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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2009-04-15 to 2009-09-04
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material : manganese chloride tetrahydrate
- Molecular formula : MnCl2
- Physical state: Liquid
- Storage condition of test material: Bulk test item was stored in its shipping container at ambient temperature (approximately 15-30 °C), as per the instructions accompanying the test item. A 1 gram reserve sample of the neat test item was stored at ambient temperature (approximately 15-30 °C)

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC USA
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: 18.9-22.2 g
- Assigned to test groups randomly: yes under following basis: Animals were stratified by body weight and assigned to a dose group such that mean animal weights across dose groups were approximately equal.
- Housing: Animals were housed individually housed in polycarbonate cages with absorbent hardwood bedding (Betachip, Northeastern products Corp., Warrensbury, NY USA). Animals were transferred to clean cages weekly.
- Diet : Purina Certified Rodent Chow 5002 (Ralston Purina, St. Louis, MO, USA) available ad libitum.
- Water : Reverse-osmosis water was provided ad libitum using plastic water bottles with stainless steel sipper tubes. Fresh water was supplied weekly.
- Acclimation period: All animals were examined by a veterinarian or other appropriate persons during the acclimation period to assess the health status. All animals were re-examined at the end of the acclimation period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: physiol. saline
- Amount of vehicle : 10 mL/kg BW
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing solutions were made from manganese chloride tetrahydrate and concentrations calculated as manganese (Mn2+). The manganese chloride dosing solutions were freshly prepared in 0.9 % saline on each day of treatment.

DOSE ADMINISTRATION
The animals were dosed on days 1 and 2 via oral gavage in a single dose using a stainless steel or disposable gavage needle. Each dose was administered 24 ± 0.5 hours after the first dose.
Duration of treatment / exposure:
Single oral exposures
Frequency of treatment:
Animals were dosed twice within 24 hours
Post exposure period:
46 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
25 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
50 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
200 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5 animals were included in each dosing group
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control : cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 25 mg/kg/day in 0.9 % saline

Examinations

Tissues and cell types examined:
Blood was collected from each mouse for analysis. 350 µL of MicroFlowPLUS kit Solution B (anticoagulant) was aliquoted into an appropriately labelled sterile microcentrifuge tube for each animal. One day prior to collection of blood, 2 mL of MicroFlowPLUS Solution A (fixative) was aliquoted into two appropriately labelled 15 mL polypropylene conical tubes for each study animal. Immediately prior to use, each tube containing Solution B was shaken immediately before bleeding each animal to coat the inside of the tube. Following anaesthesia by isofluorane, blood was collected from the retro-orbital sinus by heparinized hemocrit. 120 (± 20) µL of blood was dispensed into the corresponding microcentrifuge tube containing Solution B and mixed by inverting several times. The stabilised blood samples were maintained at room temperature and fixed within 5 hours of collection.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Results from a range-finding study.

METHOD OF ANALYSIS: Blood samples were fixed by 180 µL of each blood-Solution B sample was withdrawn from the tube. All tubes of fixed cells were quickly transferred back to the freezer and stored at -75 ± 10 °C for at least 24 hours prior to processing for flow cytometry analysis of reticulocytes. Flow cytometry of Mn was performed using MicroFlowPLUS (Mouse) kit reagents (Litron Laboratories, Rochester NY) according to the kit’s instructional manual with minimal modification, 1) 1 mL of fixed cells for each experimental sample was transferred to a tube containing 12 mL of Solution C and washed in preparation for labelling and 2) volumes of labelling solution I, labelling solution II and DNA stain were adjusted as appropriate each day to minimise the use of excess reagents. The MicroFlowPLUS method utilises CD71 and CD61 antibodies as well as a fixed malaria biostandard that contains DNA in the same size range as Mn to properly configure the flow cytometer to exclude platelets (CD61+) and only count micronucleus events in erythrocytes. For each peripheral blood sample, 20,000 immature (CD71+) reticulocytes were analysed to determine the frequency of both MN-RET and micronucleated mature (CD41-) normochromatic erythrocytes (NCE). A BD FACSCalibur™, a four-colour, dual laser benchtop system, was used for MN-RET and MN-NCE analysis.
Evaluation criteria:
Positive control:
The reference control (positive control) cyclophosphamide, is expected to induce an increase in the frequency of Mn-RET at p < 0.05.

Test material:
For negative studies, the highest test item concentration evaluated for MN induction must, unless precluded by the use of the limit dose of 2000 mg/kg, induce bone marrow or animal toxicity or be a dose only slightly lower than that which would be expected to induce mortality.

Criteria for a positive response:
A decision by an experienced scientific investigator to classify a test item as negative, equivocal, or positive for genotoxicity in this assay is based on the biological relevance of the equivocal, or positive for genotoxicity based on the biological relevance of the results, taking into consideration the appropriateness of the concurrent control data, the results of the statistical analysis of the experimental data and the extent of cytotoxicity.
Statistics:
Statistical analysis was conducted on the frequency of MN-RET, MN-NCE and RET. Using individual animal data, the analysis involved the use of the Shapiro-Wilk test with a confidence level of 95 % to determine the normality of the MN-RET data in the vehicle control group.

Normally distributed MN-RET frequency data were then analysed for linearity and variance between treatment groups using linear regression and one-way ANOVA analyses, respectively. The Dunnett multiple comparison test was used to determine if a treatment groups was significantly different (p < 0.05) from vehicle controls. A one-tailed independent t-test was used to verify a positive response to the control compound, cyclophosphamide.

