Diantimony trioxide

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2021 to Mar 2022

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Diantimony trioxide

EC Number:

215-175-0

EC Name:

Diantimony trioxide

Cas Number:

1309-64-4

Molecular formula:

Sb2O3

IUPAC Name:

oxostibanyl stibinate

Test material form:

not specified

Details on test material:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Campine
- Purity, including information on contaminants, isomers, etc.: 100% as received


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: between 18 - 24*C
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Stable
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Stable
- Reactivity of the test material with the incubation material used (e.g. plastic ware): None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): None
- Preliminary purification step (if any): None

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: N/A

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

The CD-1 mouse was chosen as the animal model for this study as it is an accepted rodent species for genetic toxicology testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: 8–9 weeks
- Weight at study initiation: 20g to 40g

- Assigned to test groups randomly: Yes
Animals assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Animals in poor health or at extremes of body weight range not assigned to groups. Individual body weights at randomization within ± 20% of the mean.

- Housing: Males individually housed because male mice are often aggressive and not considered social

- Diet: Ad libitum, except during acclimation to nose-only restraint tubes and during nose-only inhalation exposure periods.

- Water: Freely available to each animal via an automatic watering system, except during acclimation to nose-only restraint tubes and during nose-only inhalation exposure periods.

- Acclimation period: Each animal will be inspected by qualified personnel upon receipt. Animals judged to be in good health will be placed in acclimation for at least 7 days. All animals acclimated to nose-only restraint tubes a total of five times (1 acclimation/day) prior to the first animal exposure as follows: 1st day-0.5 hour, 2nd day-1 hour, 3rd day-2 hours, 4th day-3 hours, and 5th day-4 hours; times are approximate. Following the restraint period, each animal will be observed for clinical signs of injury or stress.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26
- Humidity (%): 30 -70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Administration / exposure

Route of administration:

inhalation: aerosol

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Standard material
- Concentration of test material in vehicle: Test substance dosing formulations prepared at appropriate concentrations to meet dose level requirements. The prepared formulations not adjusted for purity. See tables below for concentraitons
- Amount of vehicle (if gavage or dermal): N/A
- Type and concentration of dispersant aid (if powder): N/A

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

Each exposure conducted using a conventional flow nose-only exposure unit of appropriate size. The system operated in a dynamic mode. Airflow rate through the system will be set based on output from the generation device and the dilution airflow and will provide sufficient volumes for the number of animals to be exposed and for exposure atmosphere sampling. The airflow rates for each nose-only system may be monitored by measuring the pressure drop between the ports of a venturi tube using a Dwyer Magnehelic® Indicating Transmitter pressure gauge. Each gauge will be calibrated for conversion from pressure to airflow in standard liters per minute. If venturi-based methods for measuring system airflow rate cannot be used, recorded values will be calculated from calibration curves for the aerosol generation device and flowmeters

Exposure atmospheres are sampled and aerosol concentrations measured using standard gravimetric methods.

Further details are in the attached study plan

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily, 4 hours per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Remarks:

Micronucleus Oral Gavage

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Micronucleus Oral (Gavage)

Doses / concentrations 3

Dose / conc.:

0 mg/m³ air

Remarks:

Inhalation - Comet Assay

Doses / concentrations 4

Dose / conc.:

25 mg/m³ air

Remarks:

Inhalation - Comet Assay

Doses / concentrations 5

Dose / conc.:

10 mg/m³ air

Remarks:

Inhalation - Comet Assay

Doses / concentrations 6

Dose / conc.:

5 mg/m³ air

Remarks:

Inhalation - Comet Assay

No. of animals per sex per dose:

Inhalation; 4 males per dose range finding phase
Inhalation; 6 males per definitive phase
Oral gavage: 6 males per definitive phase

Control animals:

yes

Positive control(s):

Ethyl methanesulfonate

- Justification for choice of positive control(s):
- Route of administration: oral gavage
- Doses / concentrations: Once on the days of positive control dosing (Days 13 & 14 of each dosing phase)

