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Genetic toxicity: in vivo

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Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 August 2022 - 21 November 2022 (only draft report available)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Only draft report available. QA statement is missing. The summary of the study will be updated as soon as the final report is available.
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
other: not rated according to Klimisch et al.
Rationale for reliability incl. deficiencies:
other: Dose-range finding experiment
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
Adopted: 29 July 2016
Deviations:
not applicable
Principles of method if other than guideline:
This dose-range finding study is designed to select dose levels for a GLP-compliant in vivo comet assay (ILS Study Number 50227.00202; please also refer to IUCLID section 7.6.2: k_Streicker_2022_comet assay). Male and female (6 per sex) Sprague Dawley rats received gavages of 1000 or 2000 mg/kg bw/day Cesium Fluoroaluminate N or the vehicle control for two consecutive days. Approximately 3 (± 30 minutes) hours following the final dose administration, animals will be humanely euthanized. The stomach will be opened and evaluate for each animal with no tissues retained. Moreover, in-life observations were conducted, which were mortality/moribundity, clinical observations, cage-side observations, and body weights.
GLP compliance:
no
Remarks:
Dose-range finding experiment
Type of assay:
other: Dose-range finding experiment for OECD TG 489 (mammalian comet assay)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, keep dry
- Stability: Formulations were prepared once for dose administration for the study.
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat is a standard species used for toxicity studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo, an Inotiv Company (Frederick, MD)
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 265.3-278.5 g (males) and 198.1-213.5 g (females)
- Assigned to test groups randomly: yes
- Housing: 2 rats per cage in 23 cm wide by 44 cm long (1012 cm² area) and 21 cm high polycarbonate cages with micro-isolator tops; bedding: absorbent heat-treated hardwood bedding (Northeastern Products Corp., Warrensburg, NY, USA)
- Enrichment: Polycarbonate tubes (Bio Serv, Flemington, NJ) and/or Envrio-Dri (Shepard Speciality Papers, Watertown, TN)
- Cage Changes: twice per week
- Diet: Certified Purina Pico Chow No. 5001 (Ralston Purina Co., St. Louis, MO); ad libitum
- Water: Reverse osmosis treated tap water (City of Durham, NC) in polycarbonate bottles with stainless steel sipper tubes (water bottles changed once per week); ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-25°C
- Humidity: 30-70%
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 05 July 2022 To: 14 July 2022
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: deionised water
- Source: Inotiv DI System
- Concentration of test material in vehicle: 100 and 200 mg/mL
- Dose volume: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared once for dose administration for the study. Test article dose formulations were weighed out into a pre-calibrated bottle, then vehicle was added to the pre-calibrated mark and mixed using a magnetic stir bar for the desired concentration. Dose formulations were stored at room temperature and kept dry. The dosing formulations were stirred continuously on a stir plate during dose administration.

RATIONALE FOR ROUTE OF ADMINISTRATION:
The oral route of administration will be used to obtain systemic exposure and is the intended human route of administration.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Daily administrations on consecutive days
Post exposure period:
3 hours ± 30 minutes
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3 rats per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
none (not necassary)
Tissues and cell types examined:
Examinations restricted to in-life animal observations (mortality/moribundity, clinical observations, cage-side observations, and body weights)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose levels were chosen to achieve the 2000 mg/kg bw limit dose as required by the current test guideline or achieve a maximum tolerated dose.

TISSUE PREPARATION:
The stomach was opened and evaluated for each animal.

