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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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
April 13 2004 to 22 June 2004.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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 relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
1) In the range finder, 2 mice were grouped in the 2000 mg/kg/day dose level. Two mice in this group died immediately after 1st dose. The 1 animal left was grouped into the 20 mg/kg/day dose level. 2) Occasional deviations in temperature and humidity.
Qualifier:
according to
Guideline:
other: Korea National institute of Environmental Research Test Guidelines of Chemicals, Notification 1998-41, 23 December 1998
GLP compliance:
yes
Remarks:
NIER Public Notice No. 1998-41, under Toxic Chemicals Control Act, (enacted on 8 December 1996, revised on 23 December 1998) and OECD Principles of Good Laboratory Practice (as revised 1997), NV/MC/CHEM (98)17
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity: Iron dichloride (CAS No.: 7758-94-3)
Lot number: 23828CB
Purity: 98%
Storage condition: Room temperature
Appearance: Tan powder
Stability: Stable

Test animals

Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Orient Inc., Korea
- Age at study initiation: 7 weeks
- Weight at study initiation: 28.2 - 30.9 for the dose range-finding experiment, 28.0 - 31.5 g for the micronucleus experiment
- Assigned to test groups randomly: yes
- Fasting period before study: not stated in report
- Housing: Five or six animals per cage (polycarbonate cages W260 x D280 x H180 mm with bedding) unless reduced by mortality or isolation
- Diet: ad libitum access to Certified rodent diet (2014C, Teklad Global 14% Protein Rodent diet, Harlan Teklad, USA)
- Water: Free access via polycarbonate bottles (with sipper tubes) to filtered and UV treated tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): maintained within the range of 20.69 to 28.31°C
- Humidity (%): Maintained within the range 26.38 - 59.44%
- Air changes (per hr): HEPA-filtered, not recirculated
- Photoperiod (hrs dark / hrs light): 12 hours light/ 12 hours dark cycle (08:00 - 20:00). Light intensity of 150 - 300 Lux

IN-LIFE DATES: From: April 13th 2004 - Dose administration To: April 29th 2004 Slide preparation

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: not stated in report
- Concentration of test material in vehicle: 1.25, 2.5 and 5 mg/ml in micronucleus experiment
- Amount of vehicle (if gavage or dermal): not applicable
- Type and concentration of dispersant aid (if powder): none described in report
- Lot/batch no. (if required): 122KO131
- Purity: not stated in report
Details on exposure:
Injected once daily for two consecutive days
Duration of treatment / exposure:
Single exposures
Frequency of treatment:
Twice (two consecutive days)
Post exposure period:
5 days for the dose range-finding experiment
3 days for the micronucleus experiment
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2, 5, 10, 20, 50, 100 and 200 mg/ml in the dose range-finder
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1.25, 2.5 and 5 mg/ml in the micronucleus experiment
Basis:
nominal conc.
No. of animals per sex per dose:
Dose range-finder
Two males at 2000 mg/kg/day
Three males each for the 20, 50, 100, 200, 500 and 1000 mg/kg/day dose levels

Micronucleus experiment
Six males for each of 12.5, 25 and 50 mg/kg/day dose levels
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control substance: mitomycin C
- Justification for choice of positive control(s): Not stated in report
- Route of administration: Single intraperitoneal injection
- Doses / concentrations: Freshly prepared in water at a concentration of 0.2 mg/ml (dose level of 2 mg/kg)

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Polychromatic erythrocytes (PCE)
Normochromatic erythrocytes (NCE)
Micronucleated polychromatic erythrocytes (MNPCE)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The doses used in the micronucleus experiment were determined by the results of the dose range-finder

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Test article and vehicle treated mice were killed about 24 hours after the second administration. MMC-treated mice were killed about 24 hours after the single dose administration. mice were killed by cervical dislocation in the same sequence used for the dosing.

DETAILS OF SLIDE PREPARATION:
Both femurs from each animal were exposed, removed, cleaned of adherent tissue and the end removed. bone marrow was flushed from the marrow cavity with a 0.5 ml foetal bovine serum into appropriately labelled centrifuge tubes (1 tube per animal).

