 5A medium (modified) supplemented with L-glutamine (2 mM), antibiotics, and 10% fetal bovine serum (FBS). All stock and experimental cultures were maintained at 37 °C in an atmosphere of 5% CO2 in air and 95% relative humidity.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no
Metabolic activation:
with and without
Metabolic activation system:
Liver fraction (S9) from Aroclor 1254-induced male Sprague Dawley rats
Test concentrations with justification for top dose:
Without S9 (trial 1/3): 0, 0.5, 1.6, 5.0, 16.0 µg/mL
Without S9 (trial 2/3): 0, 3.0, 5.0, 7.5, 10.0 µg/mL
Without S9 (trial 3/3): 0, 3.0, 5.0, 7.5, 10.0 µg/mL

With S9 (trial 1/1): 0, 5.0, 16.0, 50.0, 160.0 µg/mL

10 or 11 dose levels at half-log intervals beginning at a high dose of 5 mg/mL (or as limited by solubility) for all 27 chemicals were used in SCE study.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DSMO was the alternative choice to serum-free culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1 x 10^6 cells seeded per 75 cm² flask 24 h prior to treatment

DURATION
- Preincubation period: 24 hours
- Exposure duration: 2 hours
- Expression time (cells in growth medium): without metabolic activation: 24 hours; with metabolic activation: 0 hours (no FBS at all)
- Fixation time (start of exposure up to fixation or harvest of cells): without metabolic activation: 24 hours; with metabolic activation: 26 hours

NUMBER OF REPLICATIONS: Positive results in initial tests were confirmed by additional tests. If both -S9 and +S9 studies gave a positive response and required confirmation, they were done sequentially (-S9 first). If the -S9 repeat was positive, the repeat +S9 study was not always performed.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Harvested cells were treated for about 3 minutes at room temperature with hypotonic KCl (75 mM), washed with fixative (3:1 methanol : glacial acetic acid, v/v), dropped onto slides, and air dried. Staining for the detection of SCE was accomplished by a modified fluorescence plus Giemsa (FPG) technique.

NUMBER OF CELLS EVALUATED: not specified

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 50 second-division metaphase cells were scored per dose for the incidence of SCE.

- OTHER: The number of chromosomes in each cell was recorded. Any cell that had fewer than 19 or more than 23 chromosomes was excluded. All slides except for the high-dose positive controls were coded.
Evaluation criteria:
An SCE frequency 20% above the concurrent solvent control value was chosen as a statistically conservation positive response. The probability of this level of difference occurring by change at one dose point is <0.01; the probability for such a chance occurrence at two dose points is <0.001. A trial with one dose showing an increase of 20% or greater was considered weak evidence of a positive response, and a trial with two doses showing an increase of 20% or greater was considered to be positive. Any cell that had fewer than 19 or more than 23 chromosomes was excluded.
Statistics:
Statisical analyses were conducted on both the slopes of the dose-response curves and the individual dose points.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Propyl gallate was a potent inducer of SCE without S9. In the presence of S9, the increase in SCE frequency was not as great, although the response was significant.
For individual results see Table 1 in box "Any other information on results incl. tables"

Table 1: SCE propyl gallate

Trial Dose (µg/mL) Total Chromosomes Total SCE SCE per Cell Harvest Time
Without S9 - Trial 1 0.0 1050 427 8.54 -
0.5 1049 469 9.38 -
1.6 1047 428 8.56 -
5.0 1047 507 10.14 -
16.0 337 311 19.44* -
Without S9 - Trial 2
0.0 1051 419 8.38 -
3.0 1050 486 9.72 -
5.0 1046 484 9.68 -
7.5 1047 587 11.74* -
10.0 755 614 17.06* -
Without S9 - Trial 3
0.0 1050 463 9.26 26.00
3.0 1048 529 10.58 26.00
5.0 1047 633 12.66* 26.00
7.5 1049 757 15.14* 31.00
10.0 1049 869 17.38* 31.00
With S9 - Trial 1
0.0 1051 488 9.76 -
5.0 1049 459 9.18 -
16.0 1046 509 10.18 -
50.0 1043 584 11.68* -
160.0 1047 591 11.82* -

* statistical significant

Conclusions:
In this SCE assay, propyl gallate was tested postive in varying concentrations up to 160 µg/mL in presesnce and absence of metabolic activation.
Executive summary:

In a mammalian cell cytogenetics assay (sister chromatid exchange), CHO cells cultured in vitro were exposed to propyl gallate at concentrations of 0, 0.5, 1.6, 3, 5, 7.5, 10, 16, 50, 160 µg/mL (solvent: DMSO) in the presence and absence of mammalian metabolic activation (S9-mix). Positive controls induced the appropriate response. In this study, propyl gallate was a potent inducer of SCE without S9. In the presence of S9, the increase in SCE frequency was not as great, although the response was significant. Thus, there was evidence of sister chromatid exchange induced over background.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
- Principle of test: Determination of DNA strand breaks by alkaline elution. The alkaline elution technique can determine single strand breaks and alkali-labile sites in cellular DNA, which may be the result of a chemical attack, or of an enzymatic reaction during the repair process.
- Short description of test conditions: Human fibroblasts (cell line GM 05757) were incubated with propyl gallate for 1 hour
- Parameters analysed / observed: Mass of DNA was determined by fluorometric assay
GLP compliance:
not specified
Type of assay:
comet assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich, Steinheim

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Propyl gallate was freshly dissolved in serum-free medium (without antibiotics) at pH 7.2.
Target gene:
Not applicable
Species / strain / cell type:
mammalian cell line, other: Human fibroblasts, cell line GM 05757
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human Genetic Mutant Cell Repository (Camden, NJ)
- Number of passages if applicable: passage 7 – 15 of monolayer cultures were used

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: minimal essential medium (MEM) supplemented with 12% fetal calf serum, vitamins, essential and non-essential amino acids, with 100 U/mL of both penicillin and 100 mg/mL streptomycin. The cells were grown at 37 °C in an atmosphere of 5% CO2/95% air, with more than 95% humidity.
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
Doses: 0.0 (control), 0.15, 0.25, 0.5 mM
Cells were also coexposed with Copper(II) chloride, although those findings are not included in this endpoint study record.
This study also included an investigation of genotoxicity (DNA strand breaks) in PM2 DNA, although those findings are not included in this endpoint study record.
Justification of top dose: not specified
Vehicle / solvent:
N.A.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
serum-free medium
True negative controls:
no
Positive controls:
no
Remarks:
A second control, incubated with 2.5 mM copper(II) chloride, was used during the investigation of coexposure effects.
Details on test system and experimental conditions:
DETERMINATION OF GENOTOXICITY:
Method: Alkaline elution technique
Subconfluently growing cells (about 0.5 x 10^6 cells per dish, 57 cm²) were incubated with the test solutions. After 1 h incubation the test solutions were removed and the cells were washed twice with ice-cold Saline A (8 g/L NaCl; 0,4 g/L KCl; 1 g/L glucose-1-hydrate; 0.35 g/L NaHCO3) and trypsinised. Two identical samples were combined in a total volume of 6 mL ice-cold saline A. The following procedure was performed in the dark. For determination of the total DNA content an aliquot of 0.5 mL was taken. The remaining cell suspension was loaded onto the surface of a polycarbonate filter (25 mm-diameter, 2 µm-pore size, Nucleopore) held in a Millipore Swinnex filter holder. The outflows from the filters were connected with vinyl tubing (0.5 mm diameter) over a peristaltic pump to a fraction collector. After the solvent had disappeared from the filter 3.5 mL lysis solution (10 mM EDTA, 0.5% Triton X-100, 2 M NaCl, pH 10) was added to the cells and pumped with a velocity of 3 mL/h through the membrane. After lysis the remaining DNA on the filter was washed with 4.5 mL buffer containing 20 mM EDTA at pH 10. Afterwards, the pump speed was reduced to 1.5 mL/h and DNA elution was started with 27 mL elution buffer (20 mM EDTA, adjusted with 20% tetraethylammoniumhydroxide to pH 12.6). The eluate of the first 20 min was discarded. The elution was continued for a further 10 h, and 10 fractions were collected. Thereafter the samples were prepared for fluorometric assay of their DNA content as follows.

