3-(2-hydroxyethyl)-p-phenylenediammonium sulphate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

3-(2-hydroxyethyl)-p-phenylenediammonium sulphate

EC Number:

298-995-1

EC Name:

3-(2-hydroxyethyl)-p-phenylenediammonium sulphate

Cas Number:

93841-25-9

Molecular formula:

C8H12N2O.H2O4S

IUPAC Name:

2-(2,5-Diaminophenyl)ethanol sulfate (1:1)

Specific details on test material used for the study:

Batch number : 36/37
Purity : 99.8a/a%

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

Deionised water

Duration of treatment / exposure:

Sacrifice time : 24 and 48 (highest dose group only) hours after the treatment.

Frequency of treatment:

Once

Post exposure period:

24 and 48 (highest dose group only) hours after the treatment.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose:

Six males per dose group except for the highest dose group where 12 males received a single dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (40 mg/kg bw/day) administered orally.

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

The animals were sacrificed with CO2 and cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fotal calf serum. The cell suspension was centrifuged and the supernatant discarded. A small aliquot of the re-suspended cell pellet was spread on a slide. The smear was air dried and then stained with May-Grunwald/Giemsa reagent. Cover slips were mounted and at least one slide was prepared from each bone marrow sample. Evaluation of the slides was undertaken using Nikon microscopes. At least 2000 polychromatic erythrocytes were analysed per animal for micronuclei. To desribe a cytotoxic effect the ratio between polychromatic and total erythrocytes was determined in the same sample and expressed as polychromatic erythrocytes per 2000 erythrocytes.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods are used as an aid in evaluating results. The primary point of consideration is the biological relevance of the results. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test item had no cytotoxic properties in the bone marrow. The urine of the animals treated with the highest dose had taken the colour of the test item indicating the bioavailability of the test item. There were no indications of an increase in the frequency of cells with micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test item were below or equivalent to the value of the vehicle control group. The positive control (cyclophosphamide) showed a statistically significant increase of induced micronucleus frequency.

Any other information on results incl. tables

Toxic Symptoms in the Main Experiment

Toxic reactions	1 hour	2 -4 hours	6 hours	24 hours	48 hours
Reduction of spontaneous activity	0	12	12	8	0
Abdominal position	0	0	3	0	0
Eyelid closure	0	0	5	0	0
ruffled fur	0	12	12	10	0

Summary of Micronucleus Test Results

Test group	Dose/[mg/kg bw]	Sampling time/[hr]	PCEs with micronuclei/%	Range	PCE per 200 erythrocytes
Vehicle	0	24	0.130	0 -7	1151
Test item	25	24	0.090	0 -4	1160
Test item	50	24	0.120	1 -4	1219
Test item	100	24	0.100	0 -4	1167
Test item	100	48	0.050	0 -2	1226
Positive control	40	24	3.010	37 -88	1155

Applicant's summary and conclusion

Conclusions:

Under the test conditions used, hydroxyethyl-p-phenylenedaimine sulfate did not induce an increase in the number of micronuclei in the bone marrow cells of mice. Hydroxyethyl-p-phenylenediamine sulfate is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

The study was performed to investigate the potential of hydroxyethyl-p-phenylenediamine sulfate to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in deionised water which was also used as the vehicle control. The volume administered orally was 10mL/kg bw. After 24 and 48 hours after a single administrationof the test item the bone marrow cells were collected for micronuclei analysis. Five male animals per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes per animal were scored for micronuclei. The following dose levels of the test item were investigated : 24 hour preparation interval (25, 50, 100mg/kg bw), 48 hour preparation interval (100mg/kg bw). The highest dose (100mg/kg bw) was estimated by a pre-experiment to be suitable. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow. The urine of the animals treated with the highest dose had taken the colour of the test item, indicating the systemic distribution of the test item and thus its bioavailability. The clear signs of systemic toxicity observed at the high dose corroborate this indication. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. Cyclophosphamide (40mg/kg bw) administered orally was used as a positive control which showed a substantial increase of induced micronucleus frequency. Under the test conditions used, hydroxyethyl-p-phenylenedaimine sulfate did not induce an increase in the number of micronuclei in the bone marrow cells of mice. Hydroxyethyl-p-phenylenediamine sulfate is considered to be non-mutagenic in this micronucleus assay.