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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro genotox/mutagenic potential of magnesium dihydrate was assessed in 3 in vitro models, i.e. the Ames test (OECD 471), the in vitro gene point mutation assay on L5178Y mouse lymphoma cells (OECD 490) and the in vitro chromosomal aberration test on human lymphocytes (OECD 473). All these 3 tests were performed following the Good Laboratory Practices (GLP). A fourth in vitro genotoxicity test (Ames test) has been performed in order to confirm or not the result of the first Ames test.

The potential clastogenic activity of magnesium dihydrate was also tested using both the in vivo micronucleus test in bone marrow (OECD 474) and the comet assay (OECD 489) in the lung and liver in the rat. This study was carried out in compliance with Good Laboratory Practice regulation.

1)     In vitro bacterial mutagenicity study (Envigo Study Number RC45DS).

Magnesium acetylacetonate (Batch No 170301, purity 98%, 9,14 % as magnesium) was tested for mutagenic potential in the Ames/microsome assay using 4 Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and the Escherichia coli strain WP2 uvrA (pKM101).

The test item was diluted in dimethylsulfoxide (DMSO) and tested up to 5000 µg/plate corresponding to the highest recommended dose in the OECD guideline No 471.

Two independent mutation tests were performed in presence and absence of liver preparations (S9mix) from rats treated with phenobarbital and 5, 6-benzoflavone. The first assay was performed according to the standard plate incorporation assay and the second assay included a pre-incubation stage.

 

Results:

          No signs of toxicity towards the tester strains were observed in the first assay following exposure to magnesium acetylacetonate while a thinning of the background lawn of non-revertant colonies (and/or a reduction in revertant colony numbers) was seen in several strains both in the absence and in the presence of S9 mix in the second assay.

               Regarding the mutagenic activity, in the first assay, a biologically significant increase in the number of revertants was observed at only 500µg/plate, only in Escherichia coli strain WP2 uvrA (pKM101), exclusively in the presence of S9-mix. No evidence of mutagenic activity was seen with any other strains.

               In the second assay performed using the pre-incubation method, a biologically significant increase in the number of revertants was again observed only in strain WP2 uvrA (pKM101) in the presence of S9 mix; A dose-effect relationship was also noted. No evidence of mutagenic activity was seen with any other strains.

               It was thus concluded that magnesium acetylacetonate showed evidence of mutagenic activity in this bacterial system under the test conditions employed.

 

2)     Bacterial Reverse mutation test on the strain of Escherichia coli WP2 uvrA (pKM101) with metabolic activation with pre-incubation using B.N. Ames technique. (Institute Pasteur study FSR-IPL 180201)

 

Purpose of the study: a previous study on the test item Magnesium acetylacetonate demonstrated positive results ion the strain of Escherichia coli WP2 uvrA (pKM 101), in presence of metabolic activation with pre-incubation (ENVIGO study RC45DS). This additional mutagenicity test aims at confirming the result of the previous test and to provide data which can explain the mechanism of the genotoxicity;

The test item used for this test is magnesium acetylacetonate, batch number: 17082101, with a purity of 98% mg content of 9,14% .

In accordance with a previous study performed with magnesium acetylacetonate dihydrate, the test item was dissolved in dimethylsulfoxide (DMSO) at a maximal initial concentration of 100 mg/ml in order to obtain the top dose of 5000 µg/plate when added at 50µL/plate.

The test item was also diluted in sterile water. The top dose of 5000µL/plate was reached when used at 500µL/plate.

               Results:

The search for any mutagenic activity of Magnesium acetylacetonate dihydrate (Batch 17082101) was studied by means of the Ames’ test (Salmonella his-/microsome system) in compliance with the Commission Regulation EC 440/2008 and the OECD Guideline 471 (1997), in only one assay in strain Escherichia coli Trp- WP2 uvrA (pKM101) in presence of S9-mix with pre-incubation, without repetition.

Moreover, in order to test the hypotheses of an instability of the test item in the test system and the implication of the release of magnesium in the positive response obtained in a previous study, the experiment was performed with the test item concurrently to Acetylacetone (according to the Amendment No.1).

Otherwise, the interference due to the nature of the solvent was also checked. To reach this goal, Magnesium acetylacetonate dihydrate and Acetylacetonate were prepared in DMSO and sterile water. Interestingly, the maximum dose of Magnesium acetylacetonate dihydrate and Acetylacetonate was the one recommended by OECD guideline 471 (1997), i.e. 5000μg/plate. Seven lower doses were also tested.