Fluorometric assay for determining the mass of DNA
1 mL of each fraction was diluted with 2 mL of elution solution and neutralized by addition of 170 mL 1M KH2PO4. The samples were treated with 100 mL Hoechst dye 33258 (40.4 mL/mL in KH2PO4-buffer from stock solution of 0.224 mg/mL in distilled water). After 30 min in the dark fluorescence was measured at an excitation of 360 nm and an emission of 450 nm. DNA remaining on the filter was calculated from the difference of the total DNA content and the DNA content in the collected fractions.

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- Any supplementary information relevant to cytotoxicity: Eight thousand cells (200 mL of a cell suspension of 40,000 cells/mL medium) were seeded in each well of a 96-well tissue culture microtiter plate. Two days after cells had achieved confluency the medium was removed and the cells were treated with the test solutions. At least four wells were used for every concentration tested. After 1 h of incubation the test solutions were removed and the cells were washed twice. MTT containing medium, which was freshly prepared by a 1:5 dilution of an MTT stock solution (5 mg/mL PBS buffer) and 100 mL were added to each well. The plate was incubated for an additional 3 h. One hundred microliters of lysis solution (20 g/L SDS, 50% v/v DMF, pH adjusted to 4.7 by adding 2.5% (v/v) of a 80 % acetic acid and 2.5 % 1 N HCl) was added to each well and the plate was shaken overnight in the dark to extract and solubilize the formazan. Formation of formazan was measured with a Biorad microplate reader using a 570 nm test wavelength and a 655 nm reference wavelength. Cell viability was calculated as the percent ratio of the absorbance of the samples to the referring control.

DETERMINATION OF INHIBITION OF CELL GROWTH

1 h incubation with the chemicals was followed by repeated washing of the cells. One part of the cells was counted immediately, whereas the other part was allowed to grow for another 48 h before counting. Cell growth was calculated from the ratio of cell number at the end of growth and cell number counted directly after treatment with the chemicals (growth 5 ln[cell number(end)/cell number(beginning)] : ln2).
Statistics:
Not specified
Species / strain:
mammalian cell line, other: human fibroblasts
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
Concentrations of 0.15 -0.5 mM propyl gallate alone did not induce strand breaks or alkali labile sites within the cells, which was measured by the alkaline elution technique. No significant effect in vell viability or cell inhibition growth was observed.
Conclusions:
In this study, propyl gallate is considered to be non-genotoxic in an alkaline elution technique.
Executive summary:

In an in vitro analysis using an alkaline elution technique, human fibroblast cells cultured in vitro were exposed to propyl gallate dissolved in serum-free medium at concentrations of 0, 0.15, 0.25, and 0.5 mM. for 1 hour.

For assessment of genotoxicity, DNA damage was measured, as induction of DNA strand breaks and alkali-labile sites. No significant effects on DNA strand breaks or induction of alkali-labile sites at the concentrations tested in human fibroblast cells were observed after incubation with propyl gallate.

Therefore, in this study, propyl gallate is not genotoxic in the alkaline elution technique.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
- Principle of test: Testing for the mutagenic potential of propyl gallate in the L5178Y tk+/- mouse lymphoma cell forward mutation assay, using procedures based upon those described by Clive and Spector (Mutat Res 44:269-278, 1975) and Clive et al. (Mutat Res 59:61-108, 1979) but certain small differences were incorporated
- Short description of test conditions: Cultures were exposed to propyl gallate for 4 hours, then cultured for 2 days before plating in soft agar with or without trifluorothymidine
- Parameters analysed / observed: Colony counting was carried out by an automated colony counter in order to determine the toxicity and mutagenic potential.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied from the National Toxicology Program Chemical Repository, Radian Corporation, Austin, TX 78766
Target gene:
thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
tk+/tk- locus
Cytokinesis block (if used):
CELLS USED
- Source of cells: Dr. D. Clive, Burroughs Wellcome Co., Research Triangle Park, NC 27709
- Suitability of cells: containing tk+/tk- locus

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Fischer's medium at 37 °C on gyratory tables. Fischer's medium (designated F0) was supplemented with 2 mM L-glutamine, sodium pyruvate, 110 µg/mL, 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v) (designated F10P). On a single occasion, within 1 week of the start of an experiment, cultures were purged of tk-/tk- mutants by exposure for 1 day to Flop containing THMG (thymidine, 6 µg/mL hypoxanthine, 5 µg/mL, glycine, 7.5 µg/mL and methotrexate, 0.1 µg/mL), then for 3 days to Flop containing THG only, (i.e., THMG without methotrexate).
Metabolic activation:
without
Metabolic activation system:
without S9
Test concentrations with justification for top dose:
Concentration ranges vary for each of the 6 experiments, ranging from 0.5 µg/mL to 1000 µg/mL:

1. trial: 0, 62.5, 125.0, 250.0. 500.0 and 1000.0 µg/mL
2. trial: 0, 50.0, 100.0, 200.0 and 300.0 µg/mL
3. trial: 0, 25.0, 50.0, 75.0, 100.0 and 125.0 µg/mL
4. trial: 0, 12.5, 25.0, 50.0, 75.0 and 100.0 µg/mL
5. trial: 0, 12.5, 25.0, 50.0, 75.0 and 100.0 µg/mL
6. trial: 0, 0.5, 1.0, 2.0, 4.0 and 5.0 µg/mL
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not specified
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: Each exposed culture consisted of 6 X 10^6 cells in a final volume of 10 mL F5p in a 30-mL screw-cap plastic tube (Sterilin Ltd.). This tube was incubated for 4 hours on a horizontal axis roller drum rotating at 10 rpm. At the end of the incubation time, the cells were sedimented by centrifugation at 500 g.av. for 10 min, washed, and finally resuspended in 20 mL F10p. These cell suspensions (3 x 10^5 cells/mL) were incubated for a 2-day expression period, the cell population density being adjusted back to 20 mL of 3 X 10^5 cells/mL after 24 hr. After 48 hours, the cell population densities were estimated and culture volumes containing 3 X 10^6 cells adjusted to 15 mL with F10p, giving a cell population density of 2 X 10^5 cells/mL.

SELECTION AGENT (mutation assays): Mutant Selection - Three aliquots (each containing 10^6 cells) of the remaining culture were distributed to 30-ml tubes, mixed with 20-ml cloning medium to give final concentrations of 0.35% Noble agar and 3 µg trifluorothymidine/mL, then poured into 90-mm Petri plates.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was expressed as either a reduction of cell population growth in suspension during the expression period or a reduction in cloning efficiency. A measure of the overall toxicity was the relative total growth (RTG), which is defined as:
RTG = ((total suspension growth X cloning efficiency) in dosed culture) / ((total suspension growth x cloning efficiency) in control culture)
Mutant fraction (MF) was calculated as follows: MF = 200 x (mutant clones per plate (usually a mean of 3)) / (total clones per plate (usually a mean of 3)) = mutants/10^6 cloneable cells.