The tubes were centrifuged at 1250x g for 5 minutes. the serum was aspirated to leave 1 or 2 drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube, and from each tube 1 drop of suspension was placed on the end of each slide labelled with the appropriate study number, animal number and date of preparation. A smear was made from the drop by drawing the end of a clean slide along the labelled slide. A slide was prepared for each animal.

METHOD OF ANALYSIS:
Slides were allowed to air-dry and then fixed for 5 minutes in absolute methanol before being stained. After air-drying, slides were stained for 60 minutes in 5% Giemsa stain in Sorenson's buffer, pH 6.8. Stained slides were rinsed for 5-6 seconds in 0.004% citric acid solution and then were rinsed 3 times in distilled water. After air-drying, randomised numbers were assigned which served as the code so analysis could be conducted blind". Slides were examined under x1000 magnification.

Evaluation criteria:
Evaluation criteria
The test article would be considered as positive if:
1) The assay was valid (please refer to acceptance criteria below) and;
2) Statistically significant increases in the frequency of MNPCE occurs at one or more levels and such increases exceeded the historical control range.

Acceptance criteria
The micronucleus test is considered valid if the following criteria are met:
1) The frequencies of MNPCE in vehicle control groups fell within or close to the historical vehicle range
2) The positive control chemical (MMC) induced statistically significant increases in the frequency of MNPCE
3) At least 5 animals for each group were available for analysis
Statistics:
After completion of microscopic analysis and decoding the data, the ratio of PCE/(PCE+ NCE) for each animal and the mean for each group were calculated, and the frequency of MNPCE for each animal and the mean for each group were also determined. This ratio was examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity.

The number of MNPCE in each treated group was then compared with the number in the vehicle control groups by using a 2 x 2 contingency table to determine chi-square. A probability value of ≤0.05 was accepted as significant. The bodyweight change in each treated group was examined using the student t-test and a probability value of ≤0.05 was accepted as significant.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 20 to 2000 mg/kg/day

- Solubility: Soluble in corn oil

- Clinical signs of toxicity in test animals: all animals of the 200, 500, 1000 and 2000 mg/kg/day dose levels and one animal at the 100 mg/kg/day dose level were dead after the first dosing. Two animals of the 100 mg/kg/day dose level and all of the 50 and 20 mg/kg/day dose levels showed piloerection after the first dosing. After the second dosing, piloerection and hypoactivity were observed in all animals of the 100 mg/kg/day dose level and piloerection was observed in all animals at the 50 and 20 mg/kg/day dose levels. One animal of 100 mg/kg/day dose level was dead two days after the second dosing. the highest level to be used in the micronucleus experiment was determined to be 50 mg/kg/day.

- Evidence of cytotoxicity in tissue analyzed: none

- Rationale for exposure: The intraperitoneal route was chosen for this study because it is the most general exposure route

- Harvest times: Cells not harvested in dose range-finder

- High dose with and without activation: not applicable for this study type

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable for this type of study

- Induction of micronuclei (for Micronucleus assay): Micronuclei not induced in mice bone marrow cells under the conditions employed for this test.

- Ratio of PCE/NCE (for Micronucleus assay): Please refer to table of ratios in the section "Any other information on results"

- Appropriateness of dose levels and route: The intraperitoneal route was chosen for this study because it is the most general exposure route

- Statistical evaluation: No significant increases in the frequency of micronucleated polychromatic erythrocytes.