Examinations

Tissues and cell types examined:

Liver, Lung (left), Nasal epithelium, and Bronchoalveolar lavage fluid (pelleted cells) tissues assessed.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: See methodology

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): see below

DETAILS OF SLIDE PREPARATION:
At least two slides per animal prepared for each organ/tissue. An aliquot of 50 µL of each cell suspension per slide mixed with 0.5% low melting agarose. The cell/agarose suspension applied to microscopic slides, commercially available pre-treated or previously coated with 1% normal melting agarose. The slides are incubated at 2-8ºC for at least 15 minutes to allow the gels to solidify.
Slides are identified with a random code that reflects the study number, group, animal number, and organ/tissue. Three wells used for scoring and the remaining slides/wells will be backup. These wells may be used in additional scoring, if deemed necessary.
Each slide submerged in a lysis solution at least overnight at 2-8ºC. The lysis solution is composed of 100 mM EDTA (disodium), 2.5 M sodium chloride, 10 mM tris hydroxymethyl aminomethane in purified water; pH10; 1% triton-X100 and 10% DMSO will be added on the day of use or commercially available lysis solution will be used after the addition of 10% DMSO on the day of use.
After cell lysis, slides are washed with neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, approximately pH 7.5) and placed in an electrophoresis chamber. The chamber reservoirs are filled with alkaline buffer (300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water, pH > 13) for approximately 20 minutes at 2-10ºC, protected from light for the unwinding of DNA. Electrophoresis will be conducted in the same buffer following DNA unwinding for 20 minutes at 0.7 volts/cm.
The slides are removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides will be dehydrated with 200-proof ethanol for at least 5 minutes, then air-dried for at least 2 hours and then stored at room temperature with desiccant.


METHOD OF ANALYSIS: standard comet assay (-FPG, +FPG)

OTHER:
12 (approx.) standard single tissue comet studies for each dosing arm.
Optimization of the experimental conditions for the batch of formamidopyrimidine [fapy]-DNA glycosylase (FPG; a base excision repair enzyme that recognizes and removes oxidized purines from damaged DNA) was required as well as generation of historical data for these tissues in CD-1 mice, using both standard conditions and the oxidative damage modification of the comet assay

Evaluation criteria:

Comet Assay
The DNA damage data (% tail DNA) in the negative control group (filtered air control or vehicle control) is expected to be within the historical vehicle control (negative control) range, and the positive control group must be significantly increased relative to the concurrent negative control group (p ≤ 0.05). Additionally, concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.

For each animal and organ, the median values calculated from the cells scored in each well will be averaged and presented as the individual animal value for the % tail DNA, Tail moment, and Tail migration endpoints. The mean and standard deviation of the mean values will be presented for each treatment group.

Micronucleus Assay
Criteria for a Positive Response
The test substance will be considered to be clearly positive for induction of MN if:
a) At least one treatment group exhibits a statistically significant increase in % MN-PCE as compared with the concurrent negative control (p ≤ 0.05); and
b) The increase is dose-related (p ≤ 0.05); and
c) Any increase is outside the 95% control interval of the historical negative control data.

Criteria for a Negative Response
The test substance will be considered to be clearly negative for induction of MN if:
a) No treatment group exhibits a statistically significant increase in %MN-PCE as compared with the concurrent negative control; and
b) There is no dose-related increase in %MN-PCE; and
c) All results are within the 95% control interval of the historical negative control data: and
d) Bone marrow exposure to the test substance has been demonstrated to occur.

Statistics:

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), ratio, percentages, numbers, and/or incidences will be reported as appropriate by dataset.

All statistical tests will be conducted at the 5% significance level. All pairwise comparisons will be conducted using two sided tests and will be reported at the 1% and 5% levels, unless otherwise noted.

