Registration Dossier

Administrative data

Endpoint:
genetic toxicity in vivo, other
Remarks:
In vivo mammalian Alkaline Comet Assay
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
WI(Han)/ young healthy adult
Details on species / strain selection:
iat. Rattus norvegicus
Sex:
male
Details on test animals and environmental conditions:
Body weight: males 239 – 282 g. The weight variation at the commencement of treatment did not exceed ± 20% of the mean weight.
The animals were derived from a controlled full barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare [9] the animals were bred for experimental purposes and underwent an adequate acclimatisation period after arrival (at least five days). The animals were distributed into test groups at random.
Environment: Temperature 19-25°C
Relative humidity 55  10%
Artificial light 6:00 - 18:00

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
PEG400 as vehicle of the test item was used as negative control. All control animals were handled in an identical manner to the test group subjects. The sampling time for the solvent control was 4 h after the second treatment.
Name: PEG400
Supplier: Sigma
Batch No.: MKBZ9899V
Route and frequency of
Administration: oral, twice within 24 h
Volume administered: 10 mL/kg bw
Sampling time: 28 h
Expiry date: 05/2017
Negative and solvent control groups were included to verify the test results as the assay was newly established at the testing facility with few historic control data
Details on exposure:
The maximum dose applied in the pre-experiment was 2000 mg/kg bw/d according to OECD 489 [5]. The maximum volume administered was 10 mL/kg bw. The animals received the test item twice at 0 h and 24 h after the first application.
Frequency of treatment:
oral, twice within 24 h
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5 males and/or females per dose group
Control animals:
yes
Positive control(s):
Name: EMS; ethyl methanesulfonate
CAS No.: 62-50-0
Supplier: Sigma
Batch No. BCBS6100V
Dissolved in: NaCl (0.9 %)
Dosing: 200 mg/kg bw
Route and frequency of administration: oral, single dose
Volume administered: 10 mL/kg bw
Sampling time: 4 h
Expiry date: 11/2017

Examinations

Tissues and cell types examined:
liver, duodenum and stomach
Details of tissue and slide preparation:
Cells from liver, duodenum and stomach were isolated, embedded in agarose, lysed and DNA allowed to migrate under electrophoresis conditions. 150 cells per animal tissue were evaluated if possible. DNA migration during electrophoresis was determined and expressed as tail intensity.
Evaluation criteria:
Liver, stomach and duodenum were evaluated because the former is the primary organ for the metabolism of absorbed chemicals, and the two latter are a site of first contact of chemicals after oral administration.
According to literature reference data, the acceptance criteria of the negative control group should be between 1-8% tail intensity in the liver and between 1-30% (preferably 1-20%) tail intensity in the stomach. No data are yet available from the JaCVAM trial for duodenum cells. However, the control range of stomach cells will be taken as a basis for duodenum cells as both organs consist of epithelial cells. All tail intensities of the negative and solvent control of this study were within the reference data of the JaCVAM trial. Therefore, the concurrent data were accepted for addition to the laboratory historical control data
The historical data of the negative control group showed the following values:
The evaluation of different rat cells of the negative control group revealed a tail intensity of 0.78 – 5.03% (LCL and UCL of liver cells), 4.25 – 7.00% (LCL and UCL of stomach cells) and 4.66 – 5.87% (LCL and UCL of duodenum cells) The values of the negative control group in the different organs were all within the historic ranges. The tail intensities of each treatment group and organ will be discussed in detail in the following sections.
For statistical analysis One-way ANOVA with Dunnett’s test was performed to verify the results. To test for normality, Kolmogorov-Smirnov-test was done. Statistically significant differences in DNA damage (p < 0.05) were marked with an (related to the solvent control) or Δ (related to the negative control) in the following figures. A linear trend test to determine statistical significances at the 5% level (p < 0.05) was performed to test whether there is a concentration-related increase in DNA damage.
Statistics:
All slides, including those of positive, negative and solvent controls were independently coded before microscopic analysis. The median %tail DNA for each slide was determined and the mean of the median values was calculated for each animal [18]. The mean of the individual animal means was then determined to give a group mean. Statistical analysis was done using a variety of approaches

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not valid
Negative controls validity:
not valid
Positive controls validity:
valid

Any other information on results incl. tables

   negative control  solvant control  500 mg/kg bw  1000 mg/kg bw   2000 mg/kg bw     
  liver cells  1.88%  tail intensity of 5.65% male: 5.68%   male: 6.36%  male: 2.17%    
 Stomach Cells 6.38%   9.15% male: 7.46%  male: 10.23%  male: 8.16%     
 Duodenum Cells:  5.05%  10.48%   male: 7.24% male: 4.68%  male: 4.06%     

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item 1,3-diphenylpropane-1,3-dione did not induce biologically relevant DNA-strand breaks in rodent liver, duodenum and stomach cells after oral administration of rats.
Therefore, 1,3-diphenylpropane-1,3-dione is considered to be non-DNA damaging under these experimental conditions in the in vivo mammalian Alkaline Comet Assay.
Executive summary:

This study was performed to assess the genotoxic potential of 1,3-diphenylpropane-1,3-dione by measuring the ability to induce DNA-strand breaks in different organs of rats (liver, duodenum and stomach). The organs were selected to cover two first-contact organs (stomach and duodenum) upon peroral exposure and liver as theprimary organ for the metabolism of absorbed chemicals.

The test item was suspended in PEG400and applied to 5 male rats by daily gavage at10 mL/kg bw (body weight) for two days. The organs were collected 4 h after the second application of the test item.

Basedon the outcome of a dose range finding study, a dose of 2000 mg/kg bw was selected as maximum tolerated dose (MTD).

In the main experiment three dose levels (500, 1000 and 2000 mg/kg bw/d) were used covering a range from the maximum tolerated dose to little or no toxicity.

The animals treated with a dose of 1 MTD showed slight signs of systemic toxicity (reduction of spontaneous activity). Slight diarrhea was observed in the solvent and test item dose groups in the main experiment after the second application. As the test item was resuspended in the solvent PEG400 and the symptom was also present in the solvent control group, the diarrhea was induced by the solvent and not related to the test item.

Cells from liver, duodenum and stomach were isolated, embedded in agarose, lysed and DNA allowed to migrate under electrophoresis conditions.150 cells per animal tissue were evaluated if possible.DNA migration during electrophoresis was determined and expressed as tail intensity. The tail intensities of each concentration and tissue per sex are summarised inTable 1.

 

The validity criteria were met:

Negative controls fell into the historic control range of the test facility and were within the literature reference data[18]for theacceptance criteria of the negative control group.

The tail intensities of the concurrent solvent controls were within the ranges of the reference data set[18]. Therefore, the data were accepted for addition to the laboratory solvent control data set.

Ethyl methansulfonate (200 mg/kg bw) administered orally was used as positive control, which induced a statistically significant increase in DNA damage for all evaluated organs compared to the solvent control group or negative control group.

In liver and stomach cells, the test item values were in the same range as the corresponding solvent control group. In duodenum cells, the solvent control was increased, however the tail intensities of the middle and high test item dose group were lower than the negative control. As the tail intensities induced by the test item were all within the literature reference data[18]and no biologically relevant increase of tail intensity were found after treatment with the test item in any of the dose groups evaluated as well asin absence of a dose-response,the test item did not induce DNA damage under the conditions tested.