Any other information on results incl. tables

Treatment group

Animal number

PCE:NCE counted

PCE/

PCE+NCE

MNPCE

Per 2000

Per 1000

Vehicle

10 ml/kg

M1

M2

M3

M4

M5

M6

304:196

267:233

275:228

284:216

473:238

275:228

0.61

0.53

0.55

0.57

0.67

0.55

3

1

2

7

4

6

1.5

0.5

1

3.5

2

3

Iron dichloride

12.5 mg/kg

M7

M8

M9

M10

M11

M12

314:186

293:207

349:190

384:205

389:250

276:225

0.63

0.59

0.65

0.65

0.61

0.55

5

4

4

2

2

6

2.5

2

2

1

1

3

Iron dichloride 25 mg/kg

M13

M14

M15

M16

M17

M18

284:216

255:245

305:300

287:220

292:248

396:209

0.57

0.51

0.50

0.57

0.54

0.65

3

5

1

4

5

4

1.5

2.5

0.5

2

2.5

2

Iron dichloride 50 mg/kg

M19

M20

M211

M22

M23

M24

218:282

-:-

232:268

272:227

263:237

337:225

0.44

-

0.46

0.55

0.53

0.60

3

-

2

1

3

1

1.5

-

1

0.5

1.5

0.5

Mitomycin C

2 mg/kg

M25

M26

M27

M28

M29

M30

239:261

301:199

460:345

326:275

270:230

267:238

0.48

0.60

0.57

0.54

0.54

0.53

178

189

181

176

173

217

89

94.5

90.5

88

86.5

108.5

Sex = all male

Sampling time = always 24 hours

PCE counted = always 200 except for 1 animal in the 50 mg/kg/day group (no value stated)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Iron dichloride (CAS No.: 7758-94-3) has been tested according to OECD TG 474 and under GLP. The test substance did not induce micronuclei in the bone marrow cells of mice under the conditions employed for this test.
Executive summary:

The potential of Iron dichloride (CAS no.: 7758 -94 -3) to induce micronuclei was determined in vivo in bone marrow cells of treated male mice following OECD Guideline no.474.

For the micronucleus test, a dose range finding experiment was conducted. Test article groups of two males of ICR mice were dosed at 2000 mg/kg/day and each of three males were dosed at 20, 50, 100, 200, 500 and 1000 mg/kg/day once daily for two consecutive days by intraperitoneal injection. All animals of 200, 500, 1000 and 2000 mg/kg/day dose levels and one animal of 100 mg/kg/day dose level were dead after the first dosing. Two animals of the 100 mg/kg/day dose level and all of the 50 and 20 mg/kg/day dose levels showed piloerection after the first dosing. After the second dosing, piloerection and hypoactivity were observed in all animals of the 100 mg/kg/day dose level and piloerection was observed in all animals at the 50 and 20 mg/kg/day dose levels. One animal of 100 mg/kg/day dose level was dead two days after the second dosing. the highest level to be used in the micronucleus experiment was determined to be 50 mg/kg/day.

In the micronucleus experiment, test article groups of six male ICR mice were dosed once daily for two consecutive days by intraperitoneal injection at dose levels of 12.5, 25 and 50 mg/kg/day.

Animals in the negative control group were dosed with the vehicle via the same route following the same treatment schedule as that in the test article groups. Animals in the positive control group were dosed once by intraperitoneal injection with mytomycin c at a dose level of 2 mg/kg/day.

After the first dosing, one animal of 50 mg/kg/day dose level was dead and piloerection was observed in all animals of the 25 and 50 mg/kg/day dose levels. After the second dosing, piloerection and hypoactivity were observed in 3 animals at the 50 mg/kg/day dose level. No clinical signs were observed in the 12.5 mg/kg/day dose level.

Bone marrow samples were collected from animals in the test article and negative control groups at 24 hours after the second dosing, while they were collected from the positive control animals at 24 hours after the first dosing. Bone marrow smear slides from all groups were analysed. Polychromatic erythrocytes (PCE), normochromatic erythrocytes (NCE) and micronucleated polychromatic erythrocytes (MNPCE) were counted for each animal.

All animals dose with Iron dichloride (CAS No.: 7758 -94 -3) exhibited similar PCE/(PCE+NCE) ratios and MNPCE frequencies compared to those of negative control animals.

All frequencies of MNPCE in the negative (vehicle) control groups fell within acceptable ranges, while the positive control groups induced clear increases in the frequencies of MNPCE.

It is concluded that Iron dichloride (CAS No.: 7758-94-3) did not induce micronuclei in the mice bone marrow cells under the conditions employed for this test.