Analyses will be performed on the following variables when possible but will exclude any group with less than 3 observations.
Body Weight
Body Weight Gains
Hematology Variables

Parametric/Non-parametric (Provantis)
Levene’s test will be used to assess the homogeneity of group variances.
The groups will be compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test is found to be significant, then pairwise comparisons will be conducted using Dunnett’s or Dunn’s, respectively.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Remarks on result:

other: Awaiting finalization of some results (see results discussion)

Any other information on results incl. tables

Provided are the intermediate results and progress status as related to the comet assay phase of Charles River Laboratory study #01699001

This study includes evaluation of oxidative damage in addition to the standard comet assay assessment of DNA damage in four different tissues (liver, nasal epithelium, lung, bronchoalveolar lavage fluid) of animals exposed to antimony trioxide.   Optimization of the experimental conditions for the batch of formamidopyrimidine [fapy]-DNA glycosylase (FPG; a base excision repair enzyme that recognizes and removes oxidized purines from damaged DNA) was required as well as generation of historical data for these tissues in CD-1 mice, using both standard conditions and the oxidative damage modification of the comet assay.

 

The current status of the comet assay phase is summarized in Table 1.

 

Table 1. Status of Comet Assay Progress (Inhalation Definitive Phase)

Tissue	Comet Assay Method	Slide Preparation	Slide Scoring	Projected Completion
Liver	Standard	Complete	Complete	June 2022
	-FPG, +FPG	Complete	In Progress
Lung (left)	Standard	Complete	Pending	August 2022
	-FPG, +FPG	Complete	Pending
Nasal epithelium	Standard	Complete	Pending	September 2022
	-FPG, +FPG	Complete	Pending
Bronchoalveolar lavage fluid (pelleted cells)	Standard	Complete	Pending	October 2022
	-FPG, +FPG	Complete	Pending

Abbreviation:  FPG = formamidopyrimidine [fapy]-DNA glycosylase

To date, data have been collected for liver using the standard comet assay.  Summary results are provided in Table 2 and results of the statistical analysis are detailed in Table 3.  Individual animal results are provided in Table 4.

Table 2. Summary Comet Assay Results for Liver (Standard Assay)

Dose Level (mg/m^3)	Number of Animals	Liver^a
		% Tail DNA^b	Tail Length (µ)	Olive Tail Moment	% Hedgehogs
0	5	2.29 ± 2.38	2.86 ± 0.61	0.03 ± 0.03	0.10 ± 0.23
5	5	3.78 ± 2.21	3.53 ± 0.75	0.06 ± 0.04	1.62 ± 0.68
10	5	2.77 ± 2.52	3.58 ± 0.89	0.05 ± 0.04	0.00 ± 0.00
25	5	2.71 ± 1.68	3.42 ± 0.62	0.04 ± 0.03	0.71 ± 0.59
EMS^c	5	10.36 ± 1.91*	4.81 ± 0.61	0.20 ± 0.05	0.10 ± 0.22

Abbreviations: EMS = ethyl methanesulfonate administered at 150 mg/kg/day

^a Values are group means ± standard deviation; statistical analysis was performed only on % tail DNA as per the study protocol.

^b Dunnett’s test and linear trend test

^c t-test

* p ≤ 0.05

 

Table 3. p-Values for Comet Assay of Liver (Standard Assay)

Dose Level (mg/m^3)	% Tail DNA^a
5	0.3073
10	0.6139
25	0.6309
EMS^b	0.0002*
Linear Trend	0.9544

Abbreviations: EMS = ethyl methanesulfonate administered at 150 mg/kg/day

^a Dunnett’s test and linear trend test

^b t-test

* p ≤ 0.05

 

Applicant's summary and conclusion

Conclusions:

For liver, no dose groups exhibited a statistically significant increase in DNA damage following inhalation of antimony trioxide as compared to concurrent control animals. There was no evidence of a dose response. The % tail DNA values for the vehicle and dose groups are consistent with the laboratory’s historical negative control data. Thus, under the conditions tested, the results of the standard comet assay are considered negative.

Additional results from this study are expected and once they have been received the dossier will be updated.