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Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
12 July 2019 - 28 February 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see attachment "Iron oxide category read-across concept-HH " in IUCLID section 13.2.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
2016-07-29
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Diiron trioxide
EC Number:
215-168-2
EC Name:
Diiron trioxide
Cas Number:
1309-37-1
Molecular formula:
Fe2O3
IUPAC Name:
diiron trioxide
Test material form:
solid: nanoform
Details on test material:
Appearance: red powder, odourless
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at 15-25°C, protected from light.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Consequently, all formulations were stored at 15-25°C, protected from light, and used within 2 hours of preparation. The recovery rates for iron in the formulation samples/suspensions provided information about the homogeneity of the test article in the formulations and were in the range of 45% to 147%. It is noticeable that the iron contents of the formulation samples are varying in a relatively wide range

Test animals

Species:
rat
Strain:
other: Sprague Dawley Crl:CD(SD)
Remarks:
out-bred
Details on species / strain selection:
The rat was selected as there is a large volume of background data in this species. As no gender differences in toxicity, metabolism or bioavailability have been previously identified, and gender-specific human exposure was not expected, the study was conducted solely in male animals.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd. (Margate, UK)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 242-282 g (DRF) and 234-276 g (Main Experiment)
- Assigned to test groups randomly: yes. Checks were made to ensure the weight variation of Main Experiment animals prior to dosing was minimal and did not exceed ±20% of the mean weight.
- Fasting period before study: Animals were not fasted prior to test material administration.
- Housing:
- Diet: 5LF2 EU Rodent Diet (analysed for specific constituents and contaminants); ad libitum with the exception of the Main Experiment where the animals underwent a period of fasting (approximately 21 hours) from the evening on Day 1 following dosing until animal necropsy on Day 2
- Water: Mains water (periodically analysed for specific contaminants); ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 40-70%
- Air changes: 15-20 air changes/hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 30 July 2019 To: 18 December 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: hydroxypropyl methylcellulose (medium viscosity) 0.5% (w/v)
- Justification for choice of solvent/vehicle: The vehicle was selected because the test article is not soluble in water or organic solvents.
- Concentration of test material in vehicle: 50, 100, and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were freshly prepared prior to each dosing occasion by formulating Red Ferroxide 212P in HPMC 0.5% (w/v). To ensure homogeneity, and to avoid any interaction of the nanoparticles with the magnetic stirrer, dose formulations were gently inverted prior to dosing.

RATIONALE FOR ROUTE OF ADMINISTRATION:
All treatments were given via oral gavage as this is the intended route of human exposure.
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Two administrations at 0 and 21 (Main Experiment) or 23 hours (DRF)
Post exposure period:
1 (DRF) or 3 hours (Main Experiment)
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
DRF and Main Experiment
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
DRF and Main Experiment
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
DRF and Main Experiment
No. of animals per sex per dose:
6 male rats per dose (Main experiment and 1. DRF); 3 male rats per dose (2. DRF)
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate (EMS, Sigma-Aldrich Chemical Co; Poole, UK)
- Route of administration: single oral administration
- Doses / concentrations: 200 mg/kg (20 mg/mL)

Examinations

Tissues and cell types examined:
single cells of the stomach and duodenum (site of contact tissues)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the lack of adverse effects in a sub-chronic (90 day) oral repeated dose toxicity study in rats up to the limit dose of 1000 mg/kg bw/day, the limit dose of 2000 mg/kg bw/day was selected for this in vivo comet assay, as recommended by the current test guideline. Based on this information, the first Range-Finding Experiment was conducted at 2000 mg/kg/day. However, the nanoparticles were granular and difficult to remove from the tissue at the point of processing impacting on the quality of the comet slides. It was therefore decided to perform additional Range-Finding work at 500, 1000 or 2000 mg/kg/day, along with a concurrent vehicle control group as a comparator in order to further refine the tissue processing and allow comet scoring to take place in order to obtain further data.

TREATMENT AND SAMPLING TIMES:
The test article was administered as two administrations, at 0 and 23 hours in the Range-Finder Experiment. In the Range-Finding Experiments the animals were not fasted. However, the bolus of food and test article present within the animals’ digestive system at the time of necropsy made removing the nanoparticles and processing the tissues challenging. Therefore, the time was extended between the final dose on Day 2 and necropsy for the Main Experiment animals to 0 and 21 hours (i.e from 1 hour from final dose to necropsy, to 3 hours). The EMS positive control was administered once only at 21 hours. All animals were sampled at 24 hours. In order to further reduce nanoparticle contamination of the tissues and subsequent cell suspensions, during the Main Experiment the animals underwent a period of fasting (approximately 21 hours) from the evening on Day 1 following dosing until animal necropsy on Day 2.

DETAILS OF SLIDE PREPARATION:
- Histopathology: Preserved stomach and duodenum samples were embedded in wax blocks and sectioned at 5 μm nominal. Slides were stained with haematoxylin and eosin.