In the single assay performed in the presence of Magnesium acetylacetonate dihydrate prepared in DMSO, no biologically or statistically significant increases in the mean number of revertants were noted.

The positive results obtained in a previous study were thus not confirmed under our experimental conditions.

Moreover, when tested in solution in sterile water, no biologically or statistically significant increases in the mean number of revertants were noted either.

The test item Magnesium acetylacetonate dihydrate was thus considered as not mutagenic in these conditions.

 

3)     In Vitro Mutation Test using Mouse Lymphoma L5178Y Cells (ENVIGO study number XM48LY)

 

Magnesium Acetylacetonate / N56 was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

The study consisted of a preliminary toxicity test and three independent mutagenicity assays.

The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

The test item used is magnesium acetylacetonate batch number 17030101, 98 % of purity, Magnesium content of 9,14%.

Magnesium acetylacetonate was dissolved in DMSO. A final concentration of 2000 mg/mL, dosed at 1%v/v, was used as the maximum concentration in the preliminary toxicity test. This is the standard limit concentration within this test system as recommended in the regulatory guidelines.

Following a 3-hour exposure to Magnesium Acetylacetonate / N56 at concentrations from 3.91 to 2000 mg/mL, no precipitate was observed at the end of treatment and relative suspension growth (RSG) was reduced from 106 to 9% and from 112 to 16% in the absence and presence of S9 mix respectively. Following a 24-hour exposure in the absence of S9 mix

RSG was reduced from 111 to 4%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG).

Following 3-hour treatment in the absence of S9 mix, the test item caused increases in mean mutant frequency above the mean concurrent vehicle control mutant frequency which exceeded the Global Evaluation Factor (GEF) at 750 and 1000μg/mL, with the RTG being reduced to 36 and 19% respectively. The increases were associated with a statistically significant linear trend.

Following 3-hour treatment in the presence of S9 mix, the test item caused increases in mean mutant frequency above the mean concurrent vehicle control mutant frequency which exceeded the GEF at 750, 1000 and 1500μg/mL, with the RTG being reduced to 57, 30 and 10% respectively. The increases were associated with a statistically significant linear trend.

In the 24-hour treatment, the maximum concentration tested was 500μg/mL. In light of the unequivocally positive results following the 3-hour treatments the plates prepared following 24-hour treatment were not analysed to determine mutant frequency.

In all tests the concurrent vehicle and positive control were within acceptable ranges.

It was concluded that Magnesium Acetylacetonate / N56 demonstrated mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

 

4)     In vitro mammalian Chromosome Aberration test in Human Lymphocytes. (ENVIGO study XK28XY)

A study was performed to assess the ability of Magnesium Acetylacetonate / N56 to cause structural chromosome aberrations in human lymphocytes cultured in vitro. Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin (PHA), and exposed to the test item both in the absence and presence of exogenous metabolic activation (S9 mix). Vehicle and positive control cultures were also included where appropriate. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

The study consisted of a preliminary toxicity test and a main test. In both types of tests the cells were treated for 3 and 21 hours in the absence of S9 mix and for 3 hours in the presence of S9 mix. Additional main tests were performed for 21 hours in the absence of S9 mix and 3 hours in the presence of S9 mix. The mitotic index was assessed for all cultures to determine cytotoxicity. Based on the data from the preliminary toxicity test, test item concentrations were selected for the main test. In the main test, justification for the highest analyzed concentration was based on cytotoxicity.

The test item used was magnesium acetylacetonate batch No 17030101, purity of 98% and magnesium content of 9,14%.

The following test item concentrations were selected for metaphase analysis:

In the absence of S9 mix, 3-hour treatment: 250, 1250 and 1500μg/mL.

In the presence of S9 mix, 3-hour treatment: 100, 200 and 500μg/mL.

 

In the absence of S9 mix following 3-hour treatment, Magnesium Acetylacetonate / N56 caused statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at 1250 and 1500μg/mL (excluding gaps) when compared with the vehicle control that were associated with a linear dose-concentration relationship. The mean proportion of cells with chromosomal aberrations in test item treated cultures was above the laboratory historical 95% confidence limits.

