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Genetic toxicity in vitro

Description of key information

Piperonal tested uniformly negative in 6 bacterial reverse mutation assays, including a key study that was consistent with the current OECD Test Guideline 471 (OECD, 1997a). Negative results also were reported in a chromosome aberration assay in Chinese hamster ovary (CHO) cells and a gene mutation assay in mouse lymphoma L5178Y TK+/- cells, both of which were conducted in the presence and absence of external metabolic activation, and in a dominant lethal assay in mice. Positive results were reported in 2 chromosome aberration assays that were judged to be unreliable due to significant methodological flaws, while conflicting data were reported in 3 studies (2 of which were GLP-compliant) that evaluated the effect of piperonal on unscheduled DNA synthesis in isolated rat hepatocytes. Based on the weight of the available evidence, the overall conclusion is that piperonal is not genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(reliability scoring based on 1997 guideline)
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 and Escherichia coli WP2 uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
polychlorinated biphenyl (PCB) induced S9 from male Sprague-Dawley rats
Test concentrations with justification for top dose:
300, 600, 1200 and 2400 μg/plate in the presence or absence of external metabolic activation.

Vehicle / solvent:
Dimethyl sulfoxide (DMSO)

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
furylfuramide
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Without S9: in agar (plate incorporation)
With S9: preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 to 72 hours at 37°C

NUMBER OF REPLICATIONS: 3 to 5 plates per test

Source of test system: Dr. Matsushima, Institute of Medical Science, University of Tokyo, Tokyo
Evaluation criteria:
No data.
Statistics:
Not required.
Key result
Species / strain:
other: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 and Escherichia coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Bacterial killing was reported at the highest test article concentration.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Piperonal was not mutagenic under the conditions of the assay.
Executive summary:

No genotoxicity was observed in any bacterial tester strain incubated with piperonal in the presence or absence of external metabolic activation. Cytotoxicity was reported at the highest concentration tested, suggesting that piperonal was evaluated up to its cytotoxic limit. Positive control substances evaluated in the presence and absence of external metabolic activation demonstrated the sensitivity of the assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 16 June 1988 to 21 November 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Study without detailed documentation; At least 200 metaphases per concentration were not examined.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
-At least 200 metaphases per concentration were not examined.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 from the livers of Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
63, 125, 250, 500 µg/ml were selected for testing in the non-activated system.
625, 1250, 2500, 5000 µg/ml were selected for testing in the activated system.
Vehicle / solvent:
Dimethylsulfoxide (DMSO).
Untreated negative controls:
yes
Remarks:
Untreated cells in growth medium were used as the negative control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
triethylenemelamine
cyclophosphamide
Remarks:
(triethylenemelamine (0.5 µg/ml; -S9); cyclophosphamide (50 µg/ml; +S9) Migrated to IUCLID6: at a concentration of 0.5 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 14 hours in the non-activated study; 2 hours in the activated study
- Fixation time (start of exposure up to fixation or harvest of cells): 16-hour harvest in the non-activated study; 12-hour harvest in the activated study

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 10 µg/ml
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: Duplicate flasks/dose

NUMBER OF CELLS EVALUATED: A minimum of 100 metaphase spreads (50 per duplicate flask) were examined

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
All conclusions were based on sound scientific basis; however, as a guide in interpretation of the data, the test article was considered to induce a positive response when the percentage of cells with aberrations were increased in a dose-responsive manner with one or more concentrations being statistically elevated relative to the solvent control group ( p≤0.05). A significant increase at the high dose only with no dose response was considered suspect. A significant increase at one dose level other than the high dose with no dose response was considered equivocal.
Statistics:
Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At the time of harvest, high dose levels in presence or absence of metabolic activation were observed to be slightly toxic upon microscopic examination of the cell monolayer.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
The solubility and osmolality of the test article were assessed prior to study initiation. For osmolality, the test article in solvent was diluted 50 µl to 5 ml medium and the osmolality was measured and recorded (Osmette S osmometer). In the event the osmolality reading exceeded 425 mOSM/kg or the test article precipitated when added to aqueous medium, 1:2 dilutions of test article in solvent were prepared and tested until a soluble concentration yielding an osmolality reading no greater than 425 mOSM/kg was obtained.

