Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
1st Phase (preliminary test with TA100): 10.0, 33.3, 66.7, 100.0, 333.3, 666.7, 1000.0, 3333.3, 6666.7, and 10000.0 µg/plate with or without S-9 Activation.
2nd Phase with the 1st sample of DAP:
- mutagenicity assay with all five strains: 50, 100, 250, 500, and 1000 µg/plate with or without S9.
- repeat mutagenic assay: 250, 500, 1000, 1500, and 3000 µg/plate without S9.
3rd phase with the 2nd sample of DAP:
- mutagenicity assay: 150, 300, 600, 1500, 3000, and 6000 µg/plate without metabolic activation.
25, 50, 100, 250, 500, and 1000 µg/plate with metabolic activation.
Vehicle / solvent:
Dimethylsulphoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
5 µg/plate Migrated to IUCLID6: TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
75 µg/plate Migrated to IUCLID6: TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
5 µg/plate Migrated to IUCLID6: TA1538; TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Anthramine for all strings
Remarks:
4 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: ovenight at 37 ± 3 ˚C
- Exposure duration: 48 - 72 h at 37 ± 3 ˚C
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: not applicable
Evaluation criteria:
For the test article to be considered positive, it must cause at least a doubling in the mean number of revertants per plate of at least one strain. This increase in the mean umber of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article. For TA1537 or TA1538 revertants less than three-fold, the response must be reproducible.
Statistics:
For each triplicate plating, an average and standard deviation were calculated.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
The positive response of TA 1535 to DAP exposure was more than 2-fold and repeated in all tests performed for both samples.
Remarks on result:
other: strain/cell type: TA 1535
Conclusions:
Interpretation of results: positive without metabolic activation For one out of five strains tested

Diallyl phthalate caused a positive response in S. typhimurium strain TA1535.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 January to 13 April 2016 (Experimental period). Study finalised 29 July 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mouse spot test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot No.of test material: Osaka Soda Co., Ltd / 15092
- Expiration date of the lot: 13 September 2016 (one year from manufacture)
- Purity test date: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool (1 to 15ºC), light protection/well-sealed
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Soluble and stable for 15 days in corn oil (at 25 ºC or below). At concentrations of 0.1 and 100 mg/ml stable and homogenous for 9 days at room temperature.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test material mixed with a small volume of vehicle, made to total volume and continuously stirred until use
Species:
mouse
Strain:
other: CD2-LacZ80/HazfBr (Trangenic strain) : CD2F1/Slc [SPF] (Non-trangenic animals of same strain)
Details on species / strain selection:
CD2-LacZ80/HazfBR mice are commonly used as transgenic animals in in vivo gene mutation assays. CD2F1/Slc mice are used as the non-transgenic animals of same rodent strain in the in vivo gene mutation assay
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan Laboratory Animals, Inc (combination assay animals)
Japan SLC, Inc (Positive control animals for micronucleus test).
- Age at study initiation: 9 weeks
- Weight at study initiation: 24.3 to 28.1g (combination assay) and 25.4 to 26.7g (micronucleus assay positive control group)
- Assigned to test groups randomly: yes, under following basis: LATOX-F/V5 (Fujitsu Advanced Engineering) computer system package
- Fasting period before study: no
- Housing: polymethylpentene (TPX) cage (W:18.2 cm / D: 26.0 cm / H: 2.8 cm)
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26
- Humidity (%): 35 to 70
- Air changes (per hr): ≥12
- Photoperiod (hrs dark / hrs light): 12 (7am to 7pm)

IN-LIFE DATES: From: To: 13 January 2016 (study start) to 13 April 2016 (study completion)
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Justification for choice of vehicle: At concentrations of 0.1 and 100 mg/mL DAP was stable and homogenous in corn oil after 9 days at 1-30°C
- Concentration of test material in vehicle: 10, 20 and 40 mg/mL
- Amount of vehicle (if gavage or dermal): Dosing volume 10 mL/kg/day
- Lot/batch no. (if required): WEF2972
- Purity: Biochemistry grade
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

2000 mg of test substance was weighed to a volumetric flask, and mixed with a little vehicle. Vehicle was added to make a total volume of 50 mL and a formulation (40.0 mg/mL) prepared by stirring continuously using a magnetic stirrer.