In the presence of S9 mix following 3-hour treatment, Magnesium Acetylacetonate / N56 caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any analyzed concentration, when compared with the vehicle control and there was no evidence of a linear dose-concentration relationship. The mean proportion of cells with chromosomal aberrations in test item treated cultures at 200 and 500µg/mL were outside the laboratory historical 95% confidence limits.

No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, under any treatment condition, when compared with the vehicle control.

As a clear statistically significant response was displayed at analyzed Magnesium Acetylacetonate / N56 treatment concentrations in the absence of S9 mix following 3-hour treatment, with mean values which clearly exceeded the laboratory historical control range, no further metaphase analysis (of cultures treated for 21 hours in the absence of S9 mix) was conducted.

Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

It is concluded that Magnesium Acetylacetonate / N56 has shown evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

 

5) In Vivo Mammalian Erythrocytes Micronucleus Test Performed in Rat Bone MarrowCombined to THE In Vivo Mammalian Alkaline Comet Assay on Lung and Liver. (Institute Pasteur de Lille, StudyFSR-IPL 180502)

 

Purpose of the study: as the results of the in vitro genotoxicity studies were ambiguous even contradictory, it was decided to complete the genotoxicity of magnesium dihydrate by carrying out an in vivo genotoxicity study by combining a micronucleus test in bone marrow with a 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.

The study consisted of a toxicity assay including both a preliminary toxicity assay and a confirmation toxicity assay, and a main test.

Toxicity assay:

Preliminary test:

ü Number of animals per group: 2 males and 2 females

ü Doses tested: 7.5 mg/kg/day (x 3)

ü 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.

 

Confirmation toxicity assay:

 

ü Number of animals per group: 5 males and 5 females per group (i.e. vehicle control and 7.5 mg/kg treated group)

ü Doses tested: 7.5 mg/kg/day (x 3)

ü 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:

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

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

ü Treatment schedule: 3 daily treatments at 24-hour intervals

ü Number of sampling times: one:

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

o  for the positive control group (cyclophosphamide): 24 hours after the single treatment (micronucleus test)

o  for the positive control group (methylmethane sulfonate): 2-3 hours after the third treatment (comet assay)

ü Positive reference substances:

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

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

 

Micronucleus 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.

Considering 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.

Considering 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.

 

Overall conclusion of the genotoxicity 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.

 

Discussion:

 

In final five genotoxicity tests have been carried out.

 

ü Two Ames tests,

o  Test 1: the low level of increase in revertant colonies associated with the fact that there is no clear dose-response relationship since the number of revertants decreases after 500 µg/plate, suggests that the result in favor of genotoxicity under the conditions of this test is not obvious.

o  In order to remove the doubt, we performed a second AMES test (Institute Pasteur study FSR-IPL 180291) performed but this time only on EC strains. In this second test the results are clearly negative.

 

ü In Vitro Mutation Test using Mouse Lymphoma L5178Y Cells

o  It was concluded in the report that Magnesium Acetylacetonate / N56 demonstrated mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

o  Comments on this report:

§ The biologically significant response is observed from high concentration of 500µg/ml.

§ Strangely, in the absence of metabolic activation, the number of large colonies (LC rather indicative of mutational events) is greater than the number of small colonies.

§ Conversely, in the presence of metabolic activation the small colonies are greater than the large colonies. (Small colonies are rather indicative of clastogenic events).(And this effect has not been observed in the chromosomal aberration test with metabolic activation).

 

ü In vitro mammalian Chromosome Aberration test in Human Lymphocytes

o  It was concluded in the report that Magnesium Acetylacetonate / N56 has shown evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

o  Comments on this report:

§ The dose-response relationship is not consistent with that expected for a known genotoxic substance

§ The statistic significant effect is exclusively due to the increase in the chromatid break and not to other aberrations.

§ There is no exchange-type lesion characteristic of a real clastogenic activity.

 

ü An in vivo genotoxicity test with a micronucleus in bone marrow combined with a comet assay in lung and liver in rat.

o  Magnesium acetylacetonate dihydrate induced no in vivo genotoxic activity under these experimental conditions.

 

Justification for classification or no classification:

 

Based on the entire database of genetic toxicity studies it is concluded that in summary, magnesium dihydrate do not appear to be a genotoxic substance.

