Bis(pentane-2,4-dionato-O,O')magnesium

Genetic toxicity: in vivo

Administrative data

Endpoint:

genetic toxicity in vivo, other

Remarks:

In vivo mammalian erythrocytes micronucleus test combined to in vivo mammalian alkaline comet assay on lung and liver

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: combined mammalian comet assay and mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Bis(pentane-2,4-dionato-O,O')magnesium

EC Number:

237-857-7

EC Name:

Bis(pentane-2,4-dionato-O,O')magnesium

Cas Number:

14024-56-7

Molecular formula:

C10H14MgO4

IUPAC Name:

magnesium;4-oxopent-2-en-2-olate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

As the search for systemic exposure through the determination of the ratio PCE/NCE as done in the
confirmatory toxicity assay demonstrated a statistically significant decrease exclusively in female rats , the main assay was performed only in female rats that demonstrated a higher sensitivity to the test
item

Administration / exposure

Route of administration:

intratracheal

Vehicle:

Sterile water.

Details on exposure:

Treatment took the form of 3 successive administrations at 24-hour intervals by endotracheal. Samples
were taken 2 to 3 hours after the last treatment.

Duration of treatment / exposure:

3 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

7.5 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

3.75 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1.875 mg/kg bw/day

No. of animals per sex per dose:

7 females for the higher dose, 5 females for the other doses

Positive control(s):

5 females exposed to positive controle (cyclophosphamide 25 mg/kg IP
5 females exposed to Methylmethane sulfonate 100 mg/kg/day (x2) +70 mg/kg/day PO (x1)

Examinations

Tissues and cell types examined:

bone marrow lung and liver.

Details of tissue and slide preparation:

The femurs were removed, and the bone marrow was extracted with foetal calf serum (1 mL per animal).
The cell suspensions were centrifuged for 5 minutes at 1000 rpm. The supernatants were removed. After
homogenisation, the centrifugate was spread on slides. The smears were stained using a technique,
derived from the May Grunwald Giemsa technique (Schmid, 1975) (see also § 12.1), which makes it
possible to distinguish between polychromatic (PCE) and normochromatic erythrocytes (NCE): PCE are
purple whereas NCE are red.
After blind coding of slides by a person not involved in the reading, two slides per animal were scored by
two independent operators; for each animal, the number of polychromatic erythrocytes having one or more
Howell-Jolly bodies (micronuclei) was determined from the microscopic examination of 4000 polychromatic
erythrocytes.
The proportion of immature among total (immature and mature) erythrocytes was determined for each
animal. The polychromatic/normochromatic erythrocyte ratio was determined from the microscopic
examination of 1000 erythrocytes per animal. When analysing slides, the proportion of immature
erythrocytes among total erythrocytes should not be less than 20% of the control value.

Evaluation criteria:

The results are reported in the form of tables giving the number of micronucleated cells per 4000
polychromatic erythrocytes for each animal, together with the total and the statistical analysis.
The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE), calculated from 2000 polychromatic
erythrocytes is also established for both treated and control animals.
The statistical comparison for the polychromatic/normochromatic erythrocyte ratio was performed using
Student's t test.
Statistical analysis was performed for micronucleus number using a non-parametric test, the Mann Whitney
U rank test, recommended by UKEMS (Lovell et al., 1989). Statistical analysis for micronucleus number
was conducted.

Results and discussion

Test results

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

The preliminary toxicity assay performed on male and female rats at the highest dose of 7.5 mg/ kg/ day (x3) by endotracheal route induced neither clinical signs, nor mortality, and was thus retained for the confirmatory toxicity assay.

Vehicle controls validity:

valid

Remarks:

The sponsor has chosen to perform the check of the concentration of Magnesium acetylacetonate dihydrate in dosing formulations in a GLP-compliant test site. The stability was studied in the validation study CitoxLab No. 46707 VAS.