1000 mg of test substance was weighed to a volumetric flask, and mixed with a little vehicle. Vehicle was added to make a total volume of 50 mL, and a formulation (20.0 mg/mL) prepared by stirring continuously using a magnetic stirrer.

500 mg of test substance was weighed to a volumetric flask, and mixed with a little vehicle. Vehicle was added to make a total volume of 50 mL, and a formulation (10.0 mg/mL) prepared by stirring continuously using a magnetic stirrer.
Duration of treatment / exposure:
Administered over 28 consecutive days
Frequency of treatment:
Once daily
Post exposure period:
3 days (post last dose) manifestation period for the gene mutation assay) and 24 hours (post last dose) for the micronucleus assay
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Six treated animals (5 evaluated) per negative, test substance and positive control groups
Control animals:
yes
Positive control(s):
TGR assay:
N-ethyl-N-nitrsourea (ENU) Toronto Research Chemicals Inc. Lot No. 4-RFS-48-2 at 100 mg/kg/day
- Justification for choice of positive control(s): Accepted positive control as stated in OECD 488 guideline
- Route of administration: Oral by gavage
- Doses / concentrations: 100 mg/kg/day (10 mg/mL)

Micronucleus assay:
Mitomycin C (MMC) Kyowa Hakko Kirin Co., Ltd. Lot No. 575AEA
- Justification for choice of positive control(s): Accepted positive control as stated in OECD 474 guideline
- Route of administration: Oral by gavage
- Doses / concentrations: 0.5 mg/kg/day (0.05 mg/mL)
Tissues and cell types examined:
Macroscopic examination of the liver, stomach, femur, testes and vas deferens/cauda epididymis. All samples were stored in liquid nitrogen. Germ cells were collected and stored in case of future analysis of germ cell mutagenicity.
Details of tissue and slide preparation:
Animals were euthanized by inhalation of carbon dioxide, the liver, stomach, femora, testes and vas deferens/cauda epididymis removed and observed macroscopically. Liver weights were measured. Organs were removed and stored according to standard methods and procedures.

Liver: Two samples were prepared from the left lateral lobe and each separately frozen in liquid nitrogen. Remaining lobes were stored in liquid nitrogen.

Stomach: The greater curvature of the stomach was incised and stomach contents removed using physiological saline. It was then divided into forestomach (two pieces) and glandular stomach and frozen in liquid nitrogen.

Femur: The right and left femora were collected and frozen in liquid nitrogen.

Germ cell: The seminiferous tubules and vas deferens/cauda epididymis were cut, placed in cold Dulbecco’s phosphate-buffered saline (PBS) to migrate, then filtered and cell suspensions frozen in liquid nitrogen.

Frozen tissues were stored in an ultra-low temperature freezer (set temperature: -80°C, acceptable range: -90 to -60°C).

Extraction of genomic DNA in the liver, forestomach and bone marrow was conducted. Examination for germ cell mutagenicity was not performed. In the liver and forestomach, 3 mL of buffer (containing RNase) were poured into a homogenizer and ice cooled. Each frozen tissue sample was homogenized with a pestle.
Homogenized fragments were poured into an ice-cooled centrifuge tube containing 3 mL of 0.5 mol/L sucrose solution, and centrifuged (3000 rpm/10 minutes). The supernatant removed and 3 mL of cooled RNase-containing Dounce buffer added and mixed (nuclear/cell suspension).

Bone marrow cells were flushed with RNase-containing dounce buffer into a homogenizer and homogenized with a pestle (nuclear/cell suspension). 3 mL of proteinase K solution was added, mixed by inversion, and incubated at 50°C for ca. 3 hours until clear.