IN-LIFE OBSERVATION:
- Observation of mortality/moribundity: Twice daily
- Clinical Observations: Observed for assignment to a dose group, daily prior to administration, and at termination.
- Cage-Side Observations: Observed 1 hour ± 30 minutes after dosing each day.
- Body Weights: Measured for assignment to a dose group, prior to administration, and at termination.
Evaluation criteria:
Effects on in-life observation will be evaluated in order to determine appropriate doses for the subsequent main study. The maximum dose in the main study will be the recommended limit dose of 2000 mg/kg bw or the maximum tolerable dose.
Statistics:
No statistical tests were performed.
Sex:
male/female
Genotoxicity:
not determined
Toxicity:
yes
Vehicle controls validity:
not applicable
Negative controls validity:
not examined
Positive controls validity:
not examined
Remarks on result:
other: This dose-range finding experiment was not designed for genotoxicity evaluations.
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Males and females of both sexes survived to termination with very slight body weight loss (-6.1 and -7.7%, respectively; please refer to "Attachments": Results_Body weight) and minor clinical signs of toxicity (including piloerection and chromodacryorrhea; please refer to "Attachments": Results_Clinical observations). Whitish fluid was present in the stomach of the high dose animals and is presumed to be test article preparation.
Conclusions:
Males and females of both sexes survived to termination with very slight body weight loss (-6.1 and -7.7%, respectively) and minor clinical signs of toxicity (including piloerection and chromodacryorrhea). Thus, a dose of 2000 mg/kg bw/day was chosen as limit dose based on the recommendations of OECD TG 489 (2016). As no gender differences in toxicity have been identified study was conducted solely in male animals.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
Adopted: 29 July 2016
Deviations:
no
GLP compliance:
yes
Remarks:
QA statement will be reported as soon as the final report is available
Type of assay:
mammalian comet assay

Test material

Constituent 1
Reference substance name:
Cesium fluoroaluminate
EC Number:
604-086-6
Cas Number:
138577-01-2
IUPAC Name:
Cesium fluoroaluminate
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in report): Cesium Fluoroaluminate N
- CAS Number: 138577-01-2
- EC Number: 604-086-6
- Source: Honeywell Specialty Chemicals Seelze GmbH (Germany)
- ILS Repository Number: 22-86
- Description: Solid, white powder (UVCB)
- Correction Factor: The formulations were not corrected for purity.
- Water solubility: 19.1 g/L (22℃)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, keep dry
- Stability: Formulations were prepared daily for dose administration for the study. Test article formulations in the vehicle were validated out to 30 days. (Impact Analytical R220823-1r0).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat is a standard and accepted species for toxicity studies. Moreover, the rat has been extensively validated for the in vivo comet assay (OECD TG 489).
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo, an Inotiv Company (Frederick, MD, USA)
- Age at administration: 7-10 weeks of age
- Weight at administration: 274.3-310.4 g
- Assigned to test groups randomly: yes, under following basis: The animals were assigned to a dose group using a procedure that stratified animals across groups by body weight, such that the mean body weight of each group was not statistically different from any other group using analysis of variance (ANOVA). Only clinically healthy animals were assigned to the study.
- Housing: 2-3 rats per cage in 23 cm wide by 44 cm long (1012 cm² area) and 21 cm high polycarbonate cages with micro-isolator tops; bedding: absorbent heat-treated hardwood bedding (Northeastern Products Corp., Warrensburg, NY, USA)
- Enrichment: Animals were provided items such as treats, shelter, or nesting material except when interrupted by study activities.
- Cage Changes: twice per week
- Diet: certified Rodent Diet 5002 pellets (Ralston Purina Co., St. Louis, MO, USA); ad libitum
- Water: reverse osmosis treated tap water (City of Durham, NC, USA) in polycarbonate bottles with stainless steel sipper tubes (water bottles changed once per week); ad libitum
- Acclimation period: ≥ 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21.4-24.0°C
- Humidity: 42.90-53.40%
- Air changes: 12-20 changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 22 August 2022 To: 01 September 2022

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: deionised water
- Source: Sigma-Aldrich
- Concentration of test material in vehicle: 50, 100, and 200 mg/kg bw/day
- Dose volume: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared daily for dose administration for the study. Test article dose formulations were weighed out into a pre-calibrated bottle, then vehicle was added to the pre-calibrated mark and mixed using a magnetic stir bar for the desired concentration. Formulations were then split and vialed for daily dosing. Dose formulations were stored at room temperature and kept dry. Dose formulations were placed on a stir plate ≥ 30 minutes before dosing and continuously stirred if necessary.