Results and discussion

Test results
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
Animal number 23 which showed hunched posture at the 1 hour post dose observation on Day 1 of the study. All other animals were found to be normal throughout the study
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 125-1000 mg/kg bw
- Clinical signs of toxicity in test animals: Please refer to Range finding study under section: Remarks on results including tables and figures for full details.

RESULTS OF DEFINITIVE STUDY
- Micronucleus Assay Results (incl. statistical analysis): The percent of micronucleated cells among 20,000 RET and ≥900,000 NCE from the peripheral blood of B6C3F1 mice treated with the vehicle, reference chemical, and test material as analysed by flow cytometry. Based on a one-way ANOVA (p = 0.415) test and Dunnett pair wise comparison of each dose group against the concurrent control, the group %MN-RET means comparison of each dose group against the concurrent control, the group %MN-RET means were not significantly increased by treatment with manganese at dose levels 25, 50, 100 or 200 mg/kg bw. No dose response was indicated by linear regression analysis (p = 0.509). There was also no impact on treatment with manganese on %MN-NCE (p = 0.3939) or %RET (p = 0.7375). Treatment with 25 mg/kg/day of the reference control, cyclophosphamide, induced a statistically significant increase in MN-RET (p < 0.0001) as well as MN-NCE (p = 0.0012) and resulted in a 40.6% decrease in %RET.
- Clinical signs of toxicity in test animals: Cage side observations were conducted prior to dosing and at 1 and 4 hours post-dose each day. All animals in the definitive study were considered normal throughout with the exception of animal number 23 which showed hunched posture at the 1 hour post dose observation on Day 1 of the study.

Any other information on results incl. tables

Range finding study:

Male and female mice were treated with Mn2 + at 1000, 500, 250 and 125 mg/kg bw. A few minutes after dosing, the fist animal (male) in the 1000 mg/kg treatment group exhibited seizures and convulsions. This animal was euthanised and no further mice were treated in this dose group. At 1 -hour post-treatment observation period, male and female mice in the 500 mg/kg treatment groups exhibited lethargy, uncoordinated movement, and abnormal breathing. These mice were euthanised after the 1-hour observation period. Also at the 1 -hour post-treatment observation period, the mice in the 250 mg/kg treatment group exhibited hunched posture, decreased movement and piloerection. At the 4-hour post-treatment observation, the mice in the 250 mg/kg group continued to exhibit a hunched posture, decreased movement and lethargy, with one animal found dead. These mice were immediately euthanised. The control mice and the mice in the 125 mg/kg treatment group were normal throughout both days of treatment.

Based on the results of the first day of treatment, the study was discontinued and restarted using 175, 200 and 225 mg/kg bw. At the 1 -hour post-treatment observation, the female mice in the 225 mg/kg group exhibited a hunched posture, decreased movement and piloerection. These mice were immediately euthanised. All other animals were considered normal throughout the study.

Based on the range-finding study, the selected doses for the definitive study were 25, 50, 100 and 200 mg/kg manganese. Female mice were dosed chosen for the definitive test.

Definitive test:

Table 2: Frequency of RET, MN-RET and MN-NCE in peripheral blood reticulocytes of female B6C3F1 mice administered manganese and cyclophosphamide by oral gavage

Dose (mg/kg)

% MN-RET

SEM

% MN-NCE

SEM

% RET

SEM

Vehicle

0

0.202

0.006

0.118

0.001

1.725

0.172

Manganese

25

50

100

200

0.173

0.202

0.197

0.180

0.012

0.017

0.019

0.008

0.120

0.113

0.122

0.112

0.007

0.005

0.003

0.004

1.527

1.565

1.697

1.675

0.087

0.191

0.067

0.105

Cyclophosphamide

25

1.057

0.020

0.144

0.004

1.025

0.077

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Manganese administered twice at 24 ± 0.5 hour intervals to female B6C3F1 mice by oral gavage at 25, 50, 100 or 200 mg/kg bw did not induce chromosomal damage.
Executive summary:

The potential of the test material, manganese dichloride, to induce chromosomal damage in vivo was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 476.

The assay was performed in two phases. The first phase (dose range-finding phase), designed to assess the toxicity of the test material and set dose levels for the definitive study consisted of a toxicity study followed supplemental toxicity study. The second phase was the definitive micronucleus study.

In the range finding phase, male and female mice were treated with Mn2 + at 1000, 500, 250 and 125 mg/kg bw. However, based on the high levels of toxicity observed on the first day of treatment, the study was discontinued and restarted using doses of 175, 200 and 225 mg/kg bw. At the 1-hour post-treatment observation, the female mice in the 225 mg/kg group exhibited a hunched posture, decreased movement and piloerection. These mice were immediately euthanised. All other animals were considered normal throughout the study. Therefore, based on the range-finding study, the selected doses for the definitive study were 25, 50, 100 and 200 mg/kg manganese. Female mice only were dosed in the definitive test.

In the definitive micronucleus study there were no significant increases in micronucleated polychromatic erythrocytes in test material-treated groups relative to the respective vehicle control groups was observed in the female mice at any treatment level.

The results of the assay indicate that under the conditions of the study, administration of the test material at doses up to 200 mg/kg bw did not induce a significant increase in micronucleated polychromatic erythrocytes in female mice. Therefore, the test material was considered to be negative in this mouse micronucleus assay.

Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.