Negative controls validity:

not specified

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test item Magnesium acetylacetonate dihydrate (batch 18030702), was
investigated for genotoxic potential by the means of the in vivo micronucleus test in bone marrow
combined with the in vivo comet assay under alkaline conditions (SCGE) in the Lung and Liver, in
female OFA Sprague-Dawley rats, according to OECD Guidelines (Nos. 474 and 489, 2016). Animals
were treated endotracheally at dose levels of 7.5, 3.75 and 1.875 mg/kg once a day for 3 consecutive
days, 24 hours apart, followed by one sampling time 2-3 hours after the last treatment.
The results of assays for Magnesium acetylacetonate dehydrate in treatment formulations were
conform. No test item was found in the vehicle control. The results were thus satisfactory.
The validity criteria for the results were considered as fulfilled. The study is thus valid.
Under these experimental conditions, the test item did not present DNA strand breaks and/or alkalilabile
sites inducer activities toward the lung and liver from OFA Sprague-Dawley female rats.
Furthermore, Magnesium acetylacetonate dihydrate induced no genotoxic activity in bone marrow
cells.
As a conclusion, Magnesium acetylacetonate dihydrate induced no in vivo genotoxic activity under
these experimental conditions.

Executive summary:

Study initiation date (date Study Director signed Study Plan): 21/06/18

PURPOSE

The potential clastogenic activity of Magnesium acetylacetonate dihydrate provided was tested using both the in vivo micronucleus test in bone marrow and the comet assay in the lung and liver in the rat. The actual treatment was carried out by endotracheal route, using 1 daily treatment for 3 days.

METHOD

Animals ( strain, species) : OFA Sprague Dawley rat

Feedstuff : A04C-10 from SAFE (batch 17318)

Form administered : suspension

Route : endotracheal

Dose volume : 0.75 mL/kg b.w.

Vehicle : sterile water (Fresenius, batch 13MCP211)

Stability in vehicle : unknown at the preparation dates (dosing formulations were thus prepared just prior to use)

Limiting factors for the top dose used in the toxicity assay and the main assay : solubility of the test item in sterile water (i.e. 10 mg/mL) and maximal volume that can be administered endotracheally to the rats (ca. 150 μL/rat, i.e. 0.7 mL/kg)

TOXICITY ASSAY

Preliminary toxicity assay

Number of animals per group : 2 males and 2 females weight: 220 g and 205 g (males) 190 g and 185 g (females)

Doses tested : 7.5 mg/kg/day (x 3)

Treatment schedule : 3 daily treatments at 24-hour intervals

The preliminary toxicity assay performed on male and female rats at the highest dose of 7.5 mg/ kg/ day (x3) by endotracheal route induced neither clinical signs, nor mortality, and was thus retained for the

confirmatory toxicity assay.

Confirmatory toxicity assay

Number of animals per group : 5 males and 5 females per group (i.e. vehicle control and 7.5 mg/kg treated group weight: 183 g to 210 g (males) 185 g to 202 g (females)

Doses tested : 7.5 mg/kg/day (x 3)

Treatment schedule : 3 daily treatments at 24-hour intervals

The confirmatory toxicity assay performed on 5 male and 5 female rats at the highest dose of 7.5 mg/ kg/day (x3) by endotracheal route induced neither clinical signs, nor mortality, and was thus retained for the

main assay. Two inferior doses of 3.75 and 1.875 mg/kg/day (x3) were also tested.

Otherwise, in accordance with the Sponsor, the proportion of immature among total (immature and mature) erythrocytes was determined for each animal used in the confirmatory toxicity assay. To reach this goal,

bone marrow removal, slide preparation and counting were carried out as depicted in §8.1 (the number of polychromatic erythrocytes (immature) having one or more micronuclei was not determined). To assess an

eventual decrease in the ratio PCE/NCE, vehicle control groups of 5 animals of each sex treated with the vehicle only were also included in the confirmatory toxicity assay.

The ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE) was thus established at the highest dose level tested of 7.5 mg/kg/day (x3). A statistically significant decrease in the ratio PCE to NCE was noted in the Magnesium acetylacetonate dihydrate treatment group when compared to the negative control group, only in female rats. The main assay was thus performed in female rats only as they were

considered as the probably the most sensitive gender.

