Neodecanoic acid, iron salt

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Micronucleus Tests in mice on 39 food additives and eight miscellaneous chemicals

Author:

Hayashi M. et al.

Year:

1988

Bibliographic source:

Fd Chem Toxic Vol 26 (6), 487-500

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Iron sulphate

EC Number:

231-753-5

EC Name:

Iron sulphate

Cas Number:

7720-78-7

Molecular formula:

Fe.H2O4S

Specific details on test material used for the study:

- Name of the test substance: Ferrous sulphate
- Purity: 87.65%

Test animals

Species:

mouse

Strain:

other: ddY

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka, Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: no data
- Diet (e.g. ad libitum): food pellets CE-2 (Japan Clea, Tokyo), ad libitum
- Water (e.g. ad libitum): ad libitum

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: water

Details on exposure:

Six male mice per dose group were used and the test item was administered by intraperitoneal injection.

Duration of treatment / exposure:

Experiment 1 and 2: single exposure plus 24 h post treatment incubation
Experiment 3: multiple dosing (4 times) at 24 h intervals plus 24 h post treatment incubation

Frequency of treatment:

Experiment 1 and 2: once
Experiment 2: 4 times (not specified in detail) at 24 h intervals

Post exposure period:

24 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

single dosing (experiment 1)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

single dosing (experiment 1)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

single dosing (experiment 1)

Doses / concentrations 4

Dose / conc.:

180 mg/kg bw/day (nominal)

Remarks:

single dosing (experiment 1)

Doses / concentrations 5

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

single dosing (experiment 2)

Doses / concentrations 6

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

single dosing (experiment 2)

Doses / concentrations 7

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

multiple dosing (experiment 3)

Doses / concentrations 8

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control, experiment 1 & 2

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

- Mitomycin C, 2.0 mg/kg bw

Examinations

Tissues and cell types examined:

- Tissue: Bone marrow
- Cell type examined: Erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The maximum dose level was set at the supposed maximum tolerated dose referring to the LD50.

DETAILS OF SLIDE PREPARATION:
Mice were killed by cervical dislocation at the appropriate time after administration. Femoral marrow cells were flushed out with foetal bovine serum and smeared on clean glass sides. Cells were fixed with methanol for 5 min and stained with Giemsa.

METHOD OF ANALYSIS:
The preparations were coded and analysed without any knowledge of the treatment. One thousand polychromatic erythrocytes per mouse were scored using a light microscope and the number of micronucleated polychromatic erythrocytes (MNPCE) was recorded. The proportion of polychromatic erythrocytes among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide.

Evaluation criteria:

The frequencies of MNPCEs in concurrent negative and positive control groups were compared with the control charts of the historical data to confirm the technical validity of the experiment.

Statistics:

The dose-response relationships were tested using the Cochran-Armitage trend test. A positive dose-response was considered significant at p< 0.05.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

4 mortalities out of 6 at 180 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

For detailed results please refer to Table 1 in box "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: Result of the micronucleus test using mouse bone marrow cells

No. of doses	Time between doses [h]	Sampling time [h]	Dose level [mg/kg bw]	MNPCE [%]	PCE [%]	Mortality	Trend Test*	Judgement
1	-	24	0	0.20 ± 0.13	53.8 ± 7.7	0/6	NS	negative
			25	0.23 ± 0.23	48.9 ± 16.8	0/6
			50	0.28 ± 0.16	50.0 ± 9.3	0/6
			100	0.40± 0.17^a	54.2 ± 12.7	0/6
			180	0.10 ± 0.14	53.8 ± 0.7	4/6
			MMC: 2.0	7.25 ±2.33^a	45.6 ± 7.8	0/6		positive
1		24	0	0.27 ± 0.20	71.8 ± 9.2	0/6	NS	negative
			100	0.23 ± 0.26	64.3 ± 7.8	0/6
			150	0.30 ± 0.10	65.4 ± 17.7	0/6
4	24	24	50	0.23 ± 0.18	43.2 ± 6.7	0/6		negative

* dose-response relationships were tested using the Cochran-Armitage trend test. A positive dose-response was considered significant at p<0.05

MNPCE = micronucleated polychromatic erythrocytes

PCE = polychromatic erythrocytes

NS = not significant

^asignificantly different from historical control (p< 0.01)

Applicant's summary and conclusion

Conclusions:

Under the reported experimental conditions iron sulphate did not induce structural and/or numerical chromosomal damage in the bone marrow cells of the mouse.

Executive summary:

In a ddY mouse bone marrow micronucleus test conducted similar to OECD guideline 474, six male mice per dose were treated intraperitoneally with iron sulphate (87.65 % purity) at doses of 0, 25, 50, 100 and 180 mg/kg bw (experiment 1), in experiment 2 at doses of 0, 100 and 150 mg/kg bw and at 50 mg/kg bw (experiment 3). The animals were injected intraperitoneally with the test substance once (experiments 1 and 2) or four times at 24 h intervals (experiment 3) with an additional 24 hours period after the last dosing. The vehicle was water. Iron sulphate did not increase the level of micronuclei in comparison to the concurrent vehicle control. Thus, the substance is considered to be non-mutagenic according to the results of the in vivo micronucleus test reported.