OTHER: Methods of incubation - The agar was gelled at 4 °C for 5-10 min, then the plates were incubated for 11-14 days in 5% C02:95% air at 37°C. Methods of colony counting - Colonies were counted using an Artek 880 Automated Colony Counter, with the colony size discriminator control in the “off” position.
Evaluation criteria:
Positive response (+): The dose-related trend and the response at one of the three highest acceptable doses were statistically significant.
Negative response (-): Two categories were used. In both there was a) no dose-related trend, b) no statistically significant response at any dose, c) an acceptable positive control response.
Nontoxic, negative response (=): There was an RTG among the acceptable doses of >30% (approximately), higher toxicities being unattainable due to intrinsic properties of either the compound or the system.
Toxic, negative response (-): There was either an RTG of 30% (approximately) at the maximum acceptable dose, or the lethal concentration was no greater than 1.5 x a lower concentration at which the RTG was >30%.
Inconclusive (i): There was a) no dose-related trend and a statistically significant dose was any other than one of the highest three doses b) a response which would have been negative, but the lowest RTG acceptable doses was >35%, c) a response which would have been negative, but there were no acceptable positive controls.
Questionable (?): There was either a) no dose-related trend, but a statistically significant response occurred at one of the highest three doses, or b) a statistically significant dose-related trend, but none of the acceptable doses was statistically three doses, or significant on its own.
Statistics:
The statistical analysis was based upon the mathematical model proposed for this system (Lee and Caspary, 1983) and consisted of a dose-trend test (Barlow et al., 1972) and a variance analysis of pair-wise comparisons of each dose against the vehicle control. In Tables IV-LXXV, significant differences from concurrent vehicle control values at the 5% level are indicated by underlines of the average mutant fractions at the appropriate concentrations. Where a statistically significant response occurred, the lowest observed effective dose (LOED) was noted.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Significant mutagenic responses were observed in the absence of S9 mix in six experiments at all tested concentrations. The lowest concentration was 50 µg/mL in five experiments and 5 µg/mL in the sixth. The five higher dose range experiments were judged to be questionable because the dose-related response was inverted. In the remaining experiment, covering a lower dose range, the inversion effect was avoided. Because of the reproducibility of the effect, propyl gallate was evaluated as positive and the unusual dose-response relationship was considered to be real, although the mechanism causing this type of response is unknown.

For individual results see table 1 in box "Any other information on results incl. tables".

Table 1: Test results of the MLA with propyl gallate

Without S9 Trial 1
Concentration µg/mL CE RTG MC MF AVG MF
DMSO 109 131 170 52 66
DMSO
0
91 91 204 75
73 79 166 76
80 99 149 62
62.5 71 19 968 452 469
72 15 1051 485
125.0 74 11 615 276 278
73 12 617 280
250.0 69 7 230 112 113
73 7 248 114
500.0 63 4 256 135 132
50 3 193 128
1000.0 lethal
lethal
EMS 250 µg/mL 70 79 453 217 235
56 79 420 252
MMS 15 µg/mL 41 28 514 415 397
37 25 419 379
Without S9 Trial 2
Concentration µg/mL CE RTG MC MF AVG MF
DMSO
0
108 106 131 40 38
85 101 85 34
77 98 70 30
82 95 114 46
50.0 80 17 456 190 210
75 16 518 231
100.0 67 13 297 147 136
70 11 262 125
200.0 56 7 171 102 100
70 7 203 97
300.0 lethal
lethal
EMS 250 µg/mL 71 60 346 162 158
77 64 354 154
MMS 15 µg/mL 46 27 233 171 179
45 27 250 187
Without S9 Trial 3
Concentration µg/mL CE RTG MC MF AVG MF
DMSO
0
80 93 106 44 51
86 107 102 40
64 94 121 63
72 113 122 56
25.0 65 22 810 419 425
42 19 698 431
50.0 42 18 685 541 508
51 20 727 475
75.0 63 17 813 428 438
48 12 640 448
100.0 60 14 711 393 455
45 12 691 517
125.0 60 18 631 354 390
46 18 592 427
MMS 15 µg/mL 14 12 260 634 640
19 17 371 645
Without S9 Trial 4
Concentration µg/mL CE RTG MC MF AVG MF
DMSO
0
82 103 153 62 65
66 88 121 61
78 103 168 72
contaminated
12.5 70 46 446 211 184
65 42 306 157
25.0 45 18 568 425 379
82 17 815 333
50.0 62 18 446 240 278
52 15 491 317
75.0 57 9 527 309 334
63 7 673 358
100.0 38 8 360 314 313
31 8 287 312
MMS 15 µg/mL 62 12 347 445 426
21 11 256 406
Without S9 Trial 5
Concentration µg/mL CE RTG MC MF AVG MF
DMSO
0
79 84 117 50 48
99 107 114 38
73 97 127 58
70 103 98 46
12.5 73 21 542 247 249
83 24 625 251
25.0 93 16 284 102 108
91 17 312 115
50.0 52 9 226 146 138
54 10 210 130
75.0 57 10 527 309 334
63 8 673 358
100.0 38 8 360 314 313
31 8 287 312
MMS 15 µg/mL 23 27 288 411 383
27 29 285 354
Without S9 Trial 6
Concentration µg/mL CE RTG MC MF AVG MF
DMSO
0
64 96 127 66 60
75 98 103 46
75 100 166 73
65 110 108 55
0.5 72 61 215 100 112
77 62 287 124
1.0 64 23 378 198 182
76 24 381 166
2.0 72 19 360 166 171
58 22 307 175
4.0 57 17 283 167 166
55 17 271 165
5.0 90 35 342 127 156
59 26 325 184
MMS 15 µg/mL 21 18 279 439 432
25 17 321 425

CE: cloning efficiency

RTG: relative total growth

MC: mutant colony count

MF: mutant fraction

AVE MF: group average mutant fraction

Conclusions:
In this study under the given conditions, propyl gallate was evaluated as positive in the mouse lymphoma assay.
Executive summary:

In a mammalian cell cytogenetics assay (mouse lymphoma assay) using procedures based upon those described by Clive and Spector (Mutat Res 44:269-278, 1975) and Clive et al. (Mutat Res 59:61-108, 1979), L5178Y mouse lymphoma cells were exposed to propyl gallate dissolved in DMSO at concentrations of 0 to 1000.0 µg/mL without metabolic activation in six experiments. The lowest concentration was 50 µg/mL in five experiments and 5 µg/mL in the sixth. Significant mutagenic responses were observed in all tested concentrations. The five higher dose range experiments were judged to be questionable because the dose-related response was inverted. In the remaining experiment, covering a lower dose range, the inversion effect was avoided. Because of the reproducibility of the effect, propyl gallate was evaluated as positive and the unusual dose-response relationship was considered to be real, although the mechanism causing this type of response is unknown. Positive controls induced the appropriate response.