RANGE-FINDING/SCREENING STUDIES:
Dose levels for the chromosome aberration assay were selected following a preliminary toxicity test based upon reduction in mitotic index after treatment relative to the solvent control. CHO cells were exposed to solvent alone and to nine concentrations of test article ranging from 0.5 to 5000 µg/ml in the presence and absence of an S-9 reaction mixture. The test article was partially soluble in medium at 1510 and 5000 µg/ml. All dilutions in the solvent were soluble. The osmolality of the highest concentration tested, 5000 µg/ml, was 412 mOSM/kg. Based upon the findings of the preliminary toxicity study, dose levels of 63, 125, 250 and 500 µg/ml in the non-activated system and 625, 1250, 2500, and 5000 µg/ml in the S-9 activated system were selected for the main study. The harvest time was set at 16 hours in the non-activated system and 12 hours in the S-9 activated system in order to assure that cells were evaluated in first division metaphase after treatment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of metabolic activation, the 500 µg/ml dose level was observed to be slightly toxic upon microscopic examination of the cell monolayer. The mitotic indices at all dose levels were moderately reduced relative to the solvent control.
In the presence of metabolic activation, the 5000 µg/ml dose level was observed to be moderately toxic upon microscopic examination of the cell monolayer. The mitotic indices at all dose levels were slightly reduced relative to the solvent control. At the time of analysis, no mitotic figures were observed at 5000 µg/ml.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

No increase in chromosome aberrations was observed in the non-activated or S-9 activated test systems. The test item was concluded to be negative in the CHO cytogenetics assay.
Executive summary:

The test article was tested in the chromosome aberration assay using Chinese hamster ovary cells. The assay was conducted both in the absence and presence of an Aroclor-induced S-9 activation system. Dose levels of 63, 125, 250 and 500 µg/ml were selected for testing in the non-activated system and 625, 1250, 2500 and 5000 µg/ml in the S-9 activated system. Metaphase cells were collected for microscopic evaluation at 16 and 12 hours after treatment in the non- activated and S-9 activated systems, respectively. In the S-9 activated system, the test article could not be evaluated for chromosome aberrations at 5000 ug/ml due to toxicity. No effect on chromosome aberrations were observed at any test article concentration in the presence or absence of external metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 29th April 1982 to 13th September 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study without detailed documentation; Information on purity of test substance not provided.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Information on purity of test substance not provided.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The cells were maintained in Fischer’s mouse leukemia medium supplemented with pluronic solution, L-glutamine, sodium pyruvate, antibiotics, and horse serum (10% by volume). Cloning medium consists of the preceding growth medium minus pluronic, with the addition of agar to a final concentration of 0.35% to achieve a semisolid state. Selection medium is cloning medium containing 100 µg/ml of 5-Bromo 2’-deoxyuridine (BrdU) or 3 µg/ml 5-trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver microsomes (S9)
Test concentrations with justification for top dose:
2 trials under non-activated conditions were carried out; in the first, the test material was assayed from 200 µg/ml to 1000 µg/ml in duplicates; in the second, the test material was assayed from 600 µg/ml to 1000 µg/ml in triplicates.
In the presence of metabolic activation, the test material was assayed from 62.5 µg/ml to the testing limit of 1000 µg/ml.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test substance was insoluble in water at 100 mg/ml but formed a clear, colorless liquid in DMSO.
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: used for nonactivation study
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: dimethylnitrosamine
Remarks:
for activations study
Details on test system and experimental conditions:
1) Non-activation Assay
The procedure used is based on that reported by Clive and Spector (1975) and is summarized as follows. The cells were exposed to the test chemical for four hours and are then washed and placed in growth medium for two or three days to allow recovery, growth and expression of the induced TK-/- phenotype. Cell counts are determined daily and appropriate dilutions are made to allow optimal growth rates. At the end of the expression period, 5 doses are usually selected for mutant analysis.
After the doses for cloning are chosen, 3 x 10^6 cells for each selected dose are seeded in soft agar plates with selection medium; resistant (mutant) colonies are counted after approximately 10 days incubation. To determine the actual number of cells capable of forming colonies, a portion of the cell suspension is also cloned in normal medium (non-selective). The ratio of resistant colonies to total viable cell number is the mutant frequency.