RATIONALE FOR ROUTE OF ADMINISTRATION:
The oral route of administration will be used to obtain systemic exposure and is the intended human route of administration.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Daily administrations on consecutive days
Post exposure period:
3 hours ± 30 minutes
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 male rats per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
ethylmethanesulphonate
- Justification for choice of positive control(s): For the positive control, previous use has shown that an EMS dose level of 150 mg/kg bw/day for 2 days induces DNA damage in mice and rats without producing adverse signs of clinical toxicity.
- Route of administration: oral: gavage
- Doses / concentration: 15 mg/mL / 150 mg/kg bw/day

Examinations

Tissues and cell types examined:
single cells of the stomach (site of contact tissue; n=150 cells per animal), duodenum (site of contact tissue; n=150 cells per animal) and liver (n=150 cells per animal)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The selected dose levels were based on the results of a range finder study (ILS Study Number 50227.00202RF) in which male and female rats received up to 2000 mg/kg bw/day of the test article for 2 days.
Males and females of both sexes survived to termination with very slight body weight loss (-6.1 and -7.7%, respectively) and minor clinical signs of toxicity (including piloerection and chromodacryorrhea). Thus, a dose of 2000 mg/kg bw/day was chosen as limit dose based on the recommendations of OECD TG 489. As no gender differences in toxicity have been identified study was conducted solely in male animals

TREATMENT AND SAMPLING TIMES:
Twenty-five (25) male Sprague Dawley rats were allocated to one of 5 designated groups. For 2 consecutive days, the vehicle (deionised water) or one of 3 dose levels of the test article was administered once daily to the animals using oral gavage. Positive control animals were administered EMS once daily for 2 days, using oral gavage. Animals were not fasted. All animals were humanely euthanized 3 hours ± 30 minutes after the final dose administration. Liver, glandular stomach, and duodenum tissue were collected for evaluating DNA damage with the comet assay and for potential histopathology. Blood was collected for potential bioanalytical evaluation. The remainder of the carcass was appropriately disposed of without further analysis.

TISSUE COLLECTION:
Following exsanguination, portions of the liver, stomach, and duodenum were collected for potential analysis.
- The left lobe of the liver was cut longitudinally into two large sections. Two small sections were further cut from one large section and kept cold and moist with mincing solution for comet analysis. Each small section of liver tissue was placed in a separate microcentrifuge tube containing 1 mL of mincing solution (Mg and Ca free Hanks Balanced Salt Solution, 10% v/v DMSO, and 20 mM EDTA pH 7.4-7.7) and rapidly minced to generate duplicate samples. The samples were kept on wet ice until processed. The second large section of the left lobe was fixed in 10% neutral buffered formalin (NBF), trimmed, and paraffin-embedded for possible histopathology evaluation. A sample of the left lobe was stored frozen in cryotubes at ˗80°C for potential bioanalytical evaluation. The other lobes of the liver tissue were discarded.
- The stomach was removed, cut open, and washed free from stomach contents using cold mincing buffer. Presence of residual test item in glandular stomach was recorded. The forestomach was removed. A 5 mm wide strip of glandular stomach was fixed in 10% NBF, trimmed, and paraffin-embedded for histopathology evaluation. A portion of each strip of the glandular stomach was placed into cold mincing buffer and incubated on ice for 15 to 30 minutes. After incubation, the surface epithelia were gently scraped two times using a scalpel blade or a Teflon scraper. This layer was discarded, and the gastric mucosa rinsed with the cold mincing buffer. The stomach epithelia were carefully scraped 4 to 5 times (or more, if necessary) in 500 µL mincing solution with the back of a scalpel blade or Teflon scraper to release the cells. The mincing solution containing released epithelial cells was divided into two microfuge tubes and placed on ice.
- The duodenum was removed, cut open, and flushed with mincing solution to remove any intestinal contents. Presence of residual test item in duodenum was recorded. A 5-mm length of the proximal duodenum (region closest to stomach) was further cut from which two small sections were placed into microcentrifuge tubes containing 1 mL of mincing solution and rapidly minced while kept on wet ice. An additional 5 mm length of duodenum was fixed in 10% NBF, trimmed, and paraffin-embedded for potential histopathology evaluation. The remaining portions of the small intestine were discarded.
All tubes containing tissue samples were flash frozen in liquid nitrogen and stored in a ˗80°C freezer until processing.