 

Based on the above information, the classification criteria for germ cell mutagenicity according to regulation (EC) 1272/2008 are not met, thus no classification required.

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 system

Test concentrations with justification for top dose:

5 µg
15 µg
50 µg
150 µg
500 µg
1500 µg
5000 µg
No evidence of toxicity was obtained following exposure to the test substance. A maximum exposure concentration of 5000 µg/plate was, therefore, selected.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: the solubility of the test substance was assessed at 50mg/ml in water, DMSO, ethanol and acetone. It dissolved in DMSO, which was therefore used as the vehicle for this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene, 5 µg/plate for strains TA100 and 10 µg/plate for strain WP2 uvrA (pKM101), in the presence of S9 mix.

Details on test system and experimental conditions:

First Test
Aliquots of 0.1 mL of the test item solutions (seven concentrations up to 5000 µg/plate), positive control or vehicle control were placed in glass tubes. The vehicle control was DMSO. S9 mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 34 to 39C for 48 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).
Any toxic effects of the test item may be detected by a reduction in mean revertant colony numbers to ≤50% of the concurrent vehicle control count, by a sparse or absent background bacterial lawn, or both. In the absence of any toxic effects, the maximum concentration selected for use in the second test is the same as that used in the first. If toxic effects are observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate is observed on the plates at the end of the incubation period, at least four non-precipitating concentrations should be included in the second test, unless otherwise justified by the Study Director.

Second Test
A variation to the test procedure was used for the second test; the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 34 to 39°C for 30 minutes with shaking before the addition of the agar overlay was performed. The maximum concentration chosen was again 5000 µg/plate.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 500µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Executive summary:

The in vitro gene mutation study in bacteria test was performed according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay)

The test showed evidence of mutagenic activity.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase locus (TK+/-)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The test substance was found to be soluble at 200 mg/mL in dimethyl sulphoxide (DMSO). A final concentration of 2000 g/mL, dosed at 1%v/v, was used as the maximum concentration in the preliminary toxicity test. This is the standard limit concentration within this test system as recommended in the regulatory guidelines.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

1) 3-hour Treatment in the Absence and Presence of S9 Mix
The procedure for preparing the cell suspension was the same as for the preliminary toxicity test. Cultures contained a total of 1.2 x 10^7 cells in a final volume of 20 mL. The final concentration of the S9 fraction was 2% v/v, if present. Duplicate cultures were prepared throughout for each concentration of test item and positive control. Quadruplicate cultureswere prepared for vehicle controls. Aliquots of 200 µL of test item dilution (at 100 times the desired final concentration), vehicle or positive control were added, and then all cultures were incubated, with continuous shaking, for 3 hours at 34 to 39C. At least four serial dilutions of the test item were tested.
Following the 3-hour treatment, the cells were washed once, re-suspended in R10p to nominally 2 x 10^5 cells/mL (assuming no cell loss) and incubated for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary. After 48 hours cultures with a density of more than 1 x 105 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cultures that were assessed were chosen at the Study Director’s
discretion. Cloning efficiency was assessed by plating 1.6 cells/well, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 10^3 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 34 to 39°C in an atmosphere of 5% CO2 in air.
After the plates had been incubated for 10 to 12 days, the number of empty wells was assessed for each 96-well plate (P0). P0 was used to calculate the cloning efficiency (CE) and mutant frequency (MF). The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants. The maximum concentrations assessed for mutant frequency in the main test were 1000 and 1500 µg/mL in the absence and presence of S9 mix respectively.

2) 24-hour Treatment in the Absence of S9 Mix
A test with a 24-hour treatment in the absence of S9 mix was carried out. Duplicate 20 mL cultures containing 6 x 10^6 cells were treated for 24 hours with 200 µL of test item or positive control. Quadruplicate cultures were prepared for vehicle controls. At the end of the exposure period, the cells were washed once, re-suspended in 20 mL R10p and counted to ascertain treatment growth. The cultures were then diluted to 2 x 10^5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension.
After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary. Following this, the procedure was the same as in the 3-hour treatment.

