Alkanoic acid and polyoxomolybdate borate alkanoate clusters

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 7 July 2021 to 20 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 7 July 2021 to 20 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

CD Sprague Dawley rat

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Saint-Germain-surl’Arbresle, France
- Age at study initiation: young adult 8 weeks old
- Weight at study initiation: between 187 g and 226 g
- Not fasted at the treatment time
- Housing: animals were dispatched in polypropylene cages by random-distribution
- Diet: A04C-10 from SAFE
- Drinking water: Softened by reverse osmosis and filtered on 0.22 μm membrane, provided ad libitum
- Acclimatization period: 7 days, the animals received a clinical examination in order to retain only those which were healthy.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3°C
- Humidity: 30-70 %
- The cages were placed in a ventilated system in the animal room, which was also ventilated.
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: gavage

Vehicle:

corn oil (Sigma, batch MKCM3364 for the preliminary toxicity assays; batch MKCM9808 for the main assay)

Details on exposure:

In the main genotoxicity assay, three emulsions of the test item at the concentrations of 60 - 30 and 15 mg/mL were prepared, giving final doses of 600 - 300 and 150 mg/kg, respectively when administered at 10 mL/kg.

Duration of treatment / exposure:

Treatment took the form of 3 successive administrations at 24-hour intervals by oral route (gavage).
Samples were taken 2 to 6 hours after the last treatment.

Frequency of treatment:

3 successive administrations at 24-hour intervals

Post exposure period:

Samples were taken 2 to 6 hours after the last treatment.

Dose / conc.:

600 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males per dose, 7 males for highest dose

Control animals:

yes, concurrent vehicle

Positive control(s):

5 animals were treated orally with Methylmethane sulfonate (MMS; Aldrich, batch MKCL6261) at the doses of 100 mg/kg b.w. /day (x 2) and 70 mg/ kg b.w. /day PO (x1), 48, 24 hours and 2 to 6 hours before sacrifice, respectively.

Tissues and cell types examined:

liver, glandular stomach and duodenum cells

Details of tissue and slide preparation:

CELL ISOLATION:
A total 5 males of each group were assigned for cell isolation and assessed for DNA fragmentation.
Single cell preparation was done within one hour after animal sacrifice.
A 'V' shaped incision was made from the centre of the lower abdomen to the rib cage. The skin and muscles were removed to reveal the abdominal cavity.
A portion of the liver, glandular stomach, duodenum and gonad was removed and washed in the cold mincing buffer until as much blood as possible has been removed. The portion was minced with a pair of fine scissors to release the cells. The cell suspension was stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected.
Cells were enumerated on a haemocytometer, and sufficient cells to obtain 25± 5 x 103 viable cells per slide were harvested from each cell suspension for proceeding to slides preparation.

DRIED SLIDES PREPARATION (PRE-LAYERING):
Conventional slides were dipped in hot 1.5 % normal melting point agarose in PBS. After gentle removal, the underside of the slides was wiped in order to remove excess agarose. The slides were then laid in a tray on a flat surface to dry.

SLIDE PREPARATION:
Before use, a volume of 85 μL of 0.8% of Normal Agarose (NA) was added on the microscope slide prelayered with 1.5% of NA and then covered with a glass coverslip. Slides were placed at +2-8°C until the agarose layer hardens. The cells of the different doses tested were mixed with 0.5% of Low Melting Point Agarose (LMPA) (75 μL/slide) kept at 37 °C and added on the microscope slide after gently sliding off the coverslip. The slides were then covered with a new glass coverslip, and were placed once again at +2-8°C.
Seven slides per animal were prepared for the Comet assay.

