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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well-documented

Data source

Reference
Reference Type:
publication
Title:
Genotoxic activities in vivo of cobaltous chloride and other metal chlorides as assayed in the Drosophila wing spot test
Author:
Ogawa, H.I et al.
Year:
1994
Bibliographic source:
Mutation Research 320: 133 - 140.

Materials and methods

Principles of method if other than guideline:
STUDY 1 (Ogawa et al., 1994):
Iron dichloride and iron trichloride were subjected to the wing spot test of Drosophila melanogaster. In the test, larvae trans-heterozygous for the wing-hair mutations mwh and flr were orally treated at the third instar stage with a test compound and the wings were inspected at the adult stage for spots expressing phenotypes of the markers.
GLP compliance:
not specified
Type of assay:
somatic mutation and recombination test in Drosophila

Test material

Constituent 1
Reference substance name:
STUDY 1: iron dichloride & iron trichloride
IUPAC Name:
STUDY 1: iron dichloride & iron trichloride
Test material form:
other: STUDY 1: no data
Details on test material:
STUDY 1:
1) Molecular formula (if other than submission substance): FeCl2 (purchased from Wako Pure Chemical Industries, Ltd., Osaka; reagents of the highest grade)
2) Molecular formula (if other than submission substance): FeCl3 (purchased from Wako Pure Chemical Industries, Ltd., Osaka; reagents of the highest grade)

Test animals

Species:
other: STUDY 1: Drosophila melanogaster
Strain:
other: STUDY 1: genotypes mwh jv; spa^pol, and flr³/TM3, Ser
Sex:
male/female
Details on test animals or test system and environmental conditions:
STUDY 1:
D. melanogaster with genotypes mwh jv; spa^pol (from Dr. T. Ayaki, Nagasaki Univ.), and flr³/TM3, Ser (Graf et al., 1989*; from Dr. F.E. Würgler, Zurich Univ.) were used as parental stocks. The markers mwh and flr³ are recessive wing-hair mutations located on the third chromosome at 0.0 and 38.8, respectively. Wing-hair cells homozygous for the mutation mwh produce three or more hairs per cell instead of a single smooth hair. Those cells homozygous for the mutation flr³ produce misshapen, flare-like hairs. TM3 is a multiply inverted third chromosome marked with dominant visible mutation Ser (Beaded-Serrate) that manifests a notched wing phenotype.

*Reference:
- Graf, U., H. Frei, A. Kägi, A.J. Katz and F.E. Würgler (1989) Thirty compounds tested in the Drosophila wing spot test, Mutation Res., 222. 359 - 373.

Administration / exposure

Route of administration:
other: STUDY 1: oral: feed
Vehicle:
STUDY 1:
- Vehicle(s)/solvent(s) used: distilled water
Details on exposure:
STUDY 1:
Wing spot test:
Virgin mwh jv; spa^pol females and flr³/TM3, Ser males were mated, and their F1 progeny were sampled as third instar larvae at 72 - 96 hours after oviposition. The larvae were introduced in 2.3 cm X 9.5 cm polycarbonate vials containing 1.2 g per vial of Drosophila Medium (Formula 4-24, Carolina Biological Supply Co., Barlington, NC, USA) hydrated with 3.3 mL of test solution or distilled water. They were allowed to develop to adulthood in the same vials. Emerged adult flies were scored, fixed and preserved in 70% ethanol.
Adult flies were of two types, mwh jv/frl³; spa^pol/+ and mwh jv/TM3, Ser; spa^pol/+. Hereafter, these are referred to as mwh/frl and mwh/TM3, respectively.
All the experiments were carried out at 25 ± 1°C.
Duration of treatment / exposure:
STUDY 1:
not clearly stated
Frequency of treatment:
STUDY 1:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
STUDY 1: 20, 40, 60, and 80 mM (iron dichloride & iron trichloride were tested at the concentrations)
Basis:
nominal in diet
No. of animals per sex per dose:
STUDY 1:
no data
Control animals:
other: STUDY 1: vehicle control
Positive control(s):
STUDY 1:
no data

Examinations

Tissues and cell types examined:
STUDY 1:
Wing spot test:
Permanent wing preparations were examined microscopically at a magnification of X400. Spots comprising only mwh or frl hairs (single spots) and those with neighbouring mwh and flr spots (twin spots) were scored as mutant clones. The spots were reclassified into two classes according to the number of mutant hairs per spot, namely, small spot with one or two mutant hairs and large spots with three or more mutant hairs.
Details of tissue and slide preparation:
STUDY 1:
Wing spot test:
Wings of the mwh/frl flies were sampled and rinsed in 99.5% ethanol for 5 - 10 minutes. The dehydrated wings were mounted on slide glasses using a 1:1 mixture of nail polish and acetone.
Evaluation criteria:
STUDY 1:
Please refer to the field "Statistics" below.
Statistics:
STUDY 1:
Wing spot test:
Frequency of spots, F, was calculated for each of the two classes following the equation:
F=S/W
where S is the number of spots detected and W the number of wings inspected.
The data obtained for the frequencies of spots per wing were analysed following a procedure proposed by Frei and Würgler (1988) which tests the hypotheses that (i) the frequency in the treated series is not higher than that in the control series and (ii) the frequency in the treated series is no less than m times the control species. The m value adopted was 2 for small spots and 5 for large spots. Both hypotheses were tested at the 5% significance level by the χ² test with the Yate's correction. Conclusions drawn from this multiple-decision procedure are as follows: positive (the first hypothesis rejected/the second accepted), weak positive (both hypotheses rejected), negative (the first accepted/the second rejected) and inconclusive (both accepted).
The dose-response data and other information were also taken into account in making a final judgement about the genotoxic activity of the test compound.

Toxicity test:
The survival, S, of treated larvae was determined at each dose of test compound following the equation:
S =St/So
where St is the surviving fraction in the treated vial, i.e., the ratio of the number of emerged flies to the number of treated larvae and So is the surviving fraction in the control vial.

*Reference:
- Frei, H., and F.E. Würgler (1988) Statistical methods to decide whether mutagenicity test data from Drosophila assays indicate a positive, negative, or inconclusive results, Mutation Res., 203, 297 - 308.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
STUDY 1: iron dichloride & iron trichloride; please refer to the field "Additional information on results" below
Toxicity:
yes
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
STUDY 1:
- LD50 (larval toxicity; iron dichloride) = 64 mM
- LD50 (larval toxicity; iron trichloride) = 75 mM
- iron dichloride and iron trichloride were negative in the present assay.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: STUDY 1: negative
STUDY 1:
According to the authors, iron dichloride and iron trichloride were negative in the Drosophila wing spot test.