O-ethylhydroxylamine

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (only two dose tested, only 1000 cells scored for PCE)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(Temperatures fell to between 9 and 13°C due to failure of the room heating), only two dose tested, only 1000 cells scored for PCE

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

O-ethylhydroxylamine

EC Number:

402-030-3

EC Name:

O-ethylhydroxylamine

Cas Number:

624-86-2

Molecular formula:

C2 H7 N O

IUPAC Name:

O-ethylhydroxylamine

Details on test material:

- CTL reference number: Y05460/002/001
- Name of test substance as cited in report: Substance H109360
- Identification of test sample as cited in report: 50% (w/v) aqueous solution (45% Substance H109360 and approximately 5% ethanol)
- Appearance: clear aqueous solution
- Lot #: EFI-100
- Analytical purity: not specified

Test animals

Species:

mouse

Strain:

C57BL

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Name as cited in report: C576L/6J/Alpk mice
- Source: Animal Breeding Unit, Imperial Chemical Industries PLC, Cheshire, UK
- Age at study initiation: 9-12 wks
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: 10/sex/cage on mobil racks
- Diet: Porton Combined Diet PCD provided ad libitum
- Water: filtered tap water (via an automatic water system) provided ad libitum
- Acclimation period: performed, but duration not specified


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-57
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: physiol. saline

Details on exposure:

DOSING VOLUME: 10 ml/kg bw

Duration of treatment / exposure:

24 hours (all doses + positive control)
48 and 72 hours (control + 432 mg/kg bw)

Frequency of treatment:

single dose application

Post exposure period:

no

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
270 and 432 mg/kg bw
Basis:
actual ingested
(test article containing 50% of H109360)

Doses / concentrations 2

Remarks:

Doses / Concentrations:
135 and 216 mg/kg bw substance H109360:ethyl alcohol [90:10])
Basis:
actual ingested
(normalised to the actual percentage of H109360 in the test article which is 50%

No. of animals per sex per dose:

5/sex/dose/sampling time

Control animals:

yes, concurrent vehicle

Positive control(s):

- Name: Cyclophosphamide
- Justification for choice of positive control(s): as cited in the guideline OECD 474. Cyclophosphamide, is an established clastogen was used as a positive control in order to assess the sensitivity of the test system
- Route of administration: oral gavage
- Doses / concentrations: 65 mg/kg bw
- Vehicle: physiol. saline

Examinations

Tissues and cell types examined:

Bone marrow and erythrocytes

Details of tissue and slide preparation:

SAMPLING TIMES
Time after dosing:
- 24 hours: negative control, 270 and 432 mg/kg bw, postive control
- 48 hours: negative control and 432 mg/kg bw
- 72 hours: negative control and 432 mg/kg bw

DETAILS OF SLIDE PREPARATION:
- Procedure: Bone marrows were flushed from the marrow cavity of the femur with bovine solution (6 % in Physiol. saline). Bone marrows were placed on slides.
- Staining: The prepared slides were stained with polychrome methylene blue and eosin


METHOD OF ANALYSIS:
- Conducted "blind": yes
- No. of PCE scored for micronucleus: 1000
- Parameters checked: Ratio of PCE to NCE (%PCE) in 500 cells (measure of bone marrow toxicity), frequency of micronucleated PCE (%MNPCE)

Statistics:

The results were analysed for significant difference from the corresponding negative control group using a one-sided Students -t- test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No reduction in %PCE compared to controls. Tested was performed at the MTD

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range 1: 20 (10), 200 (100) and 2000 (1000) mg/kg bw
- Dose renge 2: 500 (250), 1000 (500) and 1500 (750) mg/kg bw
- Duration: 4 days
- No. of animals: 2 females/dose (trial 1) and 5/sex/dose (trial 2)
- Frequency of treatment: single oral dose
- Mortality: In first trial, 2/2 at 2000 mg/kg bw. In second trial; 1/sex at 500 mg/kg bw; 5/sex at 1000 mg/kg bw) and 5/5 males at 1500 mg/kg bw (due to the high toxicity of the test sample seen in males at 1500 mg/kg bw, the corresponding females were not dosed at this level.

The MLD/4 was determined graphically to be 540 (270)mg/kg. From this the 80% and 50% MLD levels were calculated to be 432 (216)mg/kg and 270 (135) mg/kg. These dose levels were then administered in the main study.

RESULTS OF DEFINITIVE STUDY
Clinical signs: At the 432 (216)mg/kg dose level of the test sample of Substance H109360 one male and one female were found dead, and three males and three females were killed in extremis. (These animals were replaced by the satellites)

Induction of micronuclei (Incidence MN/1000 cells):
- 24 hours: 2.1, 1.8, 2.0 and 14.0** for control, 270 mg/kg bw, 432 mg/kg bw and postive control, respectively (** p< 0.01)
- 48 hours: 0.8 and 2.0* for control and 432 mg/kg bw (* p< 0.05)
- 72 hours: 1.5 and 1.6 for control and 432 mg/kg bw

Ratio of PCE/NCE (% PCE):
- 24 hours: 41.8, 38.6, 38.1 and 31.0 * for control, 270 mg/kg bw, 432 mg/kg bw and postive control, respectively (* p< 0.05)
- 48 hours: 38.3 and 34.9 for control and 432 mg/kg bw
- 72 hours: 41.7 and 38.3 for control and 432 mg/kg bw

- Appropriateness of dose levels and route: The test is valid as the positive control illicited a significant elevation of the MNPCEs. In addition, the test substance was tested at the maximum tolerable dose as illustrated by the number of deaths observed with the test sample of Substance H109360 at a dose level of 432 (216)mg/kg

- Statistical evaluation: A statistically significant increase in the frequency of MNPCE was observed in animals treated with the test sample of Substance H109360 at a dose level of 432 (216) mg/kg bw at the 48 hour sampling time. Such a response is considered to be of questionable biological significance, since the level of MNPCE observed in this treatment group was well within the expected control levels of MNPCE, and is lower than the levels of MNPCE observed with the negative control group at the 24 hour sampling time, which according to the guideline OECD 474 vis the most appropriate sampling time after single dosing

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of this study, te test substance (Substance H109360) was not mutagenic (clastogenic) in mammals.