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Main mutation test - 3 hours t reatment in the presence and absence of S9mix

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Executive summary:

The in vitro mammalian chromosome aberration test was performed according to the OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

The test showed evidence of mutagenic activity.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Test concentrations with justification for top dose:

The test item was found to be soluble at 200 mg/mL which provided a maximum final concentration of 2000µg/mL when dosed at 1% (v/v).
Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, the osmolality of the test item in medium was tested at 2000 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control. No fluctuations in pH of the medium were observed at 2000 µg/mL of more than 1.0 unit compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 2000 µg/mL as this is the standard limit concentration within this test system as recommended in the regulatory guidelines.
Magnesium Acetylacetonate was formulated and diluted in DMSO (ACS reagent grade), shortly before dosing. The final volume of DMSO added to the cultures was 1% v/v.
All concentrations cited are expressed in terms of Magnesium Acetylacetonate / N56 as received.
Preliminary toxicity test: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
Main tests: -S9 mix (3 hours) 250, 1250 and 1500 µg/mL
+S9 mix (3 hours) 100, 200 and 500 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Executive summary:

The In Vitro Mammalian Chromosome Aberration Test in Human Lymphocytes test was performed according to the OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test).

It is concluded that the test item has shown evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A previous study on the test item Magnesium acetylacetonate dihydrate demonstrated positive results in the strain of Escherichia coli WP2 uvrA (pKM101) in presence of metabolic activation with preincubation
only.
The present study aimed at confirming or not such results.
Moreover, in order to test the hypotheses of the chemical instability of the test item in the solvent and the implication of the possible release of magnesium in the positive response, the experiment was performed
with the test item concurrently to Acetylacetone. Otherwise, the interference due to the nature of the solvent was checked. Therefore, the treatment preparations of both Magnesium acetylacetonate dihydrate and Acetylacetone were performed in DMSO and in an aqueous solvent. The latter solvent that was compatible with the test systemand allowed reaching a final dose of 5000 μg/plate for both Magnesium acetylacetonate dehydrate and Acetylacetone.
As a negative result was obtained in the 1st step with Magnesium acetylacetonate dehydrate, the hypothetical oxidative pathway was not investigated.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Remarks:

Test done on a single bacterial strain.

Type of assay:

bacterial reverse mutation assay

Target gene:

tpr gene

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with

Test concentrations with justification for top dose:

In accordance with a previous study performed on Magnesium acetylacetonate dihydrate, the test item was dissolved in dimethylsulfoxide (DMSO) at a maximal initial concentration of 100 mg/mL in order to
obtain the top dose of 5000 μg/plate when added at 50 μL/plate (with S9-mix, with pre-incubation).
In accordance with the Final Study Plan and Study Plan Adherence (see § 10), the test item was also prepared in another solvent. The test item was not soluble in ethanol, tetrahydrofuran, acetone, formamide, acetonitrile, cyclohexane and dimethylformamide. In return, Magnesium acetylacetonate dihydrate was freely soluble in sterile water at 10 mg/mL. This aqueous solvent was thus chosen. The top dose of 5000 μg/plate was reached when used at 500 μL/plate.
Lower concentrations were also prepared with DMSO and sterile water by serial dilutions and used at either 50 or 500 μL/plate, respectively.

Acetylacetone was also prepared in the DMSO and sterile water at initial concentrations of 100 and 10 mg/mL, and used at 50 or 500 μL/plate, respectively to reach the top dose of 5000 μg/plate. Lower concentrations were also prepared with DMSO and sterile water by serial dilutions and used at either 50 or 500 μL/plate, respectively.

Vehicle / solvent:

DMSO and sterile water (because those are vehicles in which the test substance was soluble).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

The following solutions were added in this order: the bacterial strain to be tested (100 μL), S9-mix (500 μL; see § 3.4) and at the end the test item solution (appropriate volume, depending on the solvent used). The mixture was preincubated with stirring at ca. 37°C for 60 minutes prior to adding to 2 mL of top agar supplemented with 10 % of 0.5 mM tryptophan solution and 5% of Nutrient broth No. 2, maintained in a state of superfusion at ca. 45°C. The content of each tube was agitated then spread out in a Petri plate containing 20 mL of minimum agar. Three plates were used per dose. The plates were then incubated at ca. 37°C for approximately 44 h*. At the end of the expression time, colonies of revertants were counted for each plate.
*As the plates could not be read immediately, they were kept at ca. +4°C up to 48 hours before reading.

Solvent controls (6 plates per solvent) and assay for the control of media were performed like in the mutagenicity assay.
The positive control was benzo[a]pyrene 2.5 μg/plate.
The test was not repeated.