DETAILS OF SLIDE PREPARATION:
Frozen tissue samples (one of the duplicate tubes/animal) were removed from the freezer, thawed appropriately, and kept cold during processing. A portion of the cell suspension of each tissue sample was empirically diluted with 0.5% NuSieve GTG low melting point agarose (Lonza, Durham, NC, USA) dissolved in phosphate buffer (Ca, Mg and phenol free) at 37±2°C and layered onto at least two commercially available CometSlides™ or Flare™ slides (R&D Systems, Minneapolis, MN, USA). The volume of the cell suspension did not decrease the percentage of low melting point agarose by more than 10% (i.e., not below 0.45%). The slides were immersed in chilled lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10, with 10% DMSO and 1% Triton X-100 added fresh) overnight in a refrigerator under light-proof conditions. After this incubation period, the slides were rinsed in purified water or neutralization solution [0.4 M Trizma base (pH 7.5)] to remove residual detergent and salts prior to the alkali unwinding step. Slides were randomly placed onto the platform of a submarine-type electrophoresis unit and cold electrophoresis solution (300 mM NaOH, 1 mM Na2EDTA; pH >13) added. The slides were incubated at ≤10°C for 20–25 minutes to allow DNA unwinding, then electrophoresed at ≤10°C for 20 minutes at 25 V (0.7-1.0 V/cm), with a current of approximately 300 mA. After electrophoresis, slides were neutralised with 0.4 M Trizma base (pH 7.5) for ≥5 minutes and then dehydrated by immersion in absolute ethanol (≥99.6%) for ≥5 minutes and allowed to air dry. Air-dried slides were stored in a desiccator at room temperature with a relative humidity of ≤60% until they were stained and scored. Stained slides were stored desiccated.

METHOD OF ANALYSIS:
After staining slides (200 µL of stain per slide and stained for at least 20 minutes before scoring) with SYBR™ Gold, 150 cells (50-75 cells per slide well, if possible) were scored according to SOP at 200x magnification per sample. Slides were coded and scored without knowledge of their identity. The extent of DNA migration, measured as the %Tail DNA, was detailed by animal and treatment group. “Hedgehogs” (i.e., small or no visible head or the head and tail appear separate) were tabulated to determine a frequency, but not scored for migrated DNA. For each animal, the median value calculated from the cells scored in each well was averaged and presented as the individual animal value. The arithmetic mean of the individual animal means was determined to obtain the group mean, which is the basis for the statistical comparison.

OTHER:
- Observation of mortality/moribundity: Twice daily
- Clinical Observations: Observed for assignment to a dose group, prior to administration, and at termination.
- Cage-Side Observations: Observed 1 hour ± 30 minutes after dosing each day.
- Body Weights: Measured for assignment to a dose group, prior to administration, and at termination.
- Plasma Collection: At termination, all animals in the vehicle and test article dose groups had ~0.5 mL blood collected by puncturing the heart or caudal vena cava. Blood was placed into a K2EDTA tube, centrifuged at 1500x g for 15 min at 4°C, and the plasma removed. Plasma was stored frozen in cryotubes at -80°C for potential bioanalytical evaluation.
- Dose formulation analysis: The testing was completed using inductively coupled plasma – optical emission spectrometry (ICP-OES) to measure total
cesium and aluminum. A long-term stability study was also completed concurrently with the dose formulation analysis. A one-month time point was added to the stability study previously performed during the method validation testing.
Evaluation criteria:
- Negative: The test article as considered clearly negative and did not induce DNA strand breakage in the tissue(s) evaluated if:
1) no dose level exhibited a statistically significant increase in %Tail DNA compared to the concurrent negative control group;
2) there was no dose-related increase when evaluated with an appropriate trend test;
3) all results were within the 95% confidence limits of the distribution of the laboratory’s historical negative control data; and
4) direct or indirect evidence occurred to support exposure or toxicity to the tissue(s) evaluated.
When all of these criteria are met, the test article is then considered able to induce DNA strand breakage in the tissues studied in this test system.