Evaluation criteria:

IMAGE ANALYSIS:
Slides were examined with a 200 x magnification, using a fluorescent microscope (Leica Microsystems SAS - DM 2000, Heerbrugg, Switzerland), equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm, connected through a gated monochrome CCD IEEE1394 FireWire video camera (Allied Vision Technologies), to the Comet Assay IV Image Analysis System, version 4.11 with Windows XP Pro Software (Perceptive Instruments Ltd, Suffolk, UK).
Only the slides from the set processed with a 20-minutes electrophoresis from liver, glandular stomach and duodenum were analysed. Indeed, in accordance with the final study plan, as no decrease in the percentage of DNA in tail was evidenced, the slides from the 40-minute electrophoresis were not analysed.
Moreover, as no primary DNA damage was noted in liver, glandular stomach and duodenum, the assessment of genotoxicity towards gonadal cells was not carried out.
For these groups, three slides were analysed with 50 nuclei per slide randomly scored. Five animals were retained per group, i.e. 15 slides per group, 750 analysed nuclei per group.

EXPRESSION OF THE RESULTS:
The results obtained in the different treatments are presented giving:
- the median per slide of the percentage of DNA in tail for at least 50 cells
- the mean of medians of the percentage of DNA in tail per animal (i.e. 3 slides, 150 cells)
- the mean of median per concentration (i.e. 5 animals, 750 cells)
In addition, each slide was also examined for presence of hedgehogs (possible indicator of toxicity and or apoptosis). Ghost cells were excluded from image analysis data collection. However, determining their frequency might be useful for data interpretation. Therefore, the percentage of hedgehogs was recorded for each slide per animal and per organ actually studied.

Statistics:

In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software GraphPad Version 3.10.
As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian distribution (E. Bauer et al., 1998), the non-parametric, one-way Kruskall-Wallis test was performed. This method is based on the analysis of variance by ranks for testing equality of population medians among groups.
The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the vehicle control in order to determine statistical significance of differences in group median values between each group versus the vehicle control. This test was also used to compare vehicle control and positive control to
determine acceptable criteria of a valid test.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

liver from rats

Toxicity:

yes

Remarks:

clinical signs

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

glandular stomach from rats

Toxicity:

yes

Remarks:

clinical signs

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

duodenum from rats

Toxicity:

yes

Remarks:

clinical signs

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CLINICAL SIGNS:
In the main experiment, the dose of 600 mg/kg elicited a slight or a moderate decrease in spontaneous motor activity in 2 and 3 animals, respectively, 15 minutes after the 1st treatment. Between 2 and 4 hours after the 1st treatment slight decrease in spontaneous motor activity was observed in 3 animals.
Fifteen minutes after both the 2nd and the 3rd treatment, a slight decrease in spontaneous motor activity was observed in 1 animal. Noteworthy, this animal was replaced by 1 of the 2 supplementary animals.
In return, no clinical sign was noted at the 2 inferior doses of 300 and 150 mg/kg b.w. /day (x 3).

MAIN ASSAY FOR GENOTOXICITY IN RATS LIVER CELLS:
An increase in the mean of medians of percentage of tail DNA in was noted at the intermediary analysed dose of 300 mg/kg b.w. /day (x 3) with a value of 2.51 vs. 0.36% in the relative control. This value was over the intervals of historical data for the vehicle control (i.e. 1.10). However, the Kruskall-Wallis test was not significant, no statistically relevant effect was noted when using the Mann-Whitney test and no dose effect relationship was observed. Two animals out of 5 demonstrated a percentage of tail DNA over the intervals of historical data: animal No. 1451 and 1455 (Table 15a, Appendix No. 3a) with values of 7.91 and 3.68%.An out of range value of 17.17% for the slide No. 84A of the animal 1451 was noted, explaining the relative high a value for the percentage of DNA in tail for this animal. For the 3 other animals, percentage of DNA in tail were comparable to the mean of medians obtained in the vehicle control with values ranging from 0.25 to 0.38% vs. 0.36%.
From the overall data, the test item was thus considered as not genotoxic in liver from rats.

MAIN ASSAY FOR GENOTOXICITY IN RATS GLANDULAR STOMACH CELLS:
A statistically significant increase in the mean of medians of percentage of tail DNA was observed only at the intermediary analysed dose of 300 mg/kg b.w. /day (x 3), with a value of 3.96% vs. 0.90% for the vehicle control. Despite the Kruskall-Wallis test was significant, as this value was clearly within the intervals of historical data for vehicle control (i.e. 3.75-7.47% for the 95th confidence intervals) and as no clear dose effect relationship was noted, this effect was considered as not biologically relevant and the test item was considered as not genotoxic in glandular stomach from rats.