2) Activation Assay
The activation assay can be run concurrently with the non-activation assay. The only difference is the addition of the S9 fraction of rat liver homogenate and necessary cofactors (CORE) during the four-hour treatment period. CORE consists of NADP (sodium salt) and isocitric acid.
Evaluation criteria:
The suspension growth of each culture is calculated as (Day 1 Cell Count/3) x (Day 2 Cell Count/3) x (day 3 Cell Count/3) when the cultures are split back to 3 x 10^5 cells/ml, the culture is not split back and the cell count is substituted for 3 in the denominator of the next daily count. In most assays, 3 - day expressions are not used, so only the first two factors in the preceding calculation are used. The suspension growth is calculated for each solvent control and then averaged. Relative suspension growth values by the average solvent control value and multiplying by 100%.
The average cloning efficiency for the negative controls in an assay is the average number of viable colonies for the solvent and untreated controls, divided by 300 and multiplied by 100%.
A percent relative growth value is calculated as (relative suspension growth) x (relative cloning efficiency/100). Corrected values for the relative cloning efficiency are used in the cases where the number of cells seeded for viable colonies differs from 300.
The mutant frequency is calculated as the ratio of mutant colonies to viable colonies times 10^-4. This calculation is unaffected by changes in the number of cells seeded for viable count because the number of cells seeded for mutant selection is changed by the same factor. Thus, as an example, if 250 cells are seeded for viable count, 2.5 x 10^6 cells are seeded for mutant selection; the 10^-4 factor remains constant.
Statistics:
Information not reported.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Three trials of the mutation assays were performed, but Trials 2 and 3 were only performed without activation. The non-activation portion of Trial 1 was not used in the evaluation because of erratic background frequencies (solvent and untreated controls).
Under non-activation conditions, most of the assayed treatments induced moderate toxicities (64.9% to 31.4% relative growth), but one treatment at 1000 µg/ml was highly toxic (percent relative growth, 4.0%). The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 36.9 x 10^-6 and three treatments (one 300 µg/ml and duplicate 1000 µg/ml treatments) induced mutant frequencies that exceeded the minimum criterion. Such a weak response required confirmation; another non-activation assay was therefore performed.
In the second non-activation assay, none of the assayed treatments induced mutant frequencies that exceeded the minimum of 31.1 x 10^-6. Moderate to high toxicities were induced (percent relative growths, 33.1% to 6.5%). Since the increases observed in Trial 1 were not repeatable, the test material was considered non-mutagenic without activation in this assay.
In the presence of metabolic activation, no evidence of mutagenic activity was observed. Although the observed toxicities only ranged from non-detectable to moderately toxic, according to the study authors (percent relative growths, 154.6% to 26.5%), the test material was assayed up to the testing limit of 1000 µg/ml where it was insoluble. The test material was therefore considered non-mutagenic with activation in this assay.

The test material was assayed with and without activation up to the testing limit of 1000 µg/ml and no significant increases above the background (average of solvent and untreated control mutant frequencies) were observed. Without activation, a wide range of toxicities were induced but with activation highly toxic treatments were not available for analysis.

