Reaction mass of ethoxylated N-oleyl-1,3-propanediamine, hydrofluorides of and ethoxylated N-palmityl-1,3-propanediamine, hydrofluorides of and ethoxylated N-stearyl-1,3-propanediamine, hydrofluorides of and olaflur

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

Study period:

2001-09-27 to 2001-09-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Mammalian Erythrocyte Micronucleus Test

Test material

Test material information

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 960
- Expiration date of the lot/batch: 2002-04

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: Mouse
Strain: NMRI
Stock: Crl:NMRI BR
Age (at start of adaptation): 22 d (male); 24 d (female)
Body Weight (at administration): 18-23g (male); 17-22g (female)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- 0.8 % aqueous hydroxypropyl-methylcellulose gel (also used as negative control; administered orally by gavage)
- Aqua ad iniectabilia (used as vehicle for positive control Cyclophosphamide)

Details on exposure:

Olaflur and the negative control (vehicle only) were administered orally via gavage, the administration volume was 10 mL/kg bw. The positive control (Cyclophosphamide) was administered by intraperitoneal injection, the administration volume was 20 mL/kg bw.

Duration of treatment / exposure:

Sampling times (main study) = 24 and 48 h after administration
(except positive control: only 24h)

Frequency of treatment:

one single administration

Post exposure period:

Observation period was 3 d

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

The dose levels employed were based on a preliminary DRF-study of Olaflur in mice. Dose levels tested 30, 100, 300, 1000 and 2000 Olaflur/ kg b.w.

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Five male and five female NMRI mice were employed per sampling interval and group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (27 mg/kg b.w.) was employed as a positive control and administered once by intraperitoneal injection.

Examinations

Tissues and cell types examined:

- bone marrow
- erythrocytes

Details of tissue and slide preparation:

Preparation of bone marrow
The mice were sacrificed. After having removed some of the muscle, the femurs were excised below the knee and at the iliac joint. The bone marrow was flushed out and centrifuged at 150 xg , 3-5 minutes. The supernatant was removed and the sediment resuspended in a drop of calf serum. Then a smear of 30-60 mm length was prepared.
One dry, the preparations were immediately fixed in solvent methanol for 5 minutes. Cells were stained (filtered Mayers Haemallum), rinsed with cold tap water, further stained in 0.5% w/v ethanolic eosin solution. The slides were left to air-dry before being dipped in xylene and mounted.

Analysis
2000 polychromatic erythrocytes/ animal were scored for incidence of micronuclei via microscope. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) was determined /animal by counting a total of 1000 erythrocytes.

Evaluation criteria:

Evaluation of data
After completion of scoring, the ratio of PCE/NCE for each animal and the mean for each group was calculated, the individual and group mean of micronucleated PCE/1000 also determined.
PCE/NCE ratios were determined to evaluate possible bone marrow toxicity.
Data analysis was carried out by a comparison of treatment and vehicle control groups, using a chi-square test corrected for continuity according to YATES (COLQUHOUN, 1971) as recommended by the UKEMS guidelines.
The PCE/NCE ratios and frequencies of micronucleated PCE in vehicle control animals were compared to historical control data.
The numbers of micronucleated PCE in each treated group (males and females, separately and combined) were compared with the numbers in vehicle control groups.

Statistics:

Neither PCE/NCE ratios nor number of micronucleated PCE were influenced by the treatment with the test substance.
The numbers of micronucleated PCE were also similar to those seen in historical controls.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

1000 mg/kg bw: on 2.day after dosing, 2 males died prematurely; remaining mice: no clinical signs of toxicity

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Clinical signs (male and females):
- 100 mg/kg bw: no clinical signs of toxicity
- 300 mg/kg bw: no clinical signs of toxicity
- 1000 mg/kg bw: on 2.day after dosing, 2 males died prematurely; remaining mice: no clinical signs of toxicity

Any other information on results incl. tables

Pre-test DRF study

Dose levels = 30, 100, 300, 1000 and 2000 mg Olaflur/kg b.w. p.o:

No clinical signs of toxicity were noted in this test up to the dose of 1000 mg/kg bw, 2000 mg/kg bw was within the lethal range.

Hence, 1000 mg/kg bw was considered to be the maximum tolerated dose and was used as top dose level in the main study.

Main study

No clinical signs of toxicity at 100 and 300 mg/kg bw. Treatment with 1000 mg/kg bw p.o. led to premature death of 2 males of the 20 mice treated.

Micronucleus assay

No substance–related increase in micronucleated polychromatic erythrocytes (PCE) was observed in the treated groups after 24 and 48 h (sampling times), compared with corresponding negative control group. even at doses considering lethality in some male test animals (1000 mg/kg bw p.o.). The positive control group receiving Cyclophosphamide exhibited significant increase in number of micronucleated polychromatic erythrocytes.

Applicant's summary and conclusion

Conclusions:

Under the present test conditions, Olaflur did not reveal any indication of mutagenic properties in the mouse micronucleus test, tested up to the maximum tolerated dose level of 1000 mg/kg bw. In the same system, Cyclophosphamide (positive control) induced significant damage.

Executive summary:

Olaflur was assayed in an in vivo bone marrow micronucleus test in the mouse for the detection of damage to the chromosomes or the mitotic apparatus using the maximum tolerated dose level. The dose levels employed were based on a preliminary dose-range finding study of Olaflur in mice. The no-effect dose level was 1000 mg Olaflur/kg b.w. p.o., 2000 mg Olaflur/kg b.w. p.o. was within the lethal range.

Hence, for the main study Olaflur was administered at doses of 100, 300 and 1000 mg Olaflur/kg b.w. p.o. to groups of five male and five female mice each killed 24 and 48 h (vehicle and high dose only) after administration of the dose.

No signs of toxicity were noted at the tested dose levels of 100 and 300 mg/kg bw. Treatment with 1000 mg/kg b.w. led to premature death of 2 males of 20 mice. Immediately after sacrifice bone marrow smears were prepared.

The negative (vehicle) control in the study was 0.8 % aqueous hydroxypropyl-methyl-cellulose gel, also administered orally.

Cyclophosphamide (27 mg/kg b.w.) was employed as a positive control and administered once by intraperitoneal injection.

Two sampling times were used for Olaflur and the negative control: 24 and 48 h (vehicle and high dose only) after administration, for the positive control one sampling time (24 h) was used. 2000 erythrocytes were evaluated per animal.

The maximum tolerated dose of 1000 mg Olaflur/ kg b.w. p.o. resulted in no increase in the incidence of micronucleated polychromatic erythrocytes (PCE). The incidences were 1.7 and 1.7 micronuclei per 1000 PCEs for the 24- and 48-h sampling points, respectively (control: 2.5 and 2.6, respectively). No increase was noted in the lower dose levels, either. Cyclophosphamide (27 mg/kg b.w. i.p.) resulted in a significant increase to 10.5 micronuclei per 1000 PCEs. The ratio of polychromatic to normochromatic erythrocytes was not influenced.

However, toxicokinetic studies clearly demonstrate a good bioavailability of the test item and allow the conclusion that the target organ (bone marrow) was reached by the test item.