Acetic acid, oxo-, sodium salt, reaction products with cresol and ethylenediamine, iron sodium salts

Genetic toxicity: in vivo

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:

key study

Study period:

January-March 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed and reported GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

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:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA, CFR 40, Subpart F - In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay - 1 July 1986

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Acetic acid, oxo-, sodium salt, reaction products with cresol and ethylenediamine, iron sodium salts

EC Number:

283-041-9

EC Name:

Acetic acid, oxo-, sodium salt, reaction products with cresol and ethylenediamine, iron sodium salts

Cas Number:

84539-53-7

Molecular formula:

non specified (UVCB substance)

IUPAC Name:

non specified (UVCB substance)

Constituent 2

Reference substance name:

EDDHMA-Fe

IUPAC Name:

EDDHMA-Fe

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name of the test compound: FeEDDHMANa
Batch no: 74 : 18277
Appearance: dark red to brown solid
Purity of technical grade: 52-53%
Storage: at room T, light protected in closed containers
Expiry date: October 4, 1994

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: minimally 10 weeks (at delivery)
- Weight at study initiation: ca. 30 g
- Assigned to test groups randomly: yes, under following basis: no info
- Fasting period before study: 18 h before application
- Housing: individually
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: minimally 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3
- Humidity (%): 30-70
- Air changes (per hr): no info
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 January To: 11 March 1992

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Aqua deionized
- Vehicle; aqua deionized
- Justification for choice of solvent/vehicle: non toxic
- Concentration of test material in vehicle: 250 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: dissolved in deionized water

Duration of treatment / exposure:

single application

Frequency of treatment:

single application

Post exposure period:

24, 48 and 72 h

Doses / concentrations

Remarks:

Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

6 (of which 5 were evaluated); the 6th animal functioned as spare animal

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): no info
- Route of administration: per gavage
- Doses / concentrations: 30 mg/kg bw at 10 ml/kg bw
- Prepared on the fay of administration

Examinations

Tissues and cell types examined:

Bone marrow:
- per animal, 1000 PCE were analysed for micronuclei,
- ratio PCE/NCE to determine cytotoxicity

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: limit test based on results RF test (2 males/2 females); no toxic signs were noted

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): single application, sampled at 24, 48 and 72 h after application

DETAILS OF SLIDE PREPARATION: small drop of resuspended cells was spread on a slide; stained with May-Gruenwald/Giemsa

METHOD OF ANALYSIS: 1000 PCE per animal were analysed for micronuclei.

Evaluation criteria:

Statistically significant increases for at least one of the test points, together with biological relevance.

Statistics:

Mann-Whitney test

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 0 and 5000 mg/kg bw
- Solubility: OK
- Clinical signs of toxicity in test animals: no
- Evidence of cytotoxicity in tissue analyzed: not done
- Rationale for exposure: no info
- Harvest times: only used for determination of toxicity
- High dose with and without activation: not applicable

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable
- Induction of micronuclei (for Micronucleus assay): no increases when compared to controls
- Ratio of PCE/NCE (for Micronucleus assay): see Table below
- Appropriateness of dose levels and route: limit dose was tested
- Statistical evaluation: not needed (one dose level tested)

Any other information on results incl. tables

Micronuclei - results

Dose (mg/kg bw)	PCEs with micronuclei (%)	Range	PCE/NCE
24 h sampling time
0	0.10	0-3	1000/682
5000	0.13	0-3	1000/899
Positive control	1.45	7-34	1000/869
48 h sampling time
0	0.12	0-3	1000/748
5000	0.10	0-3	1000/814
72 h sampling time
0	0.08	0-2	1000/719
5000	0.15	0-3	1000/728

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Executive summary:

This study was performed to investigate the potential of FeEDDHMANa to induce micronuclei in polychromatic erythrocytes (peE) in the bone marrow of the mouse. The test article was dissolved in aqua deionized. This solvent was used as negative control. The volume administered orally was

20 ml/kg bw. 24 h, 48 hand 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (peE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 peE.

The following dose level of the test article was investigated: 24 h, 48 h and 72 h preparation interval: 5000 mg/kg bw.

In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed no toxic reactions.

After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding" negative controls thus indicating no cytotoxic effects. In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FeEDDHMANa is considered to be non-mutagenic in this micronucleus assay.