The same volume (ca. 6 mL) of Ph/Cl mixture was added, mixed by inversion, then rotator mixed for 10 minutes and centrifuged (2500 rpm/10 minutes). The upper water layer was transferred into another centrifuge tube and the same volume of Ph/Cl mixture added to the solution. This was again mixed by inversion, rotator mixed for 10 minutes, and centrifuged (2500 rpm/10 minutes). After collecting the water layer, the same volume of chloroform/isoamyl alcohol was added and the contents mixed by inversion, rotator mixed for 10 minutes and centrifuged (2500 rpm/10 minutes). The water layer was transferred into another centrifuge tube. Genomic DNA was extracted by gradual addition of ethanol and the extracted genomic DNA transferred into a microtube containing 70% ethanol, stood for ca. 10 minutes, centrifuged (13000 rpm/10 minutes) and the supernatant removed. The tube remained at room temperature to allow ethanol evaporation.
An appropriate volume of TE buffer was added to the tube and stood overnight at room temperature to dissolve DNA residues. The DNA solution was then stored refrigerated.

The concentration of DNA in each genomic DNA solution was spectrophotometrically measured (and within a concentration range ca. 250 to 600 µg/mL). All DNA solutions were discarded after Sponsor’s consent.

For the preparation of test strains, 30 mL (LB broth), 300 µL (maltose solution, 200 mg/mL), 30 µL (ampicillin solution, 50 mg/mL), and 30 µL (kanamycin solution, 20 mg/mL) were poured into a 200-mL flask. A 50 µL suspension of thawed Escherichia coli C (lacZ--, gal E-) was inoculated into the flask and incubated overnight at 37°C with a shaker (120 strokes/min) as the pre-incubation culture.
One hundred millilitres of LB broth and 1 mL (maltose solution, 200 mg/mL) were poured into a 500-mL flask. The pre-incubation culture (1 mL) was inoculated into the flask and incubated for ca. 2 hours (OD: approximately ≥0.8) under the same pre-incubation conditions. The bacterial suspension was centrifuged at 1000 rpm for 10 minutes, the supernatant removed and cells suspended in LB broth containing 10 mmol/L magnesium sulfate (E. coli suspension).

Packaging of genomic DNA was conducted in accordance with instructions ‘Transpack packaging extract’. One red tube of packaging extract was thawed and 10 µL of genomic DNA solution (concentration range ca. 250 to 600 µg/mL) transferred to the red tube. The packaging reaction was pipette mixed and the tube incubated (30°C for 90 minutes). The blue tube of packaging extract was thawed and 10 µL transferred to the red tube containing a packaging reaction and mixed as previously detailed. It was then incubated (30°C for 90 minutes) and diluted with 700 µL of SM buffer, and mixed (packaged DNA sample).

For plating of packaging DNA, 1 mL of E. coli suspension for calculating total number of plaques (for tittering) and 2 mL for calculating mutant frequency (for selection) were dispensed into each tube. The entire volume of packaged DNA sample (ca. 700 µL) was added to the E. coli suspension in the selection tube (total ca. 2700 µL), mixed and incubated (room temperature/ca. 30 minutes) to allow phage to infect E. coli. Thirty microliters of the content were diluted 10-fold with 270 µL of LB broth (containing 10 mmol/L magnesium sulfate). Thirty microliters was transferred to the tube for tittering, and stirred.
The magnesium sulfate solution (1 mol/L) was added to the agar solution at 2:100 volume ratio to make top agar for tittering. The P-gal solution was added to the agar solution at 2:100 volume ratio of to make the top agar for selection.
Then, 17 mL of top agar were added to the tube for tittering and mixed and the contents were poured over a LB agar plate. To the tube for selection, 16 mL of top agar were added and the contents were poured over a LB agar plate in the same manner as above. The agar plate was incubated (37°C overnight).
In the case of liver, forestomach and bone marrow the above packaging procedure was repeated until the total number of plaques per animal reached 300,000.
Evaluation criteria:
The test was considered valid if:

- The mutant frequency for the liver, forestomach or bone marrow in the positive control group markedly increases with a statistically significant difference from the concurrent negative control group.
- The frequency of MNRET in the positive control group increases with a statistically significant difference from the concurrent negative control group.
Statistics:
TGR: The mutant frequency (MF) of the negative control gp vs each test substance gp were tested (Bartlett’s test for homogeneity of variance, two-sided (significance 0.05). If not significant, a Dunnett’s multiple comparison test, two-sided, significance 0.05, was performed. If no homogeneity, a Steel’s test, a two-sided, significance 0.05) was performed.