GENOTOXICITY ASSAY

Number of animals per group : 5 females for both the micronucleus and the comet assays

weight: 179 g to 205 g

Doses tested : 7.5 – 3.75 – 1.875 mg/kg/day (x 3)

Treatment schedule : 3 daily treatments at 24-hour intervals

Number of sampling times : one:

for the negative control and the 3 treated groups: 2-3 hours after the third treatment

for the positive control group (cyclophosphamide): 24 hours after

the single treatment (micronucleus test)

for the positive control group (methylmethane sulfonate): 2-3

hours after the third treatment (comet assay)

Positive reference substances : cyclophosphamide (Baxter, batch 5K044J) in NaCl at 0.9% in distilled water (Aguettant, Batch 170 6841), 25 mg/kg, intraperitoneal administration

methylmethane sulfonate (Aldrich, batch MKCD8572) in sterile water (Fresenius, Batch 13MCP211), 100 mg/kg/day (x2) – 70 mg/kg/day (x1), gavage

Micronucleus test in bone marrow

Number of polychromatic

erythrocytes analysed

for each animal : 4000

No statistically significant increase in the number of micronuclei was noted at the doses of 7.5 – 3.75 and 1.875 mg/kg/day (x3) by endotracheal route in female rats.

Comet assay in the Lung

Number of cells observed

per animal : 150

Number of cells observed

per dose : 750

No statistically significant increase in the mean of medians of percentage of DNA in tail per group was observed at the tested doses of 7.5 – 3.75 and 1.875 mg/kg/day (x3) by endotracheal route of Magnesium

acetylacetonate dihydrate in Lung from OFA Sprague-Dawley female rats.Taking into account the overall results, it was concluded that Magnesium acetylacetonate dihydrate is not

genotoxic toward lung cells from OFA Sprague-Dawley female rats as investigated by the in vivo Comet assay.

Comet assay in the Liver

Number of cells observed

per animal : 150

Number of cells observed

per dose : 750

No statistically significant increase in the mean of medians of percentage of tail DNA per group was observed at the tested doses of 7.5 – 3.75 and 1.875 mg/kg/day (x3) of Magnesium acetylacetonate

dihydrate in Liver from OFA Sprague-Dawley female rats. Statistically significant decreases in the mean of medians of percentage of tail DNA per group were

observed at the tested doses of 7.5 and 1.875 mg/kg/day (x3). They are however without any significance in terms of genotoxicity hazard.

Taking into account the overall results, it was concluded that Magnesium acetylacetonate dihydrate is not genotoxic toward hepatocytes from OFA Sprague-Dawley female rats as investigated by the in vivo Comet assay.

The test item Magnesium acetylacetonate dihydrate (batch 18030702), was investigated for genotoxic potential by the means of the in vivo micronucleus test in bone marrow

combined with the in vivo comet assay under alkaline conditions (SCGE) in the Lung and Liver, in female OFA Sprague-Dawley rats, according to OECD Guidelines (Nos. 474 and 489, 2016). Animals

were treated endotracheally at dose levels of 7.5, 3.75 and 1.875 mg/kg once a day for 3 consecutive days, 24 hours apart, followed by one sampling time 2-3 hours after the last treatment.

The results of assays for Magnesium acetylacetonate dehydrate in treatment formulations were conform. No test item was found in the vehicle control. The results were thus satisfactory.

The validity criteria for the results were considered as fulfilled. The study is thus valid. Under these experimental conditions, the test item did not present DNA strand breaks and/or alkalilabile

sites inducer activities toward the lung and liver from OFA Sprague-Dawley female rats.

Furthermore, Magnesium acetylacetonate dihydrate induced no genotoxic activity in bone marrow cells.

As a conclusion, Magnesium acetylacetonate dihydrate induced no in vivo genotoxic activity under these experimental conditions.