Based on these results, propyl gallate is considered to be mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
- Principle of test: A chromosome aberration assay with Chinese hamster ovary cells were conducted in vitro using propyl gallate, with and without metabolic activation.
- Short description of test conditions: Propyl gallate was tested in concentrations of 0.25 to 1.5 mM in plugged flasks for 3 hours
- Parameters analysed / observed: Chromosome aberrations were counted.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Purity: 98%

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: propyl gallate sourced from Tokyo Kasei Kogyo Inc. (Tokyo)
- Treatment of test material prior to testing: Prior to use, propyl gallate was dissolved in dimethyl sulfoxide
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
K1
Details on mammalian cell type (if applicable):
CELLS USED
- Methods for maintenance in cell culture if applicable: grown at 37 °C in plugged tissue-culture flasks

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F-12 medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, USA), penicillin (100 units/ml), and streptomycin (100 µg/mL)

Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Colcemid
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Test concentrations with justification for top dose:
0, 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s): DMSO; solvent concentration in medium was always 1 vol.%
Untreated negative controls:
yes
Remarks:
medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 6 x 10^5 cells

DURATION
- Preincubation period: 48 hours
- Exposure duration: the cells were exposed to test chemical with or without the metabolic activation mixture for 3 hours, washed, and incubated in dark for 27 hours.
- Expression time (cells in growth medium): after 25 hours' incubation, cells were treated with 0.1 µg/mL Colcemid for 2 hours.
- Selection time (if incubation with a selection agent): n.a.
- Fixation time (start of exposure up to fixation or harvest of cells): 30 hours

SELECTION AGENT (mutation assays): n.a.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: hypotonic treatment, fixation, and preparation followed by staining with fluorescent plus Giemsa (FPG). Slides stained with Hoechst 33258 were exposed to UV light in 0.065 M Sorensen's buffer (pH 6.8) and after that stained with 2% Giemsa solution.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): scored 100 metaphases per chemical dose

DETERMINATION OF CYTOTOXICITY
- Method: cell-cycle delay was used as an indicator of cytotoxic effects by calculating the percentage of metaphases with differently staining sister chromatids
Evaluation criteria:
Not specified
Statistics:
Chi-squared test to assess statistical significance, compared to controls.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
MMC (0.04 µg/mL) increased SCEs but not CAs; CP increased CAs
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Lowering the pH (6.8) of the medium or a 5% CO2 atmosphere inhibited propyl gallate autoxidation (even at lowest dose of propyl gallate (0.1 mM), induced CAs and ERDs, and intensified cytotoxicity so much that no harlequin chromosomes were harvested. The pH of the medium increased from 7.5 to 8.0 during treatment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Other observations when applicable: evaluated cell-cycle delay as an indicator of cytotoxic effects by calculating the percentage of metaphases with differently staining sister chromatids (DSC)

In the absence of exogenous metabolic activation, propyl gallate induced chromosomal aberrations at ≥0.5 mM with dose dependency up to 0.75 mM. (This includes gaps, breaks, exchanges, and dicentrics.) The positive control MMC (0.04 µg/mL) increased SCEs but not chromosomal aberrations or endoreduplications.

The positive control cyclophosphamide (0.009 mM) induced not chromosomal aberrations with 4.5% S9. With 6% S9, cyclophosphamide increased the percentage of cells containing chromosomal aberrations from 4 to 20% including 13% with exchanges and 6% with gaps.

Without S9, 0.5 mM propyl gallate significantly increased chromosomal aberrations (15 and 7%, including and excluding gaps, p≤0.01 and p≤0.05, respectively. At lower S9 concentrations, propyl gallate significantly increased chromosomal aberrations. With 3% S9, exchange-type aberrations occurred at the rate of 12%, about equal to the rate caused by 0.009 mM cyclophosphoramide (positive control) with 6% S9. Induction of chromosomal aberrations by propyl gallate decreased at the middle and higher S9 concentrations, and gap-free chromosomal aberrations were no longer induced.

Conclusions:
Propyl gallate is considered to be genotoxic according to the results of a chromosome aberration test.
Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration assay) conducted similar to OECD guideline 473, CHO-K1 cells were exposed to propyl gallate (purity ≥98%) dissolved in DMSO at concentrations of 0, 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 mM with and without metabolic activation. Propyl gallate was tested up to 1.5 mM and induced chromosomal aberrations. The positive controls did induce the appropriate response. Based on these results, propyl gallate is considered to be genotoxic.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Principles of method if other than guideline:
- Principle of test: A sister chromatid exchange assay with Chinese hamster ovary cells were conducted in vitro using propyl gallate, with and without metabolic activation. Different doses of metabolic activation were tested.
- Short description of test conditions: Propyl gallate was tested in concentrations of 0.25 to 1.5 mM in plugged flasks for 3 hours.
- Parameters analysed / observed: Sister chromatid exchanges were counted.
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Specific details on test material used for the study:
- Purity: 98%

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: propyl gallate sourced from Tokyo Kasei Kogyo Inc. (Tokyo)
- Treatment of test material prior to testing: Prior to use, propyl gallate was dissolved in dimethyl sulfoxide
Target gene:
N/A
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
K1
Details on mammalian cell type (if applicable):
CELLS USED
- Methods for maintenance in cell culture if applicable:
grown at 37 °C in plugged tissue-culture flasks

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Ham's F-12 medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, USA), penicillin (100 units/ml), and streptomycin (100 µg/mL)

Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Colcemid
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Test concentrations with justification for top dose:
0, 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO and distilled water; solvent concentration in medium was always 1 vol.%.
Untreated negative controls:
yes
Remarks:
medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 6 x 10^5 cells

DURATION
- Preincubation period: 48 hours
- Exposure duration: the cells were exposed to test chemical with or without the metabolic activation mixture for 3 hours, washed, and incubated in dark for 27 hours.
- Expression time (cells in growth medium): after 25 hours' incubation, cells were treated with 0.1 ug/mL Colcemid for 2 hours.
- Selection time (if incubation with a selection agent): n.a.
- Fixation time (start of exposure up to fixation or harvest of cells): 30 hours

SELECTION AGENT (mutation assays): n.a.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- hypotonic treatment, fixation, and preparation followed by staining with fluorescent plus Giemsa (FPG). Slides stained with Hoeschst 33258 were exposed to UV light in 0.065 M Sorensen's buffer (pH 6.8) and after that stained with 2% Giemsa solution.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
- Scored 50 metaphases per a chemical dose

DETERMINATION OF CYTOTOXICITY
- Method: cell-cycle delay was used as an indicator of cytotoxic effects by calculating the percentage of metaphases with differently staining sister chromatids
Evaluation criteria:
not specified
Statistics:
Kruskal-Wallis test and Sheffe test to assess statistical significance, compared to controls.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Lowering the pH (6.8) of the medium or a 5% CO2 atmosphere inhibited propyl gallate autoxidation (even at lowest dose of Propyl gallate (0.1 mM), induced CAs and ERDs, and intensified cytotoxicity so much that no harlequin chromosomes were harvested. The pH of the medium increased from 7.5 to 8.0 during treatment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Other observations when applicable: evaluated cell-cycle delay as an indicator of cytotoxic effects by calculating the percentage of metaphases with differently staining sister chromatids (DSC)

At 0.25–1.25 mM, PG caused statistically significant and dose-dependent increase in the SCEs, and at 1.5 mM, no cells with harlequin chromosomes were observed. The positive control MMC (0.04 µg/mL) increased SCEs.