The MF of the negative control gp vs the positive control gp were tested by F test for homogeneity of variance (two-sided, significance 0.05). If homogeneity of variance was determined, a Student’s t test (two-sided, significance 0.05) was performed. If no homogeneity, Aspin-Welch’s t test (two-sided, significance 0.05) was performed. The result was considered positive if a statistically significant increase in MF was observed in test substance gps vs the negative control gp. Biological relevance was considered.

MN: The Conditional Binomial test was performed to compare the incidence of MNRET in the negative control gp vs each treatment gp and positive control gp (upper-tailed significance level of 0.025). The ratio of RET was analysed by Dunnett’s multiple comparison test between the negative control gp vs each test substance gp. The Aspin-Welch’s t test (two-sided significance 0.05) was used for the significant difference between the negative control group vs positive control group. The Dunnett’s multiple comparison test was conducted (two-sided significance of 0.05).

The result was considered positive if a statistically significant increase in the frequency of MNRET was observed in the test substance gps compared with the negative control gp. Biological relevance was considered. Data (minus positive control gp) were tested by Bartlett’s test for homogeneity of variance (two-sided, significance level 0.05). If not significant, a Dunnett’s multiple comparison test (two-sided, significance 0.05) was performed. If not significant on Bartlett’s test, a Steel’s test (two-sided, significance 0.05) was performed.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
This combination study incorporated a peripheral blood micronucleus assessment onto an OECD 488 gene mutation study (reporter gene LacZ) evaluating the target tissues, liver, forestomach and bone marrow. Both genotoxicity endpoints were negative, i.e. no increase in mutation frequency for gene mutations and no increase in micronuclei formation for clastogenicity/aneugenicity.

TGR Assay:

Liver: Negative control group (mean±SD)of mutant frequency (individuals) 37.6±8.3 (×10-6).

The means±SD of mutant frequencies in diallyl phthalate treated groups (100, 200 and 400 mg/kg/day) 39.5±5.7 (×10-6), 55.4±13.6 (×10-6) and 37.9±12.8 (×10-6), respectively. At 200 mg/kg/day a statistically significant group increase (p≤0.05) was observed compared with the negative control group, however the observed mutant frequencies at 200 mg/kg/day were within the acceptable historical range. Furthermore, no dose response relationship was observed across treatment groups. The observed increase at 200 mg/kg/day was therefore considered not treatment-related or biologically relevant.

 

Forestomach: Negative control group (mean±SD) of mutant frequency (individuals) 33.2±5.5 (×10-6).

The means±SD of mutant frequencies in diallyl phthalate treated groups (100, 200 and 400 mg/kg/day) were 42.3±17.7 (×10-6), 36.6±3.9 (×10-6) and 31.6±7.6 (×10-6), respectively. No statistically significant increases were observed compared with the negative control group.

 

Bone marrow: Negative control group (mean±SD) of mutant frequency among (individuals) 32.6±13.5 (×10-6).

The means±SD of mutant frequencies in diallyl phthalate treated groups (100, 200 and 400 mg/kg/day) were 31.9±10.5 (×10-6), 28.8±12.4 (×10-6) and 27.6±2.6 (×10-6), respectively. No statistically significant increases were observed compared with the negative control group.

 

The positive control groups (mean±SD) of mutant frequency (individuals) for the liver 105.5±18.9 (×10-6), forestomach 544.9±101.8 (×10-6) and bone marrow 598.9±49.1 (×10-6) were all statistically significant increased compared with the concurrent negative control group.

 

MutaTMMouse: male; negative control

Organ

No.