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material is considered inactive in the Mouse Lymphoma Forward Mutation Assay with and without metabolic activation.
Executive summary:

The test material did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. The test material was assayed with and without activation up to the testing limit of 1000 µg/ml and no significant increases above the background (average of solvent and untreated control mutant frequencies) were observed. Without activation, a wide range of toxicities were induced but with activation highly toxic treatments were not available for analysis. However, the test material was assayed up to the testing limit of 1000 µg/ml where it was insoluble. The test material is therefore considered inactive in the Mouse Lymphoma Forward Mutation Assay with and without metabolic activation.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1972
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study without detailed documentation; Oral gavage dose volume, purity not reported.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
Deviations:
yes
Remarks:
-Oral gavage dose volume, purity not reported.
GLP compliance:
no
Remarks:
Study pre-dates GLP.
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
other: ICR/Ha Swiss
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: males were 8-10 weeks old at the study initiation; females were 8-10 weeks old when mated
- Weight at study initiation: Not reported
- Assigned to test groups randomly: Not reported
- Fasting period before study: Not reported
- Housing: housed in suspended mesh caged in air-conditioned rooms with automated light-darkness cycles.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not reported
- Humidity (%): Not reported
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): Not reported
Route of administration:
other: Oral gavage (daily for 5 days) or intraperitoneal (single dose).
Vehicle:
- Vehicle(s)/solvent(s) used: The study stated that agents under test were freshly prepared in triacprylin or distilled water for single administration to male mice by intraperitoneal injection or by gavage; however, it is unclear which vehicle was used in this study.
Details on exposure:
The study stated that agents under test were freshly prepared in triacprylin or distilled water for single administration to male mice by intraperitoneal(ip) injection or by gavage (further details were not reported). No details were available regarding the day(s) of dosing relative to start of mating periods.
Duration of treatment / exposure:
IP: Single administration
Oral gavage: Once per day for 5 successive days
Frequency of treatment:
IP injection: Single administration
Oral gavage administration: Once per day for 5 successive days
Post exposure period:
Each treated male was mated with 3 females per week for 8 weeks. Females were sacrificed 13 days after the midweek of their caging and presumptive mating, without being checked for vaginal plugs.
Dose / conc.:
124 other: mg/kg
Remarks:
nominal conc.
IP injection for males only
Dose / conc.:
620 other: mg/kg
Remarks:
nominal conc.
IP injection for males only
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
oral gavage administration for males only
No. of animals per sex per dose:
124 mg/kg: 7 males per dose; 21 females per dose (each mated with 3 females per week for 8 weeks)
640, 1000 mg/kg: 9 males per dose; 27 females per dose (each mated with 3 females per week for 8 weeks)
Control group: 10 males; 30 females per dose (each mated with 3 females per week for 8 weeks)
Control animals:
yes, concurrent vehicle
Positive control(s):
A total of 174 chemicals including, but not limited to, pharmaceuticals, food additives, and pesticides in the dominant lethal assay. Some substances, including acetylaminofluorene and benzoyl peroxide, produced positive results (and therefore can be considered positive controls by default).
Tissues and cell types examined:
At autopsy, each female was scored for pregnancy, and for numbers of total implants and early fetal deaths were analyzed. Corpora lutea counts were omitted.
Details of tissue and slide preparation:
Information not reported
Evaluation criteria:
See below for information on evaluation criteria.
Statistics:
Analaysis of variance performed for the complete experiment and its replicates.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
On the basis of these procedures, heliotropin was classified as a test agent meeting screening criteria but judged non-significant by analysis of variance. See Table 1.

Table 1. Agent producing early fetal deaths and/or pre-implantation losses beyond control limits but with differences not significant by analysis of variance

Agent

Route and frequency of administration

Total weeks of mating

Dose (mg/kg)

No. of males (No. of deaths)

Parameters selected in screen (mating week)

% pregnancy

Implants per pregnancy

Early deaths per pregnancy

% pregnant females with early deaths

Heliotropine*

IP

8

124

7

-

-

-

-

 

 

 

620

9

-

-

1.80 (2)

-

 

 

8

124

7

20 (8)

-

-

-

 

 

 

620

9

19 (8)

-

1.00 (1)

-

 

 Oral

8

1000

9

-

-

-

-

*Compound meeting only less stringent screening criteria.