Conclusions:
Propyl gallate is considered to be genotoxic according to the results of a sister chromatid exchange assay.
Executive summary:

In a mammalian cell cytogenetics assay (sister chromatide exchange) conducted similar to OECD guideline 479, CHO-K1 cells were exposed to propyl gallate (purity ≥98%) dissolved in DMSO at concentrations of 0, 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 mM with and without metabolic activation. At 0.25–1.25 mM, PG caused statistically significant and dose-dependent increase in the SCEs, and at 1.5 mM, no cells with harlequin chromosomes were observed. The positive control did induce the appropriate response. Based on these results, propyl gallate is considered to be genotoxic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1987
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Very little information on methods
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Ames Salmonella/microsome mutagenesis assay uses bacteria to test whether a chemical can cause mutations in the DNA of the test organism.
- Short description of test conditions: not described
- Parameters analysed / observed: not described
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other:
Remarks:
not specified
Metabolic activation:
not specified
Test concentrations with justification for top dose:
1 000 µg/plate
Vehicle / solvent:
No data
Details on test system and experimental conditions:
Study refers to outside reference Haworth S. et al. 1983 for methods.
Rationale for test conditions:
Not reported
Evaluation criteria:
Refers to Haworth et al. 1983 and Mortelmans et al. 1984
Statistics:
Not reported
Species / strain:
S. typhimurium, other:
Remarks:
Strain not specified
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
The highest dose with a negative result is reported.
Remarks on result:
not determinable
Remarks:
Negative
Conclusions:
Based on the negative findings of the Ames Salmonella/microsome mutagenesis assay, propyl gallate is not mutagenic.
Executive summary:

In a reverse gene mutation assay in bacteria conducted according to Haworth et al. 1983 and Mortelmans et al. 1984, strains of S. typhimurium were exposed to propyl gallate at a concentration of 1000 µg/plate (highest tested concentration). There was no evidence of induced mutant colonies over background. Due to the limited information on materials and methods, this study cannot be used for classification.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1987
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
- Principle of test: Chromosome aberration induction assay in Chinese hamster ovary cells to predict rodent carcinogenicity in vitro.
- Short description of test conditions: Not specified
- Parameters analysed / observed: Not specified
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Test concentrations with justification for top dose:
5.0 µg/mL
Vehicle / solvent:
Not specified
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
Study references Galloway et al. 1985 for all details on test system and conditions.
Evaluation criteria:
Study references Galloway et al. 1985 and Galloway (no date given).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
not specified
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
The lowest positive dose tested is reported.
Conclusions:
Using a chromosome aberration assay in Chinese hamster ovary cells, 5.0 µg/mL of propyl gallate was tested and was the lowest positive dose for which chromosomal aberrations were observed.
Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration) Chinese hamster ovary cells were exposed to propyl gallate. Based on the results of this test, 5.0 µg/mL propyl gallate was the lowest positive dose and was positive. Propyl gallate is considered to be mutagenic in this test system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1987
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Mouse lymphoma L6178Y cell mutagenesis assay predicts rodent carcinogenicity in vitro
- Short description of test conditions: Not described
- Parameters analysed / observed: Not described
GLP compliance:
not specified
Type of assay:
other:
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
not specified
Test concentrations with justification for top dose:
50.0 µg/mL
Vehicle / solvent:
No specified
Details on test system and experimental conditions:
Study refers to B. Myhr, L. Bowers, W. Caspary, in Evaluation of Short-Term Tests for Carcinogens: Reports of the International Programme on Chemical Safety's Collaborative Study on in Vitro Assays, vol. 5 of Progressin Mutation Research Series, J. Ashby et al., Eds. (Elsevier, Amsterdam, 1985 pp 555-568)
Evaluation criteria:
Study refers to Myhr et al. 1985 and McGregor et al. (no date given)
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Only the lowest positive dose result is given.
Remarks on result:
not determinable
Remarks:
Negative
Conclusions:
The lowest positive dose of 50.0 µg/mL propyl gallate illicited a positive result with no exogenous metabolic activation in the mouse lymphoma L5178Y cell mutagenesis assay.
Executive summary:

In a publication from Tennant et al. (1987), results of a mouse lymphoma L5178Y cell mutagenesis assay with propyl gallate. The publication reports that the lowest positive dose of 50.0 µg/mL propyl gallate illicited a positive result with no exogenous metabolic activation in the mouse lymphoma L5178Y cell mutagenesis assay.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1987
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
- Principle of test: The sister chromatid exchange assay using Chinese hamster ovary cells detects genotoxicity of a chemical and predicts rodent carcinogenicity.
- Short description of test conditions: Not specified
- Parameters analysed / observed: Not specified
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Test concentrations with justification for top dose:
5.0 µg/mL
Vehicle / solvent:
Not specified
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
Study references Galloway et al. 1985 for all details on test system and conditions.
Evaluation criteria:
Study references Galloway et al. 1985 and Galloway (no date given).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
not specified
Genotoxicity:
positive
Remarks:
with and without exogenous metabolic activation
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Based on the results of a sister chromatid exchange assay using Chinese hamster ovary cells, 5.0 µg/mL was the lowest positive dose and was positive.
Executive summary:

In a mammalian cell cytogenetics assay (sister chromatide exchange) Chinese hamster ovary cells were exposed to propyl gallate. Based on the results of this test, 5.0 µg/mL propyl gallate was the lowest positive dose and was positive. Propyl gallate is considered to be genotoxic.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Several in vivo studies assessed the genotoxic potential of the target substance propyl gallate and have indicated that there are no genotoxic effects induced by propyl gallate observed in animals.

Two mouse bone marrow micronucleus studies, which were conducted similarly to OECD Guideline 474, reported no genotoxicity (i.e. no increased formation of micronuclei) from propyl gallate tested up to 300 mg/kg (Shelby et al., 1993; Raj & Katz, 1984). A ddY mouse comet assay (conducted similarly to OECD Guideline 489) also showed no increased DNA damage in any of the examined organs at 3 and 24 hours after exposure to 2000 mg/kg propyl gallate (Sasaki et al., 2002). Similar to mice, two genotoxicity studies in male rats performed by Litton Bionetics (1974), as cited by CIR (2007) and EFSA (2014), reported that propyl gallate tested up to 5000 mg/kg resulted in no dominant lethal effects or significant increase of bone marrow chromosomal aberrations.

Given the existing data and taking a weight-of-evidence approach, it is concluded that propyl gallate does not induce genotoxic effects in vivo.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Principles of method if other than guideline:
- Principle of test: Comet assay in mice similar to OECD guideline 489
- Short description of test conditions & parameters analysed / observed: Mice were exposed to the limit dose of 2000 mg/kg and eight organs (glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow) were removed and were afterwards processed to be analysed in a Comet assay.
GLP compliance:
not specified
Type of assay:
mammalian comet assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Kanto Chemical Co. Inc., Tokyo, Japan
Species:
mouse
Strain:
other: ddY
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Japan SLC Co., Shizuoka, Japan
- Age at study initiation: 7 weeks of age and used after 1 week of acclimatization.
- Assigned to test groups randomly: [no/yes, under following basis: ] not specified
- Fasting period before study: no
- Diet (e.g. ad libitum): commercial pellets MF (Oriental Yeast Industries Co., Tokyo, Japan), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
Duration of treatment / exposure:
Once orally
Frequency of treatment:
once
Post exposure period:
not specified
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
4 males
Control animals:
yes, concurrent vehicle
Positive control(s):
not specified
Tissues and cell types examined:
Eight organs (glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The oral dose of 2000 mg/kg was determined via preliminary simple acute toxicity experiment on 4-5 animals. No death was observed at 2000 mg/kg, so the LD50 was defined at >2000 mg/kg.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Animals were sacrificed 3 or 24 hours after treatment.