Mutant Frequency (x10-6) (mean±SD)

Range
(Min-max)

Acceptable range
(Lower-upper)

 

Liver

137

50.1±16.5

16.6

95.0

0.6

99.6

Stomach

43

46.8±10.4

31.1

84.7

15.6

78.0

Bone marrow

15

43.4±12.6

21.8

67.6

0.6

86.4

 

Acceptable range calculated by mean±3xSD

Conclusions:
Diallyl phthalate (DAP) did not induce gene mutations or micronucleus in male transgenic mice under experimental conditions. Although at 200 mg/kg/day (intermediate dose group in the gene mutation test) and 400 mg/kg/day (top dose group in the micronucleus assay) a statistically significant increases were observed from the concurrent negative controls. As no dose dependent response was observed across treatment groups with the observed significance from control values small, the biological relevance was questionable and the effects were considered as spontaneously occurring and not of biologically relevance.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The genotoxic potential of Diallyl Phthalate (DAP) has been assessed in vitro and in vivo. In bacterial assays weak positive results were observed in S. tymphimurium and E. coli WP2 in the absence and presence of metabolic activation respectively. Negative results for bacterial mutagenicity have also been reported in the absence and presence of metabolic activation. In vitro testing in mammalian cells showed unequivocally positive responses in the presence of metabolic activation In vitro, DAP induced structural chromosomal aberrations, sister chromatid exchanges and the formation of micronuclei. A positive was observed in the in vitro mammalian cell gene mutation test in the presence of metabolic activation, colony size distribution having a predominance towards small colonies indicative of clastogenicity.

In vivo, negative results in the in vivo mouse micronucleus test and equivocal results in an in vivo chromosome aberration test using mouse bone marrow cells (published literature) were reported. In the chromosome aberration test, a small yet statistically significant increase in aberration frequency at the high dose considered the dose-effect and biological relevance as questionable. With the potential of DAP to cause gene mutations having only been addressed in vitro, this in conjunction with in vitro data from bacterial reverse mutation assays and a mammalian cell gene mutation test indicate a potential alert for gene mutation which had not been addressed in vivo.

To evaluate DAP for its mutagenic potential in somatic cells, and if required, germ cells, it was considered appropriate to perform a gene mutation in vivo, the transgenic rodent gene mutation assay (OECD 488) being the most appropriate in vivo test as it detects stable mutations and evaluates gene mutation in both somatic, and germ cells. An OECD 488 was conducted using tissues from the stomach, liver and bone marrow, male germ cells were stored in the event of a positive result in somatic cells. Toxicokinetic data and repeated dose toxicity studies were considered adequate to demonstrate that DAP or its main metabolites reached relevant tissues.

It was proposed that performing a micronucleus assessment on peripheral blood samples taken from animals exposed to DAP over 28-day dosing of the OECD 488 would adequately address the concern related with clastogenicity/aneugenicity, thus removing the ambiguity related to in vivo chromosome aberration tests. Although no validated protocol was available for a combined OECD 488 and OECD 474, the latter allows the option to integrate the micronucleus test and repeated-dose toxicity studies.

An in vivo combination assay using transgenic animals to assess both gene mutation and chromosomal aberration induction in parallel would adequately evaluate in vivo genotoxicity. A “Gene Mutation-Micronucleus Combination Assay of Diallyl Phthalate in Muta Mouse” (Masumori, S., 2016) was therefore performed to evaluate the potential of DAP to induce gene mutations in the liver (primary target organ/slow dividing tissue), forestomach (initial site of contact) and bone marrow (distal site/rapidly dividing cells) and also to induce concomitantly micronucleated reticulocytes (MNRET) in peripheral blood.

Dosages of 400, 200 and 100 mg/kg/day were selected from the dose range finding test and previous studies. DAP was administered to male transgenic mice orally for 28 consecutive days, after three days the liver, stomach, femur, testes and vas deferens/cauda epididymis were removed and frozen. The mutant frequencies in the liver, forestomach and bone marrow were determined, and peripheral blood sampled 24 hours after the last dose and the incidences of MNRET determined.

In conclusion, DAP did not induce gene mutations or micronucleus formation in transgenic mice under the experimental conditions. Any statistically significant deviations from the concurrent negative control were not considered dose dependent with deviations from the negative control value considered small. These effects were spontaneously occurring deviations and were not considered biologically relevant.

Justification for classification or non-classification

Although the in vitro studies returned a positive result, the in vivo studies gave a negative result. It is on this basis that this substance will not be classified for mutagenicity.