- Comet assay:
Preparation of Cell Suspension:
The comet stomach samples were washed in Merchants solution and then incubated on ice for 15 minutes, covered in fresh Merchants solution. After incubation the stomach samples were removed and placed in 200 μL of fresh Merchants solution. In order to remove as many of the particles as possible, stomach samples from the Range-Finder Experiment and Main Experiment were placed in a honey pot containing 20 mL of fresh Merchants solution and vortex mixed for approximately 15 seconds. Cells were gently scraped from the inside surface of the stomach using the back of a scalpel blade to produce single cell suspensions. Three independently coded slides were prepared per single cell suspension per tissue. Slides were dipped in molten normal melting point agarose (NMA) such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 40 μL of each single cell suspension was added to 400 μL of 0.7% low melting point agarose (LMA) at approximately 37°C. 100 μL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on ice.

Cell Lysis:
Once gelled the coverslips were removed and all slides placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH adjusted to pH 10 with NaOH, 1% Triton X-100, 10% DMSO) overnight at 2-8°C, protected from light.

Unwinding and Electrophoresis:
Following lysis, slides were washed in purified water for 5 minutes, transferred to electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH>13) at 2-8°C and the DNA unwound for 20 minutes. At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 20 minutes. As not all slides could be processed at the same time a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run; i.e. for all animals the same number of triplicate slides was processed at a time.

Neutralisation:
At the end of the electrophoresis period, slides were neutralised in 0.4 M Tris, pH 7.0 (3 x 5 minute washes). After neutralisation the slides were dried and stored at room temperature prior to scoring.

Staining:
Prior to scoring, the slides were stained with 100 μL of 2 μg/mL ethidium bromide and coverslipped.

METHOD OF ANALYSIS:
- Comet:
Slides from the first Range-Finder Experiment were visually assessed to confirm that there were no anomalies which could confound comet scoring in the Main Experiment. However, no formal scoring was conducted. It was noted that extensive background debris was present on the slides. In the following Range-Finder Experiments (2RF-5RF) and the Main Experiment, scoring was carried out using fluorescence microscopy at an appropriate magnification and with suitable filters. All slides were allocated a random code and analysed by an individual not connected with the dosing phase of the study.
In the Main Experiment, scoring was carried out using fluorescence microscopy. A slide from a vehicle and positive control animal were checked for quality and/or response prior to analysis. All slides were allocated a random code and analysed by an individual not connected with soring of the study. All available animals per group were analysed. Measurements of tail intensity (%DNA in tail) and tail moment were obtained from 150 cells/animal/tissue. In general this was evenly split over two or three slides. The number of ‘hedgehogs’ (a morphology indicative of highly damaged cells often associated with severe cytotoxicity, necrosis or apoptosis) observed during comet scoring was recorded for each slide. Each slide was scanned starting to the left of the centre of the slide. After completion of microscopic analysis and decoding of the data the percentage tail intensity (i.e. %DNA in the tail) and Olive tail moment were calculated.

Data were treated as follows:
1. The median value per slide was calculated
2. The mean of the slide medians was calculated to give the mean animal value
3. The mean of the animal means and standard error of the mean was calculated for each group.

- Scoring Criteria for Comet Assay:
The following criteria were used for analysis of slides:
1. Only clearly defined non overlapping cells were scored
2. Hedgehogs were not scored
3. Cells with unusual staining artefacts were not scored.

OTHER:
- Clinical signs and body weight: All animals were examined at the beginning and the end (nominal) of the working day to ensure that they were in good health and displayed no signs of overt toxicity. Individual body weights were recorded on a daily base during the dose phase. Clinical chemistry parameters (aspartate aminotransferase, creatinine, alkaline phosphatase, alanine aminotransferase, potassium, sodium, inorganic phosphorus, calcium, total protein, albumin, globulin, albumin/globulin ratio, total cholesterol, glucose, urea, total bilirubin, and chloride) were assessed from plasma derived from terminal blood samples.
Evaluation criteria:
For valid data, the test article was considered to induce DNA damage if:
1. A least one of the test doses exhibited a statistically significant increase in tail intensity, in any tissue, compared with the concurrent vehicle control
2. The increase was dose-related in any tissue
3. The increase exceeded the laboratory’s historical control data for that tissue.

The test article was considered positive in this assay if both of the above criteria were met.

The test article was considered negative in this assay if neither of the above criteria were met and target tissue exposure was confirmed.

Results which only partially satisfied the criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example comparison of the response against the historical control data and consistency of response within and between dose levels.
Statistics:
In the second DRF and the Main Experiment, tail intensity data for each slide were supplied for statistical analysis. The median of the log-transformed tail intensities from each slide was averaged to give an animal summary statistic. Where the median value on a slide was zero, a small constant (0.0001) was added before taking the logarithm and calculating the average for the animal. This animal average was used in the statistical analysis.

Data was analysed using one-way analysis of variance (ANOVA) with the fixed factor for treatment group. The positive control group was excluded from this analysis. Levene’s test was used to test for equality of variances among groups. This showed no evidence of heterogeneity (P>0.01). Comparisons between each treated group and control were made using Dunnett’s test. The test was one-sided looking for
an increase in response with increasing dose. The back-transformed difference and p-value are reported. In addition, (where appropriate), a linear contrast was used to test for an increasing dose response.