DETAILS OF SLIDE PREPARATION: The liver, kidney, lung, and brain were minced, suspended in 4 ml chilled homogenizing solution (pH 7.5) containing 0.075 M NaCl and 0.024 M Na2EDTA, and then homogenized gently using a Potter–Elvehjem type homogenizer at 500–800 rpm, in ice. The glandular stomach, colon, and urinary bladder were opened and rinsed with physiological saline; the mucosa was scraped into 4 mL chilled homogenizing buffer and homogenized gently using a Potter–Elvehjem type homogenizer at 500–800 rpm, in ice. To obtain nuclei, the homogenate was centrifuged at 700×g for 10 min at 0 °C, and the precipitate was re-suspended in chilled homogenizing buffer at 1 g organ weight/mL. of the covering slide. Finally, 75 L of agarose GP-42 was quickly layered on again. Slides prepared from nuclei isolated by homogenization were placed in a chilled lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Trizma, 1% sarkosyl, 10% DMSO, and 1% Triton X-100, pH 10) and kept at 0 °C in the dark for about 1 night, then in chilled alkaline solution (300 mM NaOH and 1 mM Na2EDTA, pH 13) for 10 min in the dark at 0 °C. Electrophoresis was conducted at 0 °C in the dark for 15 min at 25 V (0.96 V/cm) and approximately 250 mA. The slides were neutralized and then stained with 50 L of 20 g/mL ethidium bromide.

METHOD OF ANALYSIS: 50 nuclei per slide at 200× magnification with the aid of a fluorescence microscope were examined and photographed. The length of the whole comet and the diameter of the head were measured for 50 nuclei per organ per animal.
Evaluation criteria:
not specified
Statistics:
Mean migration of 50 nuclei from each organ was calculated for each individual animal. The differences between the averages of four treated animals and the untreated control animals were compared with the Dunnett test after one-way ANOVA. A P value less than 0.05 was considered statistically significant.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not specified
Negative controls validity:
not examined
Positive controls validity:
not examined
Remarks on result:
other: no in vivo positive control included in the experiment
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Propyl gallate did not increase DNA damage in any of the organs studied. Additionally, no death, morbidity, or clinical signs were observed.
Conclusions:
This experimental study in a small group of male mice shows that a single oral 2000 mg/kg exposure to propyl gallate did not increase DNA damage in any of the organs studied at 3 hours and 24 hours after the exposure.
Executive summary:

In a ddY mouse comet assay conducted similar to OECD guideline 489, 4 male mice were once orally treated with propyl gallate at a dose of 2000 mg/kg bw. All mice were euthanized at 3 hours and 24 hours after exposure, at which time organs (glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow) were removed and processed to be analyzed in a Comet assay. The length of the whole comet and the diameter of the head were measured for 50 nuclei per organ per animal. Migration was calculated as the difference between length and diameter for each 50 nuclei. Mean migration of 50 nuclei from each organ was calculated for each individual animal. The differences between the averages of four treated animals and the untreated control animals were compared. The results of this study indicate that propyl gallate did not increase DNA damage in any of the organs studies at 3 hours and 24 hours after exposure.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
- Principle of test: In this publication the induction of chromosomal breakage in bone marrow cells of mice by constituents of corn oil, among them propyl gallate, was elucidated.
- Short description of test conditions: Propyl gallate was dissolved in DMSO and the mice were injected i.p. for 2 days at 24-h intervals prior to treatment with DMBA. Bone-marrow samples were collected at 24-h intervals after the last injection. Samples were collected at 24, 48, 72 and in some cases, at 0 and 96 h.
- Parameters analysed / observed: From each mouse 500 polychromatic erythrocytes (PCEs) were scored and the results expressed as the average number of micronucleated PCEs (MNPCEs) per 500 PCEs.
GLP compliance:
not specified
Type of assay:
mammalian germ cell chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source: Aldrich Chemical Co.
Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Canada Inc.
- Age at study initiation: 8-9 weeks
- Weight at study initiation: ranging from 20 to 23 g in weight
- Assigned to test groups randomly: not specified
- Diet: Purina Laboratory Rodent Chow ad libitum
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Duration of treatment / exposure:
48 hours
Frequency of treatment:
Two times at 24 h intervals
Post exposure period:
Bone-marrow samples were collected at 24-h intervals after the last injection. Samples were collected at 24, 48, 72 and in some cases, at 0 and 96 h.
Dose / conc.:
217 mg/kg bw/day
No. of animals per sex per dose:
5 female animals per treatment and sampling period
Control animals:
yes
Positive control(s):
not specified
Tissues and cell types examined:
bone-marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: According to 1970 international standards for edible maize oil recommended by a joint commission formed by The Food and Agricultural Organization (FAO) and World Health Organization (WHO), gallates up to a maximum level of 100 mg/kg of oil could be added.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 24 h after the final injection bone-marrow samples were collected at 24-h intervals (24, 48, 72, and in some cases 0 and 96 hrs).

METHOD OF ANALYSIS: Bone-marrow samples were collected according to the method of Heddle and Salamone (1981). From each mouse 500 polychromatic erythrocytes (PCEs) were scored and the results expressed as the average number of micronucleated PCEs (MNPCEs) per 500 PCEs (Salamone et al., 1980).
Evaluation criteria:
not specified
Statistics:
not specified
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Remarks:
Control results not specified
Negative controls validity:
not specified
Remarks:
Control results not specified
Positive controls validity:
not examined
Remarks on result:
not determinable
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Bone-marrow cells from the mice which received only propyl gallate showed 1.2, 1.6 and 1.0 MNPCEs/500 PCEs at 24, 48 and 72 h respectively. Although, this is not considered to be genotoxic. Propyl gallate was investigated as a potential inhibitor of genotoxicity induced by exposure to 7,12-dimethylbenz[a]anthracene (DMBA). Propyl gallate alone was not found to be genotoxic, and did not produce significantly inhibitory effects
- Ratio of PCE/NCE (for Micronucleus assay): not reported
- Appropriateness of dose levels and route: no data
- Statistical evaluation: Statistical analyses were not reported.

The MN data on propyl gallate were not presented in figures or tables within the paper.

Conclusions:
In conclusion, under the reported experimental conditions propyl gallate did not induce genotoxic effects (assessed via levels of micronuclei) in the bone marrow cells of the female mouse. Statistical significance was not determined.
Executive summary:

In a B6C3F1 mouse bone marrow micronucleus test conducted similar to OECD Guideline 474, 5 female mice per sample point were treated intraperitoneally with propyl gallate at doses of 217 mg/kg dissolved in DMSO. The animals were injected intraperitoneally with the test substance two times at 24 h intervals (24, 48, 72, and in some cases 0 and 96 hrs). The vehicle was DMSO.

Propyl gallate was investigated as a potential inhibitor of genotoxicity induced by exposure to 7,12-dimethylbenz[a]anthracene (DMBA).

Bone-marrow cells from the mice which received only propyl gallate showed 1.2, 1.6 and 1.0 MNPCEs/500 PCEs at 24, 48 and 72 h respectively. Propyl gallate alone was not found to be genotoxic, and did not produce significantly inhibitory effect.

In summary, propyl gallate is not considered to be genotoxic according to the results of the in vivo micronucleus test reported.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Principles of method if other than guideline:
In a study from Litton Bionetics (1974), as cited by CIR (2007) and EFSA (2014), three different assays, including an in vivo chromosomal aberration assay in rats, were used to evaluate the mutagenicity of propyl gallate. In particular, for the chromosomal aberration assay, male rats were exposed to various doses of propyl gallate either once or for 5 consecutive days. Rats were then sacrificed at different time intervals (e.g. 6, 24 or 48 h) after exposure. Bone marrow was removed and the chromosome preparations were scored for abnormalities.
GLP compliance:
no
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
rat
Strain:
other: albino
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10-12 weeks
Route of administration:
other: oral (gastric intubation)
Vehicle:
Not specified
Details on exposure:
Not specified
Duration of treatment / exposure:
once or for 5 consecutive days
Frequency of treatment:
once or daily for 5 days
Post exposure period:
not specified
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
5 000 mg/kg bw/day
No. of animals per sex per dose:
3 animals for control; 5 animals each for exposed groups
Control animals:
yes
Positive control(s):
Not specified
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES: For the single dose experiment, animals at each dose were killed at 6, 24, or 48 h after dosing. For the multiple dose experiment (i.e. for 5 days), animals were killed 6 hours following the last dosing.