- Positive: The test article as considered clearly positive and induced DNA strand breakage in the tissue(s) evaluated if:
1) at least 1 dose level exhibited a statistically significant increase in %Tail DNA compared to the concurrent negative control group;
2) an increase was dose-related when evaluated with an appropriate trend test; and
3) any results were outside the 95% confidence limits of the distribution of the historical negative control data.

- If the response was not clearly negative or positive, expert judgment or further evaluation may have been warranted to aid in establishing biological relevance of the result. The outcome may have been considered equivocal if the data precluded forming a conclusion regarding the response in the assay to the test chemical.
Statistics:
First, homogeneity of variance was analysed using Levene’s Test of Homogeneity of Variances and normality was assessed using a Shapiro-Wilk Test. Data that met requirements for homogeneity and normality (p > 0.05 in the respective tests) was analysed for linear trend and treated groups were compared to the concurrent vehicle control group using a one-way ANOVA with a Dunnett’s test.
Non-homogeneous or non-normal data were transformed (log, square root, and inverse transformations, in this order of priority, as appropriate) and re-evaluated for homogeneity and normality. If transformation did not successfully achieve both homogeneity and normality, then non-parametric analyses were conducted comprising a Jonckheere-Terpstra Test for a dose-response trend, and Dunn’s Test for pairwise comparisons of treated groups to the concurrent vehicle control group.
The positive control group was compared to the concurrent vehicle control group using a t-test, provided normality of the data was confirmed by a Shapiro-Wilk Test. If data transformation (log, square root, or inverse) did not successfully achieve normality, then the non-parametric Mann-Whitney (also known as Wilcoxon Rank Sum) test was used.
One-sided tests were used to evaluate for differences in %Tail DNA; two-sided tests were used to evaluate changes in body weight and body weight gain.
Results are deemed significant when p ≤ 0.05

Results and discussion

Test resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
stomach
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
duodenum
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
liver
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- In the dose range finder study (ILS Study Number 50227.00202RF; please also refer to IUCLID section 7.6.2: s_Streicker_2022_DRF comet assay), male and female rats received 1000 and 2000 mg/kg bw/day (recommended maximum dose) of the test article for 2 days.
- Males and females of both sexes survived to termination with very slight body weight loss (-6.1 and -7.7%, respectively) and minor clinical signs of toxicity (including piloerection and chromodacryorrhea). Thus, a dose of 2000 mg/kg bw/day was chosen as limit dose based on the recommendations of OECD TG 489. As no gender differences in toxicity have been identified study was conducted solely in male animals.

RESULTS OF THE DEFINITIVE STUDY
Toxicity
- Mortality and Moribundity: All animals survived to termination.
- Clinical and Cage-side Observations: Clinical signs of toxicity were exhibited throughout all dose groups except the 1000 mg/kg bw/day group. This included one male with fur discoloration and a red nose/snout in the 500 mg/kg bw/day group; two males in the 2000 mg/kg bw/day group with uncoordinated movement and five with decreased movement and piloerection.
- Body Weights: There were statistical decreases in terminal body weight (2000 mg/kg bw/day) and body weight gain (1000 and 2000 mg/kg bw/day) with decreasing dose-dependent trends (please refer to the field ‘Attachments’: Results_Body weight). The high-dose group (2000 mg/kg bw/day) showed a statistically significant decrease in terminal body weight (-9.1%). The body weight gain was decreased by 8.56±8.59 g and 27.44±7.90 g in animals dosed at 1000 and 2000 mg/kg bw/day, respectively.

