Registration Dossier

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Golpanol PAP / 2-Propyn-1-ol, compd. with methyloxirane
- Physical state: liquid
- Analytical purity: 54.7 area %
- Impurities (identity and concentrations): 33.9 g/100 g water, propargyl alcohol 0.6 area %, propylene glycol 5.3 area %, higher alcoxylated products 5.4 area %
- Lot/batch No.: 88767924U0
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor,
and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: mean value 36.3 g (SD +-1.7 g) first main-experiment; mean value 34.7 g (SD +- 1.9 g) second main-experiment
- Assigned to test groups randomly: [yes]
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: for a minimum of five days after their arrival

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2 °C
- Humidity (%): 35 - 75 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: [water]
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals.
The administered volume was 10 mL/kg b.w. including test substance.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was dissolved in sterile water. The vehicle was chosen due to its relative non-toxicity for the animals. All animals received a single standard volume orally.
Duration of treatment / exposure:
single oral application
Frequency of treatment:
once
Post exposure period:
24 or 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
125, 250 and 500 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
7 males for the test substance, 5 males for controls
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration potent inducer of micronuclei
- Doses / concentrations: 40 mg/kg bw.

Examinations

Tissues and cell types examined:
Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration.
The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of approximately 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight. The animals received the test item, the vehicle, or the positive control substance once orally. Seven males were treated per dose group and sampling time. However, one animal of the highest dose group (48 h sampling time) died 48h after administration. Five males each were treated for each vehicle and the positive control group. The animals of all dose groups, except the positive control were examined for acute clinical signs at intervals of around 1 h, 2 - 4 h, 6 h, 24 h, and/or 48 h after administration of the test item and vehicles.
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.

DETAILS OF SLIDE PREPARATION and METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
All animals per sex and test group were evaluated as described.
Evaluation criteria:
The study was considered valid as the following criteria are met:
- at least 5 animals per test group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the vehicle control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the vehicle control.

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 (nonparametric Mann-Whitney test (8)) are used as an aid in evaluating the results, if necessary. However, 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.
Statistics:
Statistical methods (nonparametric Mann-Whitney test are used as an aid in evaluating the results, if necessary.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
mortality in one animal at 500 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Two animals of each sex treated in the pre-experiments received the test item 2-Propyn-1-ol, compd. with methyloxirane dissolved in sterile water once orally. The volume administered was 10 mL/kg b.w.. The following dose levels were tested 500, 1000 and 1500 mg/kg bw.
Clincical signs observed: 500 mg/kg bw: Eyelid closure and Ruffled fur; 1000 mg/kg bw: Reduction of spontaneous activity, Eyelid closure and Ruffled fur; 1500 mg/kg bw: Reduction of spontaneous activity, Abdominal position, Eyelid closure, Apathy, Tumbling, Hunchback, Hypothermia, Ruffled fur and Death.
On the basis of these data 1000 mg/kg b.w. were estimated to be suitable as highest dose in the main experiment (which was later reduced due to overt toxicity and death). No substantial sex specific differences were observed with regard to clinical signs. In accordance with the guidelines the main study was performed using males only.


RESULTS OF DEFINITIVE STUDY
In the first main experiment for each test item dose groups 7 males received once orally administrations of 2-Propyn-1-ol, compd. with methyloxirane dissolved in sterile water. The volume administered was 10 mL/kg b.w.. The symptoms of toxicity observed following treatment were: 1000 mg/kg bw: Reduction of spontaneous activity, Abdominal position, Eyelid closure, Ruffled fur, Hunchback and death of 4/7 animals; 500 mg/kg bw: Reduction of spontaneous activity and Ruffled fur; 250 mg/kg bw: Ruffled fur. The animals treated with the negative control (sterile water) did not express any toxic reaction.
High mortality was observed after treatment with the high dose in the 48 hours group (male no. 37, 39, 40 and 41). The animals were found dead in their cage. Due to this mortality, an additional low dose and a high dose group (48 hours post-treatment) were included in a second experiment in order to fulfil the OECD guideline requirements for a valid study.
In the second main experiment for each test item dose groups 7 males received also once orally administrations of 2-Propyn-1-ol, compd. with methyloxirane dissolved in sterile water. The volume administered was 10 mL/kg b.w.. The symptoms of toxicity observed following treatment were: 500 mg/kg bw: Reduction of spontaneous activity, Ruffled fur and death; no clinical signs were observed at 125 mg/kg bw. The animals treated with the negative control (sterile water) did not express any toxic reaction.

Any other information on results incl. tables

The test item 2-Propyn-1-ol, compd. with methyloxirane was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was dissolved in sterile water, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

At least five males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

Based

on results of pre-experiments doses of 250, 500 and 1000 mg/kg b.w. were selected for the main experiment. However, due to high mortality observed at 1000 mg/kg (4 of 7 males of the 48 hours group) this dose was not appropriate for evaluation in accordance with the guidelines (less than 5 animals available for evaluation in the 48 hour group and maximum tolerated dose level clearly exceeded). Additional groups of male mice were treated including vehicle and positive control in a second main experiment in order to fulfil the OECD guideline requirements for a valid study. Finally, following dose levels of the test item were investigated and evaluated :

24 h preparation interval: 125b), 250a), and 500a)mg/kg b.w.
48 h preparation interval: 500b)mg/kg b.w.

                  a)First main experimentb)Second main experiment

In the second main experiment one of 7 males male died after treatment with the high dose (500 mg/kg b.w., 48 hours post-treatment).

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that 2-Propyn-1-ol, compd. with methyloxirane did not exert a cytotoxic effect in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with 2-Propyn-1-ol, compd. with methyloxirane were below or near to the value of the vehicle control groups. Only the micronucleus frequency found in the high dose group (500 mg/kg, 48 hours treatment) was statistically significantly higher (0.108%) compared to the concurrent vehicle control value (0.030%) which was incidentally quite low. However, the micronucleus frequency in the 500 mg/kg (48h) group as well as in all other test item treated dose groups at any preparation interval were very well within the laboratory’s historical vehicle control data (0.010-0.250%, mean 0.108%). Thus, the observed statistical significance at 500 mg/kg (48h) was not considered to have any biological relevance. 


40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item 2-Propyn-1-ol, compd. with methyloxirane did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Applicant's summary and conclusion