DETAILS OF SLIDE PREPARATION: Each animal received intraperitoneally 4 mg/kg colchicine in order to arrest bone marrow cells in C-mitosis. After the animals were killed, the bone marrow was removed for further processing.

METHOD OF ANALYSIS: The chromosome preparations were scored for abnormalities.
Evaluation criteria:
Not specified
Statistics:
Not specified
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
not specified

In an in vivo chromosome aberration test from Litton Bionetics (1974) as cited by CIR (2007) and EFSA (2014), 10-12-week-old male albino rats (3 animals/control, 5 animals/ treated groups) were dosed by gastric intubation with propyl gallate at single doses of 5, 50 and 500 mg/kg; animals were sacrificed at 6, 24 or 48 hours after one treatment. In the first trial, propyl gallate given as single doses produced a weak but significantly higher percentage of aberrations in bone marrow cells at the intermediate dose at 48 hours and at the high dose at 24 and 48 hours. In a subacute study using the same dose levels given for five days as above (with 24 hours intervals), there was a slight increase in the chromosomal aberrations at all dose levels compared to the negative control (4, 4 and 6 % cells with aberrations at 5, 50 and 500 mg/kg, respectively, vs. 2 % in the negative control). In a second assay propyl gallate was administered to rats (3 animals/control, 5 animals/ treated groups) at 5000 mg/kg both as single dose and in five repeated administrations with 24-hour intervals. In this second assay neither the variety nor the number of aberrations in treated rats differed significantly from the negative controls. The authors concluded that propyl gallate was non-genotoxic in this test. However, according to EFSA, the protocol of this study is limited as there were only 3 negative control animals and 50 metaphases per animal examined.

Conclusions:
In an in vivo chromosomal aberration assay in rats, propyl gallate was shown not to be genotoxic.
Executive summary:

In an in vivo chromosome aberration test from Litton Bionetics (1974) as cited by CIR (2007) and EFSA (2014), 10-12-week-old male albino rats (3 animals/control, 5 animals/ treated groups) were dosed by gastric intubation with propyl gallate at single doses of 5, 50 and 500 mg/kg; animals were sacrificed at 6, 24 or 48 hours after one treatment. In the first trial, propyl gallate given as single doses produced a weak but significantly higher percentage of aberrations in bone marrow cells at the intermediate dose at 48 hours and at the high dose at 24 and 48 hours. In a subacute study using the same dose levels given for five days as above (with 24 hours intervals), there was a slight increase in the chromosomal aberrations at all dose levels compared to the negative control (4, 4 and 6 % cells with aberrations at 5, 50 and 500 mg/kg, respectively, vs. 2 % in the negative control). In a second assay propyl gallate was administered to rats (3 animals/control, 5 animals/ treated groups) at 5000 mg/kg both as single dose and in five repeated administrations with 24-hour intervals. In this second assay neither the variety nor the number of aberrations in treated rats differed significantly from the negative controls. The authors concluded that propyl gallate was non-genotoxic in this test. However, according to EFSA, the protocol of this study is limited as there were only 3 negative control animals and 50 metaphases per animal examined.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
no guideline followed
Principles of method if other than guideline:
In a study from Litton Bionetics (1974), as cited by CIR (2007) and EFSA (2014), three different assays, including an in vivo dominant lethal assay in rats, were used to evaluate the mutagenicity of Propyl gallate. In particular, male rats were exposed to various doses of propyl gallate in a dominant lethal test. The males mated for 7-8 weeks with virgin females, which were then sacrificed 2 weeks after mating, and the fertility index, resorption sites, total implantations and lethal effects were determined.
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Route of administration:
other: oral (gastric intubation)
Vehicle:
not specified
Details on exposure:
not specified
Duration of treatment / exposure:
once or for 5 consecutive days
Frequency of treatment:
once or daily for 5 days
Post exposure period:
not specified
Dose / conc.:
0 mg/kg bw/day
Remarks:
Tested only in the multiple doses (i.e. 5 consecutive days) phase
Dose / conc.:
5 mg/kg bw/day
Remarks:
Tested in both single dose and multiple doses (i.e. 5 consecutive days) phases
Dose / conc.:
50 mg/kg bw/day
Remarks:
Tested in both single dose and multiple doses (i.e. 5 consecutive days) phases
Dose / conc.:
500 mg/kg bw/day
Remarks:
Tested in both single dose and multiple doses (i.e. 5 consecutive days) phases
Dose / conc.:
5 000 mg/kg bw/day
Remarks:
Tested only in the multiple doses (i.e. 5 consecutive days) phase
No. of animals per sex per dose:
10
Control animals:
yes
Positive control(s):
not specified
Tissues and cell types examined:
The uteri were examined for resorption sites, late fetal deaths, and total implantations.
Details of tissue and slide preparation:
not specified
Evaluation criteria:
not specified
Statistics:
not specified
Sex:
not specified
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
No dose-response or time-trend patterns that would suggest a dominant lethal effect from propyl gallate were observed. Propyl gallate was therefore non-mutagenic under these study conditions.
Conclusions:
Based on an in vivo dominant lethal test in rats from Litton Bionetics (1974), as cited by EFSA (2014) and CIR (2007), propyl gallate is considered to be non-mutagenic.
Executive summary:

In an in vivo dominant lethal test from Litton Bionetics (1974), as cited by EFSA (2014) and CIR (2007), propyl gallate was orally administered via gastric intubation to 10 male Sprague-Dawley rats, either once or for 5 consecutive days, at doses of 0, 5, 50, 500 or 5000 mg/kg bw (0 and 5000 mg/kg bw doses were only tested in the 5-day part of the study). Positive and negative controls were used. Afterwards, the males mated with 2 virgin females per week for 7-8 weeks. Pregnant dams were killed 14 days after separation from treated males; the uteri were examined for resorption sites, late fetal deaths, and total implantations. No dose-response or time-trend patterns that would suggest a dominant lethal effect for propyl gallate were observed. Propyl gallate was therefore considered to be non-mutagenic under these study conditions.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
- Principle of test: In this publication the induction of chromosomal aberration and micronuclei in bone marrow cells of mice by 49 chemicals, among them propyl gallate.
- Short description of test conditions: In this mouse bone marrow micronucleus test, male B6C3F1 mice were treated intraperitoneal with propyl gallate for three daily treatments. Bone marrow samples were obtained 24 hours following final exposure.
- Parameters analysed / observed: Number of micronuclei were counted in the bone marrow tissue
GLP compliance:
not specified
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: National Toxicology Program production facility at Taconic Fams
- Age at study initiation: 9 - 14 weeks
- Weight at study initiation: within a 2 g range of a mean weight between 25 and 33 g
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
Groups of 5 mice were administered the test chemical dissolved in corn oil by IP injection on 3 consecutive days. The test doses were based on toxicity/mortality observed in a dose determination study.
Duration of treatment / exposure:
3 x 24 h plus 24 h post treatment incubation
Frequency of treatment:
3 times at 24 hour intervals
Post exposure period:
24 hours
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
Dose / conc.:
75 mg/kg bw/day
Dose / conc.:
0 mg/kg bw/day
No. of animals per sex per dose:
5-7 male mice per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive controls:
- DMBA in corn oil
- MMC in PBS

- Justification for choice of positive control(s): not specified
- Route of administration: IP injection
- Doses / concentrations: "weakly active" dose (actual dose not specified) of DMBA dissolved in corn oil
Tissues and cell types examined:
bone marrow (2 slides per mouse)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The test doses for propyl gallate were based on toxicity/mortality observed in the dose determination studies.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Groups of mice were injected IP on three consecutive days. Mice were euthanized with CO2 24 hours after the third treatment.