Conclusions:
Interpretation of results (migrated information): negative
Heliotropine (piperonal) produced early fetal deaths beyond control limits, but judged non-significant by analysis of variance and is therefore considered to be negative for genotoxicity.
Executive summary:

Compared with control values, oral administration of heliotropin (piperonal) for 5 consecutive days had no effect on the incidence of pregnancies, early fetal deaths, or pre-implantation losses at any of the timepoints evaluated. Intraperitoneal administration of heliotropin (piperonal) (620 mg/kg body weight) resulted in a slight increase in early fetal deaths only during the first or second week after dosing; this effect was not statistically significant relative to control values and was therefore considered not adverse or reflective of potential genotoxicity. Based on these findings, the no-observed-adverse-effect level (NOAEL) for this study can be considered to be an oral dose level of 1,000 mg/kg body weight/day.

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

Additional information

In Vitro Studies

Six bacterial reverse mutation studies were identified for heliotropin (piperonal).

In the key study (Sekizawa and Shibamoto, 1982), which was consistent with OECD Test Guideline 471 (OECD, 1997a), heliotropin (piperonal) was evaluated in S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, and in E. coli WP2 uvrA. Heliotropin (piperonal) was evaluated in each bacterial strain at concentrations of 300, 600, 1,200, and 2,400µg/plate in both the presence and absence of external metabolic activation. The reported purity of heliotropin (piperonal) was 100%, and DMSO was used as the solvent control. No genotoxicity was observed in any bacterial tester strain in the presence or absence of external metabolic activation. Cytotoxicity was reported at the highest concentration tested, suggesting that heliotropin (piperonal) was evaluated up to its cytotoxic limit. Positive control substances evaluated in the absence [2-(2-Furyl)-3-(nitro-2-furyl)acrylamide (AF-2), sodium azide (NaN3), and 9-aminoacridine (9-AAc)] and presence [2-aminoanthracene (2-AA) and benzo(a)pyrene (BP)] of external metabolic activation demonstrated the sensitivity of the assay.

Five other studies have reported negative findings for heliotropin (piperonal) in bacterial reverse mutation assays. These include 4 studies judged to be reliable with restrictions (White et al., 1977; Kasamaki et al., 1982; Litton Bionetics, 1983; Haworth et al., 1983) and 1 study judged to be unreliable due to significant methodological flaws (Heck et al., 1989). Each of these studies evaluated heliotropin (piperonal) in one or more strains of S. typhimurium in both the presence and absence of external metabolic activation.

Three chromosome aberration studies were identified for heliotropin (piperonal).

In the key study, heliotropin (piperonal) (coded as “B29” in the study report) was evaluated in a chromosome aberration assay that was conducted in CHO cells (Microbiological Associates, 1988). The assay was not performed according to OECD Test Guideline 473 (OECD, 1997b), but many of the aspects were consistent with this guideline and the study was conducted according to GLP. In this study, heliotropin (piperonal) was evaluated at concentrations of 63, 125, 250, and 500µg/mL in the absence of external metabolic activation and 625, 1,250, 2,500, and 5,000µg/mL in the presence of external metabolic activation. These concentrations were selected based on the findings of a preliminary toxicity test. Information on the purity of heliotropin (piperonal) was not provided, and DMSO was used as the solvent control. No effects on chromosome aberrations were observed at any concentration in the presence or absence of external metabolic activation. The positive controls cyclophosphamide (CP) and triethylenemelamine (TEM) demonstrated the sensitivity of the test system in the presence and absence of external metabolic activation, respectively.