Species / strain:

E. coli WP2 uvr A pKM 101

Remarks:

Test item: Magnesium acetylacetonate dihydrate, in DMSO and in sterile water

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Remarks:

Test item: acetylacetonate, in DMSO and in sterile water

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Executive summary:

In the endpoint "bacterial reverse mutation test" rised a potential mutagenicity in the strain E. coli WP2 uvr A pKM 101, with metabolic activation.

a second AMES test on this strain, with metobolic activation was performed to confirm or not such results.

Moreover, in order to test the hypotheses of the chemical instability of the test item in the solvent and the implication of the possible release of magnesium in the positive response, the experiment was performed with the test item concurrently to Acetylacetone. Otherwise, the interference due to the nature of the solvent was checked. Therefore, the treatment

preparations of both Magnesium acetylacetonate dihydrate and Acetylacetone were performed in DMSO and in an aqueous solvent. The latter solvent that was compatible with the test system and allowed reaching a final dose of 5000 μg/plate for both Magnesium acetylacetonate dehydrate and Acetylacetone.

The test items Magnesium acetylacetonate dihydrate and acetylacetonate were considered as non mutagenic in DMSO and in sterile water, with metabolic activation. (see genetic toxicity endpoint summary for more explanations).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

5) In Vivo Mammalian Erythrocytes Micronucleus Test Performed in Rat Bone MarrowCombined to THE In Vivo Mammalian Alkaline Comet Assay on Lung and Liver. (Institute Pasteur de Lille, StudyFSR-IPL 180502)

Purpose of the study: as the results of the in vitro genotoxicity studies were ambiguous even contradictory, it was decided to complete the genotoxicity of magnesium dihydrate by carrying out an in vivo genotoxicity study by combining a micronucleus test in bone marrow with a 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.

The study consisted of a toxicity assay including both a preliminary toxicity assay and a confirmation toxicity assay, and a main test.

Toxicity assay:

Preliminary test:

 - Number of animals per group: 2 males and 2 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.

Confirmation 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)

- 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

- 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 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.

Considering 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.

Considering 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.

Overall conclusion of the genotoxicity 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.

Discussion:

In final five genotoxicity tests have been carried out.

- Two Ames tests,

- Test 1: the low level of increase in revertant colonies associated with the fact that there is no clear dose-response relationship since the number of revertants decreases after 500 µg/plate, suggests that the result in favor of genotoxicity under the conditions of this test is not obvious.

- In order to remove the doubt, we performed a second AMES test (Institute Pasteur study FSR-IPL 180291) performed but this time only on EC strains. In this second test the results are clearly negative.

-  In Vitro Mutation Test using Mouse Lymphoma L5178Y Cells

-  It was concluded in the report that Magnesium Acetylacetonate / N56 demonstrated mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

- Comments on this report:

*The biologically significant response is observed from high concentration of 500µg/ml.

*  Strangely, in the absence of metabolic activation, the number of large colonies (LC rather indicative of mutational events) is greater than the number of small colonies.

* Conversely, in the presence of metabolic activation the small colonies are greater than the large colonies. (Small colonies are rather indicative of clastogenic events).(And this effect has not been observed in the chromosomal aberration test with metabolic activation).

-  In vitro mammalian Chromosome Aberration test in Human Lymphocytes

-  It was concluded in the report that Magnesium Acetylacetonate / N56 has shown evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

- Comments on this report:

* The dose-response relationship is not consistent with that expected for a known genotoxic substance

* The statistic significant effect is exclusively due to the increase in the chromatid break and not to other aberrations.

* There is no exchange-type lesion characteristic of a real clastogenic activity.

- An in vivo genotoxicity test with a micronucleus in bone marrow combined with a comet assay in lung and liver in rat.

-  Magnesium acetylacetonate dihydrate induced no in vivo genotoxic activity under these experimental conditions.

Link to relevant study records

Reference

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)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

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

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

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

Dose / conc.:

7.5 mg/kg bw/day

Dose / conc.:

3.75 mg/kg bw/day

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)

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the entire database of genetic toxicity studies it is concluded that in summary, magnesium dihydrate do not appear to be a genotoxic substance.

 

Based on the above information, the classification criteria for germ cell mutagenicity according to regulation (EC) 1272/2008 are not met, thus no classification required.