MAIN ASSAY FOR GENOTOXICITY IN RATS DUODENUM CELLS:
A statistically significant increase in the mean of medians of percentage of tail DNA was observed only at the lowest analysed dose of 150 mg/kg b.w. /day (x 3), vs. with a value of 3.21% vs. 1.42% for the vehicle control. However, the Kruskall-Wallis test was not significant, and this value was clearly within the intervals of historical data for vehicle control (i.e. 1.82-4.74% for the 95th confidence intervals) and no clear dose effect relationship was noted. This effect was considered as not biologically relevant The test item was thus considered as not genotoxic in duodenum from rats.

DETERMINATION OF SYSTEMIC EXPOSURE:

The determination of systemic exposure was not scheduled in a first intent. Indeed, clinical signs were noted at the top dose tested of 600 mg/kg b.w. /day (x 3), demonstrating the systemic exposure of the animals to the test item.

HISTOPATHOLOGICAL STUDY:

As no biologically significant effect was noticed on the 3 target organs, histopathological study was not performed.

Conclusions:

Under the experimental conditions of this OECD 489 study, the test item is considered as not genotoxic in liver, glandular stomach and duodenum from CD Sprague-Dawley rats.

Executive summary:

The search for potential genotoxic activity of test item was investigated for genotoxic potential by the means of the in vivo comet assay under alkaline conditions (SCGE) in the liver, glandular stomach and duodenum in male CD Sprague-Dawley rats, according to OECD Guideline No 489, 2016. Animals were treated orally once a day for 3 consecutive days, 24 hours apart, up to the dose of 600 mg/kg compatible with the toxic activity.

The control of concentrations of test item in treatment preparations was performed in a GLPcompliant laboratory using a validated method. The results were shown to be compliant with the recovery acceptance criteria (85-115% vs. theoretical concentrations). In addition, test item was not detected in the solvent control.

The acceptance criteria for the assay were considered as fulfilled. The current study was valid.

Under the experimental conditions, the test item does not present DNA breaks and/or alkalilabile sties inducer activities toward the liver, glandular stomach and duodenum from male CD Sprague-Dawley rats.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

CD Sprague Dawley rat

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Saint-Germain-surl’Arbresle, France
- Age at study initiation: young adult 8 weeks old
- Weight at study initiation: between 187 g and 226 g
- Not fasted at the treatment time
- Housing: animals were dispatched in polypropylene cages by random-distribution
- Diet: A04C-10 from SAFE
- Drinking water: Softened by reverse osmosis and filtered on 0.22 μm membrane, provided ad libitum
- Acclimatization period: 7 days, the animals received a clinical examination in order to retain only those which were healthy.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3°C
- Humidity: 30-70 %
- The cages were placed in a ventilated system in the animal room, which was also ventilated.
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil (Sigma, batch MKCM3364 for the preliminary toxicity assays; batch MKCM9808 for the main assay)

Details on exposure:

In the main genotoxicity assay, three emulsions of the test item at the concentrations of 60 - 30 and 15 mg/mL were prepared, giving final doses of 600 - 300 and 150 mg/kg, respectively when administered at 10 mL/kg.

Duration of treatment / exposure:

Treatment took the form of 3 successive administrations at 24-hour intervals by oral route (gavage).
Samples were taken 2 to 6 hours after the last treatment.

Frequency of treatment:

3 successive administrations at 24-hour intervals

Post exposure period:

Samples were taken 2 to 6 hours after the last treatment.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

600 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males per dose, 7 males for highest dose

Control animals:

yes, concurrent vehicle

Positive control(s):

5 animals (males) received a single intraperitoneal injection 24 hours before sampling of the reference substance cyclophosphamide (CPA; Baxter, batch 9J125F) at a dose of 25 mg/kg under a volume of 10 mL/kg.