Comet assay
- Liver: The % Tail DNA was statistically significantly increased in the high-dose group (2000 mg/kg bw/day), when compared to the vehicle control group (please refer to the field ‘Attachments’: Results_Comet assay). Moreover, the increase showed a positive dose-response relationship. However, this positive trend was only due to an increase in the high dose group. The other dose groups showed values clearly below the response observed in the vehicle control group. Moreover, the increase observed in the high-dose group was only marginal (1.3-fold), when compared to the vehicle control group, and all responses observed (0.47-0.73% Tail DNA) were well within the 95% confidence interval of the historical control data base (0-10.8 and 0-9.3% Tail DNA; please refer to the field ‘Attachments’: Historical control data_Comet assay). Thus, the increase in %Tail DNA is considered not to be biologically relevant, and therefore, the outcome of the assay in the liver is considered negative.
- Stomach: The % Tail DNA was statistically significantly increased only in the low-dose group (500 mg/kg bw/day). However, no such increase was observed in the higher dose groups and the response was not dose dependent. Furthermore, the increase was marginal (1.6-fold), when compared to the vehicle control group and all values obtained (1.24-2.40% Tail DNA) were clearly within the 95% confidence interval of the historical control data base (0-20.4% Tail DNA). Thus, the increase in %Tail DNA in the low dose group is considered not to be biologically relevant, and therefore, the outcome of the assay in the stomach is considered negative.
- Duodenum: Groups exposed to Cesium Fluoroaluminate N did not show any statistically significant increase in the % Tail DNA and no positive dose-response relationship. All values observed (0.95-1.46% Tail DNA) were below the concurrent vehicle control group value (1.95% Tail DNA). Thus, the outcome in the duodenum is considered clearly negative,
In summary, the results are considered to be negative for all three tissues. There were no marked increased in the % Hedgehogs.

VALIDITY OF THE ASSAY
- The positive control induced statistically significant increases in % Tail DNA in the liver, stomach, and duodenum (over the current vehicle control group) that were comparable with the laboratory’s historical positive control data.
- The vehicle control data were within or comparable to the laboratory’s historical control data ranges.
Thus, the criteria were for a valid test were met and data were acceptable for inclusion in historical laboratory control data.

- Formulation analysis: The Cesium Fluoroaluminate N dose formulation samples were successfully tested for aluminum and cesium content following the previously validated method. Aluminum and cesium were detected in each dose formulation solution. The analytes of interest were found to be stable in frozen storage for the investigated time frame of one month with variance < 20% between the Day 0 and 1-Month stability sample recoveries.

Applicant's summary and conclusion

Conclusions:
All animals survived to termination. Clinical signs of toxicity were exhibited at the 500 and 2000 mg/kg/day dose groups including fur discoloration, uncoordinated movement, decreased movement, and piloerection. There were statistically significant, dose-dependent decreases in terminal body weight or body weight gain at 1000, and 2000 mg/kg/day. These findings are indicative of systemic exposure to the test article.
There were no statistically significant differences in DNA damage, measured as % Tail DNA, in the duodenum for any group administered the test article as compared to the vehicle controls and there was no evidence of a dose response. There were statistically significant increases in % Tail DNA in the liver (2000 mg/kg/day) and stomach (500 mg/kg/day) as compared to the vehicle controls. The % Tail DNA in the liver showed a statistically positive linear trend. However, the increases in % Tail DNA were only marginal (1.3- and 1.6-fold for liver and stomach, respectively), and the results were well within the laboratory’s historical negative control data (values fell below the mean) for both tissues; for stomach, the response was statistically significantly increased only in the lowest dose group. Exposure of the target tissues is demonstrated, since the stomach and duodenum are direct site-of-contact tissues and the systemic toxicity observed is indirect evidence, which is supportive for exposure of the liver. For these reasons, the statistically positive results are not considered to reflect a biologically meaningful induction of DNA damage and the results of the comet assay are interpreted to be negative for all three tissues. All validity criteria were met. The study was fully compliant with OECD 489 (2016).

The test article, Cesium Fluoroaluminate N, was judged as negative for genotoxicity based on the criteria in the OECD guideline (Test No. 489, 2016) and the lack of a biologically relevant response in the comet assay, when tested up to 2000 mg/kg bw/day (recommended maximum dose level).

Only draft report available. QA statement is missing. The summary of the study will be updated as soon as the final report is available.