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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Propyl gallate (target substance) was tested negative for mutagenicity in several bacterial reverse mutation assays. Only in one study it was reported, that propyl gallate was weakly mutagenic in the oxidative damage sensitive Salmonella typhimurium strain TA102. Moreover, in several in vitro chromosome aberration assays the target substance induced chromosome aberration and sister chromatid exchanges in the absence and presence of metabolic activation. In vitro studies conducted with human cells were negative. In an in vitro cytogenic study using human embronyic lung cells (WI-38), the target substance showed no clastogenic effect. In an in vitro alkaline elution test (Comet Assay) no induction of single strand breaks in human fibroblast cells were observed after treatment with propyl gallate. In an in vitro mammalian mouse lymphoma cell assay (MLA) the target substance was tested positive for mutagenic responses.

Link to relevant study records
Reference
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
Reference:
Composition 0
Qualifier:
equivalent or similar to
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
Test material information:
Composition 1
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:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (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, 3333, 10000 µg/L
Vehicle:
- Vehicle(s)/solvent(s) used: ET95
Negative controls:
yes
Remarks:
Potassium chloride
Solvent controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535 and TA100
Negative controls:
yes
Remarks:
Potassium choride
Solvent controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
TA98
Negative controls:
yes
Remarks:
Potassium chloride
Solvent controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA97 and TA1537
Negative controls:
yes
Remarks:
Potassium chloride
Solvent controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
all strains
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

EXPERIMENTAL PERFORMANCE
1-mL thawed bacterial culture inoculated into minimal glucose medium, supplemented with an excess of biotin and histidine. Bacterial culture was grown overnight for 12 - 15 hr at 37°C on a shaker, and their phenotypes were analyzed as recommended by Ames et al. (1975).

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 which 2.0 mL 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) nonmutagenic 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, nonmutagenic, 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 nonmutagenicity of the chemicals had been determined.
Species / strain:
other: TA1535, TA1537, TA100 and TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
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

With 10% hamster S9

With 10% rat S9

Without hamster

With 10% hamster S9

With 10% rat S9

Without hamster

With 10% hamster S9

With 10% rat S9

Without hamster

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

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item can be considered as non-mutagenic.
Executive summary:

In a reverse gene mutation assay in bacteria conducted similar to OECD test guideline 471, strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium were exposed to Propyl gallate (>98 % purity) in ET95 at concentrations of 10, 33, 100, 333, 1000, 3333 and 10000 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 gene mutation assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In the CIR, 2007 and EFSA, 2014 review publication data from two unpublished non-GLP in vivo study reports were published. In an in vivo dominant lethal test, propyl gallate did not induce any mutations in Sprague Dawley rats and based on the results from an in an in vivo chromosome aberration test in male albino rats it was concluded, that propyl gallate was not clastogenic. In the study by Shelby et al., 1993, propyl gallate was tested in a mouse bone marrow micronucleus test. It was shown, that the target substance did not induce the level of micronuclei. Similar results were reported in the publication by Raj & Katz, 1984 and Shelby et al., 1993. No induction of micronuclei were recorded in bone marrow derived cells from B6C3F1 mice. Sasaki et al., 2002 published results from an in vivo Comet assay, showing that propyl gallate did not increase DNA damage in eight investigated organs of treated male ddY mice after 3 and 24 hours of treatment.

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
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
yes
Remarks:
only 4 animals per dose group, only one dose, no in vivo positive control, only 50 cells scored per organ per animal
Principles of method if other than guideline:
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
Test material information:
Composition 1
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 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): 12h light-dark
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
Details on exposure:
4 male mice per group were administered orally 2000 mg/kg, and untreated mice served as controls. 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.
Duration of treatment / exposure:
Once orally
Frequency of treatment:
once
Post exposure period:
not specified
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
Justification of dose: LD50 was determined using simple acute toxicity "experiments on four–five animals." No death was observed at 2000 mg/kg, so the LD50 was defined as >2000 mg/kg. Whether 2000 mg/kg was nominal or the actual dose received was not specified.
No. of animals per sex per dose:
2000 mg/kg: 4 males
Control animals:
yes, concurrent no treatment
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:
Eight organs (glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow) were removed and were afterwards 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.