Examinations

Tissues and cell types examined:

bone marrow tissue - polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

At the sampling time, the animals of the Cyclophosphamide control group were sacrificed by CO2 asphyxia; the femurs were removed, and the bone marrow was extracted with foetal calf serum (1 mL per animal).
The animals also used for the comet assay were anaesthetized with isoflurane and exsanguined.
Bone marrows from treated and vehicle control animals were sampled just before the sampling of liver, glandular stomach, duodenum and gonades for the comet assay.
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), which makes it possible to distinguish between immature polychromatic (PCE) and mature normochromatic erythrocytes (NCE): PCE are purple whereas NCE are red.

Evaluation criteria:

SLIDE COUNTING:
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.

EXPRESSION OF RESULTS:
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 1000 polychromatic erythrocytes is also established for both treated and control animals.

Statistics:

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.
The trend test was applied, using the statistical software GraphPad Version 3.10.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CLINICAL SIGNS:
In the main experiment, the dose of 600 mg/kg elicited a slight or a moderate decrease in spontaneous motor activity in 2 and 3 animals, respectively, 15 minutes after the 1st treatment. Between 2 and 4 hours after the 1st treatment slight decrease in spontaneous motor activity was observed in 3 animals.
Fifteen minutes after both the 2nd and the 3rd treatment, a slight decrease in spontaneous motor activity was observed in 1 animal. Noteworthy, this animal was replaced by 1 of the 2 supplementary animals.
In return, no clinical sign was noted at the 2 inferior doses of 300 and 150 mg/kg b.w. /day (x 3).

PCE / NCE RATIO:
The ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE) was established at each dose level. No statistically significant decrease in the ratio PCE to NCE was noted in the 3 treatment groups when compared to the vehicle control group. In consequence, no proof of systemic exposure was evidenced when using this parameter.
Nevertheless, the test item induced clinical signs including decrease in spontaneous motor activity when treated under 600 mg/kg indicating that a systemic exposure probably occurred.

FREQUENCY OF MICRONUCLEATED PCE:
Regarding the frequency of micronucleated polychromatic erythrocytes, no statistically significant increase in the frequencies of micronucleated polychromatic erythrocytes was found in the animals treated with the test item at any dose. Indeed, 0.75 to 0.85 micronucleated PCE/1000 PCE were obtained vs. 0.55 in the relative vehicle control.

Any other information on results incl. tables

Results:

Sampling time (2 - 6 H after last treatment)*	Test item doses in mg/kg bw/day (x3)	Sex	PCE/NCE ratio		Micronuclei for 1000 PCE		Kruskall Wallis
			Mean	Student's t Test (p)	Mean	Mann Whitney (p)
Negative group control	Vehicle 10 mL/Kg	M	3.05		0.55
Positive group control	Cyclophosphamide (10 mL/Kg - IP route) 25 mg/kg/day (x1)	M	1.14	<0.05	21.85	p<0.01
Test item Treated groups	600	M	3.85	N.S.	0.80	N.S.	N.S.
	300	M	3.10	N.S.	0.75	N.S.	N.S.
	150	M	4.10	N.S.	0.85	N.S.	N.S.

* 24H after the sinlge treatment for the positive group

No statistically significant increase in the number of micronuclei was noted at the doses of 600 - 300 - 150 mg/kg bw/day (x3) per os in male rats.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this OECD 474 study, the test item induced no genotoxicity activity in bone marrow cells in male CD Sprague-Dawley rats.

Executive summary:

The search for potential genotoxic activity of test item was investigated for genotoxic potential by the means of the in vivo micronucleus test in bone marrow in male CD Sprague-Dawley rats, according to OECD Guideline No 474, 2016. Animals were treated orally once a day for 3 consecutive days, 24 hours apart, up to the dose of 600 mg/kg compatible with the toxic activity.

The control of concentrations of test item in treatment preparations was performed in a GLPcompliant laboratory using a validated method. The results were shown to be compliant with the recovery acceptance criteria (85-115% vs. theoretical concentrations). In addition, test item was not detected in the solvent control.

The acceptance criteria for the assay were considered as fulfilled. The current study was valid.

Under the experimental conditions, in male CD Sprague-Dawley rats, the test item induced no genotoxic activity in bone marrow cells.