The other 2 chromosome aberration studies reported positive findings with heliotropin (piperonal) when evaluated in Chinese hamster B241 cells (tissue origin unknown) (Kasamaki et al., 1982; Kasamaki and Urasawa, 1985); however, both of these studies were judged to be unreliable due to significant methodological flaws. The study reported by Kasamaki et al. (1982) did not provide information on the range of concentrations of heliotropin (piperonal) that were evaluated, did not include positive controls, did not conduct tests in the presence of external metabolic activation, did not adequately describe the statistical evaluation that was used, and did not evaluate a sufficient number of metaphases. The study reported by Kasamaki and Urasawa (1985) did not justify the apparent inclusion of a 5-day post-exposure period prior to evaluating chromosome effects, did not conduct tests in the presence of external metabolic activation, did not adequately describe the statistical evaluation that was used, and did not evaluate a sufficient number of metaphases.

Two in vitro mammalian cell gene mutation studies were identified for heliotropin (piperonal).

In the key study, heliotropin (piperonal) (coded as “B29” in the study report) was evaluated in a mouse lymphoma L5178Y TK+/- assay (Litton Bionetics, 1982). The assay was not performed according to OECD Test Guideline 476 (OECD, 1997c), but many of the aspects were consistent with this guideline and the study was conducted according to GLP. In this study, heliotropin (piperonal) was evaluated at concentrations ranging from 200 to 1,000µg/mL in the absence of external metabolic activation and from 62.5 to 1,000µg/mL in the presence of external metabolic activation. Precipitation of test article was observed at concentrations of 800 and 1,000µg/mL; thus, heliotropin (piperonal) was evaluated at up to and beyond its solubility limit in the assay. Information on the purity of heliotropin (piperonal) was not provided, and DMSO was used as the solvent control. No significant increase was observed in the mutant frequency at the TK locus in the presence or absence of external metabolic activation. The positive controls ethylmethane sulfonate (EMS) and dimethylnitrosamine (DMN) demonstrated the sensitivity of the test system in the presence and absence of external metabolic activation, respectively.

The other mammalian cell gene mutation study also reported negative results but was judged to be unreliable due to significant methodological flaws that included a lack of information on the purity of the heliotropin (piperonal) that was used and on the range of concentrations of heliotropin (piperonal) that were evaluated, and the lack of negative and positive controls (Heck et al., 1989).

Conflicting results were reported in 3 in vitro unscheduled DNA synthesis studies that were conducted with heliotropin (piperonal), 2 of which were GLP-compliant and were consistent with many aspects of OECD Test Guideline 482 (OECD, 1986).

The GLP-compliant study by Hazleton Laboratories (1988) evaluated unscheduled DNA synthesis in primary hepatocytes isolated from a male Fischer rat. In this study, heliotropin (piperonal) (coded as “B29” in the study report) was evaluated at concentrations ranging from 1 to 1,000µg/mL. Information on the purity of heliotropin (piperonal) was not provided, and DMSO was used as the solvent control. Concentrations of≥ 25.1 µg/mL were reported to be positive for unscheduled DNA synthesis in 2 separate trials, namely a preliminary trial and a confirmatory trial. The positive control 2-acetyl aminofluorene (2-AAF) produced anticipated results, confirming the sensitivity of the test system.

Conversely, unscheduled DNA synthesis was not observed in the GLP-compliant study by Microbiological Associates (1988). In this study, heliotropin (piperonal) (coded as “B29” in the study report) was evaluated at concentrations ranging from 0.15 to 1,000µg/mL in primary hepatocytes isolated from male Sprague-Dawley rats. Information on the purity of heliotropin (piperonal) was not provided, and DMSO was used as the solvent control. Results obtained with the positive control 7,12-dimethylbenz(a)anthracene (DMBA) demonstrated the sensitivity of the test system. The negative results obtained in this follow-up study suggest that the positive results obtained in the earlier study (Hazleton Laboratories, 1988) may have been spurious.