The positive control group was compared to the control group using a two-sample t-test. Levene’s test was used to test for equality of variances between the groups. This showed no evidence of heterogeneity (P>0.01). The test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p-value are reported.

Results and discussion

Test resultsopen allclose all
Sex:
male
Genotoxicity:
negative
Remarks:
stomach
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Remarks:
duodenum
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw/day (1. DRF) and 500, 1000, and 2000 mg/kg bw/day (2. DRF)
- Observations and signs of toxicity: No clinical signs of toxicity and no losses in animal bodyweight were evident. However, it was noted during the necropsy that the nanoparticles were granular and difficult to remove from the tissue at the point of processing. Subsequent visual assessment of the comet slides demonstrated that the quality of the comet slides were suboptimal and difficult to evaluate due to background debris. It was therefore decided to perform additional Range-Finding work (designated 2RF to 5RF) in groups of 3 male animals at 500, 1000 or 2000 mg/kg/day, along with a concurrent vehicle control group as a comparator in order to further refine tissue processing and allow the subsequent slides generated from these animals to be scored.
- Comet analysis: There were no marked, dose-related increases in %hedgehogs in the stomach or duodenum, thus demonstrating that treatment with Red Ferroxide 212P did not cause excessive DNA damage that could have interfered with comet analysis. However, it was noted that the group mean animal tail intensity values at 2000 mg/kg/day in stomach and at 500, 1000 and 2000 mg/kg/day in duodenum were slightly elevated. This was considered to be due to either mechanical damage as a result of over processing of these tissues or artifacts due to residual particulates remaining within the tissue.
Therefore, in order to further reduce nanoparticle contamination of the tissues and subsequent cell suspensions, during the Main Experiment the following experimental design changes were also implemented:
• The time was extended between the final dose on Day 2 and necropsy from 23 hours to 21 hours (i.e from 1 hour from final dose to necropsy, to 3 hours)
• Animals were allowed to undergo a period of fasting (approximately 21 hours) from the evening on Day 1 following dosing until animal necropsy on Day 2 (Groups 1-4 only).
On this basis, the Main Experiment was conducted at doses of 2000, 1000 and 500 mg/kg/day.

RESULTS OF DEFINITIVE STUDY
- Comet Assay:
In the stomach and duodenum, animals treated with Red Ferroxide 212P at all doses exhibited group mean and individual animal tail intensity (0.55-0.84% and 0.13-0.24%, respectively) and tail moment (0.06-0.08 and 0.04-0.06, respectively) values that were similar to the concurrent vehicle control group (stomach: 0.54% and 0.05, respectively; duodenum: 0.66% and 0.06, respectively) and all tail intensity values fell within the laboratory’s historical vehicle control 95% reference range (stomach: 0.16-7.18%; duodenum: 0.18-7.60%). There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control group and no evidence of a dose-response.
Hedgehog occurrence: There were no marked, dose-related increases in %hedgehogs in stomach or duodenum thus demonstrating that treatment with Red Ferroxide 212P did not cause excessive DNA damage that could have interfered with comet analysis.

- Observations and signs of toxicity: No clinical signs of toxicity were observed in any animal following treatments Red Ferroxide 212P. No direct effect of Red Ferroxide 212P treatment on animal body weights was observed. No clinical chemistry changes considered an effect of Red Ferroxide 212P treatment were recorded.

- Histopathology: On macroscopic examination, there were no changes which were considered related to Red Ferroxide 212P. On microscopic examination, dark, finely granular material was noted along the mucosal surface of the lumen of the stomach and duodenum which was considered to be test article. However, there were no microscopic changes which were considered to be related to treatment with Red Ferroxide 212P.

- Assay validity:
The data generated in this study confirm that:
1. The vehicle control data were comparable to laboratory historical control data for each tissue; 2. The positive control induced responses that were comparable with the laboratory’s historical positive control data and produced a statistically significant increase in TI compared to the concurrent vehicle control; 3. Adequate numbers of cells and doses were analysed; 4. The high dose was considered to be the maximum recommended dose.
With respect to target tissue exposure, as the route of administration was oral, direct exposure to the duodenum and stomach was assured. The histopathology data confirmed presence of the test article. The assay data were therefore considered valid.

- Formulation analysis: It was noted that mean %recovery for Groups 2, 3 and 4 (50, 100 and 200 mg/mL) on Day 1 and Groups 2 and 3 (50 and 100 mg/mL) on Day 2 fell outside protocol specification (85-115% nominal). It was also noted that there was some variation (%RSD >10%) between the samples in all groups with the exception of Group 3 (100 mg/mL) on Day 1. No iron was detected in the vehicle control samples.

Applicant's summary and conclusion

Conclusions:
It is concluded that Red Ferroxide 212P did not induce DNA strand breaks in the stomach or duodenum in male animals when tested up to 2000 mg/kg/day (the
regulatory maximum dose level).