DETAILS OF SLIDE PREPARATION: Bone marrow smears (two slides per mouse) were prepared, fixed in absolute methanol, and stained with acridine orange.

METHOD OF ANALYSIS: For each animal, slides were evaluated at 1000x magnification for the number of MN-PCE among 2000 PECD and for the percentage of PCE among 200 erythrocytes.

OTHER:
-In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow was scored for each dose group as a measure of toxicity.
Statistics:
Data were analysed using the Micronucleus Assay Data Management and Statistical software package (version 1.4) that employed a one-tailed Cochran-Armitage trend test across exposure groups and pairwise comparison between exposure group and concurrent control. The level of significance was set at an alpha level of 0.05.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: A preliminary dose-determination experiment was carried out. Details are not reported, but the maximum dose used was 300 mg/kg
- Solubility: not specified; test substance was dissolved in corn oil
- Clinical signs of toxicity in test animals: not specified
- Evidence of cytotoxicity in tissue analyzed: not specified
- Rationale for exposure: not specified
- Harvest times: 24 h after 3rd dose


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): test substance did not induce structural aberrations
- Induction of micronuclei (for Micronucleus assay): not reported
- Ratio of PCE/NCE (for Micronucleus assay): not reported
- Appropriateness of dose levels and route: not reported
- Statistical evaluation: Data were analysed using the Micronucleus Assay Data Management and Statistical software package (version 1.4) that employed a one-tailed Cochran-Armitage trend test across exposure groups and pairwise comparison between exposure group and concurrent control. The level of significance was set at an alpha level of 0.05.

The MN experiment with propyl gallate (0, 75, 150, and 300 mg/kg) showed negative results at all doses.

Table 1. Micronucleus Test with propyl gallate (solvent corn oil; bone marrow cells)

 Dose (mg/kg) MN-PCE/1000 (No. of animals) (a)  Pair-wise (b)  Survival (c)  %PCE (d)
 0 2.30±0.49 (5)   - 5/5  63.4
 75  2.30±0.52 (5)  0.5000  5/5  64.5
 150  1.25±0.43 (4)  0.9491  4/5  51.4
 300  3.17±0.44 (3)  0.1498  3/7  61.7

(a) Micronucleated PCEs per 1,000 PCE scored (2 Standard Error of the Means) (number of animals scored).

(b) The value of P for pair-wise comparisons between each treatment group and the concurrent solvent control group

(c) No. of animals surviving treatment over number of animals treated

(d) Percentage of erythrocytes that were polychromatic (i.e., [No. of PCE/No. of PCE + No. of NCE] x 100).

Conclusions:
In conclusion, under the reported experimental conditions test substance propyl gallate did not induce significant elevated levels of micronuclei in the bone marrow cells of the mouse.
Executive summary:

In a B6C3F1 mouse bone marrow micronucleus test conducted similar to OECD Guideline 474, 5-7 male mice per dose were treated intraperitoneally with propyl gallate at doses of 0, 75, 150, and 300 mg/kg. The animals were injected intraperitoneally with the test substance three times at 24 h intervals, and results were prepared 24 hours after 3rd injection. The vehicle was corn oil. Results were negative up to the highest dose of 300 mg/kg of propyl gallate. In summary, propyl gallate is considered to be non-mutagenic according to the results of the in vivo micronucleus test reported.

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

Additional information

There are multiple in vitro and in vivo studies that evaluated the genotoxicity potential of propyl gallate (target substance).

A number of bacterial reverse mutation assays (conducted similarly to OECD guideline 471) observed no genotoxic potential of propyl gallate in majority of the Salmonella typhimurium strains (Ishidate et al., 1984; Mortelmans et al., 1986; Tennant et al., 1987). However, one study by Fujita et al. (1988), as cited in EFSA (2014), did report a weak positive result of the Ames test in the oxidative damage-sensitive Salmonella typhimurium TA102 strain. In vitro studies in mammalian cells have demonstrated conflicting outcomes regarding genotoxicity of propyl gallate; however, this discrepancy might be a result of the cell types used in these studies. It has been shown in rodent cell lines such as CHO and CHL, propyl gallate induced an increase in chromosomal aberrations, micronuclei formation and sister chromatid exchanges in the absence or presence of metabolic activation (Ishidate et al., 1984; Gulati et al., 1989; Tennant et al., 1987). Also, positive mutagenic responses from propyl gallate were observed in mouse lymphoma assay (MLA) (Tennant et al., 1987; McGregor et al., 1988).

Fowler et al. (2012) compared the genotoxic potential of propyl gallate in various cell types including rodent-based cell lines like CHO, V79, CHL and human primary lymphocytes as well as human liver cell line (HepG2). This study reported that rodent cell lines were more susceptible to cytotoxicity and micronuclei formation than human cells due to the p53-deficiency in rodent cell lines, which might lead to increased frequency of misleading positive results. Indeed, several in vitro studies using human cells such as embryonic lung cells, fibroblasts, primary lymphocytes and liver cell line HepG2, propyl gallate did not induce genotoxic effects as determined from cytogenetic study, micronucleus assay and Comet assay.

Several in vivo studies have indicated that there are no genotoxic effects induced by propyl gallate observed in animals. Two mouse bone marrow micronucleus studies, which were conducted similarly to OECD Guideline 474, reported no genotoxicity (i.e. no increased formation of micronuclei) from propyl gallate tested up to 300 mg/kg (Shelby et al., 1993; Raj & Katz, 1984). A ddY mouse comet assay (conducted similarly to OECD Guideline 489) also showed no increased DNA damage in any of the examined organs at 3 and 24 hours after exposure to 2000 mg/kg propyl gallate (Sasaki et al., 2002). Similar to mice, two genotoxicity studies in male rats performed by Litton Bionetics (1974), as cited by CIR (2007) and EFSA (2014), reported that propyl gallate tested up to 5000 mg/kg resulted in no dominant lethal effects or significant increase of bone marrow chromosomal aberrations.

By assessing the available data in a weight-of-evidence approach, it can be concluded that the target substance was tested positive in vitro, but these results were only observed in rodent cell lines and not in human cells. Also, the positive in vitro findings were not verified in in vivo studies. Moreover, and in line with the conclusion from the EFSA (2014), the antioxidant action of the target substance and the complete hydrolysis in vivo indicates that the genotoxic activity promoted by propyl gallate in vitro is unlikely to be expressed in vivo. This conclusion was supported by the presented in vivo study data. Lastly, supporting evidence for not classifying the target substance for genotoxicity is also demonstrated in the negative outcomes of the carcinogenicity studies in mice and rats, which were conducted by NTP (1982) and are equivalent to OECD testing guideline 451.

Justification for classification or non-classification

Based on the assessment of the available data and taking a weight-of-evidence approach, it can be concluded that no classification of propyl gallate for genotoxicity is warranted.