Conflicting data were reported by Heck et al. (1989). In this study, heliotropin (piperonal) was evaluated in 3 separate experiments at concentrations ranging from 1 to 1,000µg/mL in primary hepatocytes isolated from adult male Fischer or Sprague-Dawley rats. Based on the results presented, the lowest active dose of heliotropin (piperonal) was reported to be a concentration of 502µg/mL while the highest inactive dose was reported to be a concentration of 150µg/mL. The criteria for determining a positive response were not clearly described, and there is considerable inconsistency among the cytotoxicity findings of the 3 different experiments, suggesting there may have been a problem with the experimental conditions and/or technique. For example, in one experiment, a concentration of 100µg/mL resulted in a relative survival of 58% and was deemed “too toxic to evaluate”, while in another experiment the same concentration of 100µg/mL resulted in the same relative survival of 58% but the cells were evaluated without issue (in fact, heliotropin (piperonal) concentrations of up to 502µg/mL were evaluated without issue). As such, the results of this study are considered to be ambiguous and of limited utility with regard to hazard identification. Information on the purity of heliotropin (piperonal) was not provided, and DMSO was used as the solvent control. Results obtained with 2-AAF and DMBA confirmed the sensitivity of the test system. This study is considered to be unreliable due to significant methodological flaws, inadequacies and deviations from standard methods.

In Vivo Studies

Epstein et al. (1972) conducted a dominant lethal assay in mice. The assay was not performed according to OECD Test Guideline 478 (OECD, 1984), but many of the aspects were consistent with this guideline. In this assay, heliotropin (piperonal) was administered to 9 male ICR/Ha Swiss mice by oral gavage at a dose level of 1,000 mg/kg body weight/day once per day for 5 days. Other groups of male mice received a single intraperitoneal administration of heliotropin (piperonal) at 124 mg/kg body weight (7 mice) or 620 mg/kg body weight (9 mice). Ten (10) male mice served as controls and received distilled water on the same respective schedule. All male mice were then individually caged with 3 virgin female mice which were replaced weekly for 8 weeks. The females were sacrificed 13 days after the caging and presumptive mating. Females were scored for pregnancy, number of total implants, and early fetal deaths. Compared with control values, oral administration of heliotropin (piperonal) for 5 consecutive days had no effect on the incidence of pregnancies, early fetal deaths, or pre-implantation losses at any of the timepoints evaluated. Intraperitoneal administration of heliotropin (piperonal) (620 mg/kg body weight) resulted in a slight increase in early fetal deaths only during the first or second week after dosing; this effect was not statistically significant relative to control values and was therefore considered not adverse or reflective of potential genotoxicity. Based on these findings, the no-observed-adverse-effect level (NOAEL) for this study can be considered to be an oral dose level of 1,000 mg/kg body weight/day.

Summary

Piperonal has tested negative in 6 bacterial reverse mutation assays, a cell gene mutation assay, and an in vivo dominant lethal assay in mice. A total of 3 chromosome aberration assays have been conducted; negative results were reported in a reliable study while 2 studies judged to be unreliable reported positive findings. Of the 3 unscheduled DNA synthesis assays that have been conducted, two were reliable, GLP-compliant studies that reported conflicting data (one reported positive findings and one reported negative findings) while the third study was unreliable and reported ambiguous findings considered to be of limited utility with regard to hazard identification. Based on the overall weight of evidence, piperonal is considered to not be genotoxic.

Justification for classification or non-classification

Negative results were reported in a chromosome aberration assay in Chinese hamster ovary (CHO) cells and a gene mutation assay in mouse lymphoma L5178Y TK+/- cells, both of which were conducted in the presence and absence of external metabolic activation, and in a dominant lethal assay in mice. Based on the weight of the available evidence, the overall conclusion is that piperonal is not genotoxic. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.5.