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 [9]. 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) [9] 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 [9]. 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 (Wako Pure Chemical Industries Ltd.) [9]. We examined and photographed 50 nuclei per slide at 200× magnification with the aid of a fluorescence microscope.
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.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
not examined
Negative controls valid:
not specified
Positive controls valid:
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:

The comet assay, also known as single-cell gel electrophoresis, was used to investigate the ability of Propyl gallate to induce genotoxicity in murine organs (glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow) 3 and 24 h after treatment. In the present study, 4 male ddY mice were exposed once orally to 2000 mg/kg of Propyl gallate. Untreated mice served as negative controls. All mice were euthanized at 3 hours and 24 hours after exposure, at which time the either organs 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
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
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.
GLP compliance:
not specified
Type of assay:
other: micronucleus assay
Test material information:
Composition 1
Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles Raver 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: [no/yes, under following basis: ] not specified
- Diet: Purina Laboratory Rodent Chow ad libitum
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Details on exposure:
The in vivo bone marrow micronucleus assay was adopted from Heddle, 1973; Schmid, 1973, 1975, 1976; and Matter and Schmld, 1971.

Propyl gallate was tested for induction of micronuclei in mouse bone marrow cells using 3 and in some cases 5 sampling points collected at 24-h intervals after the last injection (24, 48, 72, and in some cases 0 and 96 hr).

Groups of 5 female B6C3F1 mice were injected intraperitoneally twice at 24 h intervals with the test substance dissolved in DMSO, for each sampling point. The total dosing volume per mouse was not reported.
Duration of treatment / exposure:
2 x 24 h
Frequency of treatment:
Two times at 24 h intervals
Dose / conc.:
217 mg/kg bw/day
Remarks:
Dissolved in DMSO
No. of animals per sex per dose:
5 female animals per sampling period
Control animals:
yes
Positive control(s):
not specified
Tissues and cell types examined:
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).
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).
Statistics:
not specified
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
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.
Vehicle controls valid:
not specified
Remarks:
Control results not specified
Negative controls valid:
not specified
Remarks:
Control results not specified
Positive controls valid:
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.
- 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:
Interpretation of results: negative
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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Propyl gallate (target substance) was tested negative for mutagenicity in several bacterial reverse mutation assays. Only in one study it was reported, that propyl gallate was weakly mutagenic in the oxidative damage sensitive Salmonella typhimurium strain TA102. Moreover, in several in vitro chromosome aberration assays the target substance induced chromosome aberration and sister chromatid exchanges in the absence and presence of metabolic activation. In vitro studies conducted with human cells were negative. In an in vitro cytogenic study using human embronyic lung cells (WI-38), the target substance showed no clastogenic effect. In an in vitro alkaline elution test (Comet Assay) no induction of single strand breaks in human fibroblast cells were observed after treatment with propyl gallate. In an in vitro mammalian mouse lymphoma cell assay (MLA) the target substance was tested positive for mutagenic responses.

In vivo studies it was shown that propyl gallate did not induce genotoxicity. In both, the CIR, 2007 and EFSA, 2014 review publication data from two unpublished non-GLP in vivo study reports were published. In an in vivo dominant lethal test, propyl gallate did not induce any mutations in Sprague Dawley rats and based on the results from an in vivo chromosome aberration test in male albino rats it was concluded, that propyl gallate was not clastogenic. In the publication by Shelby et al., 1993 data derived from a mouse bone marrow micronucleus test was presented. It was shown, that the target substance did not induce the level of micronuclei in bone marrow derived cells. Similar results were reported in the publication by Raj & Katz, 1984. Sasaki et al., 2002 published results from an in vivo Comet assay, showing that propyl gallate did not increase DNA damage in eight investigated organs of treated male ddY mice after 3 and 24 hours of treatment.

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 not verified in in vivo studies. Moreover, and in line with the conclusion from the EFSA report, 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.

Supporting evidence for not classifying the target substance for genotoxicity is derived from the negative carcinogenicity studies in mice and rats, which were conducted by NTP, 192 equivalent to OECD testing guideline 451.

Justification for classification or non-classification

Based on the assessment of the available data, it can be concluded that no classification for genotoxicity is warranted.