Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-07-14 to 2005-04-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian bone marrow chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test
Sex:
male/female
Details on test animals and environmental conditions:
Strain: NMRI
Source: RCC Ltd., Animal Breeding Services; CH-4414 Füllinsdorf
Number of animals: 90 (54 males/36 females)
Initial age at start of acclimatization: Males: 5 - 8 weeks
Females: 7 - 10 weeks
Acclimatisation: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 36.0 g (SD ± 1.8 g)
females mean value 36.0 g (SD ± 1.9 g)

According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of RCC - CCR for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.
The animals were distributed into the test groups at random and identified by cage number.

Husbandry

The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
Bedding: granulated soft wood bedding
(Harlan Winkelmann GmbH, D-33178 Borchen)
Feed: pelleted standard diet, ad libitum
(Harlan Winkelmann GmbH, D-33178 Borchen)
Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
Environment: temperature 22 ± 3 °C
relative humidity 30 - 83 % artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
The test item was formulated in aqueous DMSO (30%). The vehicle was chosen to its relative non-toxicity for the animals.
Details on exposure:
Pre-Experiment for Toxicity
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 i.p. with the test item and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

Dose Selection
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
The volume to be administered should be compatible with physiological space available.
Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

Study Procedure

Test Groups:
Six males and six females were assigned to each test group.

Treatment:
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time.

Analysis of the Test Item Concentration in Blood
In order to quantify the concentration of the test item in blood 3 additional males per sampling interval were treated with 50 mg test item/kg b.w. intraperitoneally. 20 and 40 minutes as well as 1 and 4 hours after the treatment, the animals were sacrificed and their blood was collected and stored at – 80 °C until shipment on dry ice to the sponsor (Henkel KGaA,VTF-HSA, Henkelstraße 67, D-40191 Düsseldorf). Due to a technical error (no anti- coagulant was added to the blood samples), this part of the experiment had to be repeated.
The test item concentration in the blood samples was analysed in a separate study by the sponsor.
Duration of treatment / exposure:
The animals received the test item, the vehicle or the positive control substance once.
Frequency of treatment:
The animals received the test item, the vehicle or the positive control substance once.
Post exposure period:
The animals of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4h, 6 h and 24 h after administration of the test item.
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
On the basis of 8 pre-exepriments, 50 mg/kg b.w. were estimated to be suitable as the high dose. The volume administered was 10 mL/kg b.w.
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
The volume administered was 10 mL/kg b.w.
Dose / conc.:
12.5 mg/kg bw/day (nominal)
Remarks:
The volume administered was 10 mL/kg b.w.
No. of animals per sex per dose:
In the main experiment for the highest dose group 24 animals (12 males, 12 females) received i.p. a single dose of 50 mg/kg b.w. of naphthalene-1,5-diol formulated in aqueous DMSO (30%) .
For the mid dose group 12 animals (6 males, 6 females) received i.p. a single dose of 25 mg/kg b.w. naphthalene-1,5-diol formulated in aqueous DMSO (30%).
For the low dose group 12 animals (6 males, 6 females) received i.p. a single dose of 12.5 mg/kg b.w. naphthalene-1,5-diol formulated in aqueous DMSO (30%) .
The volume administered was 10 mL/kg b.w..
Control animals:
yes, concurrent vehicle
Positive control(s):
Name: CPA; Cyclophosphamide
Supplier: Sigma-Aldrich Vertriebs GmbH
82041 Deisenhofen
Catalogue no.: C 0768 (purity: > 98%)
Dissolved in: deionised water
Dosing: 40 mg/kg b.w.
Route and frequency of administration: intraperitoneally, once
Volume administered: 10 mL/kg b.w.
Solution prepared on day of administration.
The stability of CPA at room temperature is sufficient. At 25°C only 3.5 % of its potency is lost after 24 hours (7).

Examinations

Tissues and cell types examined:
The animals were anaesthetised with CO2 and sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded.
Details of tissue and slide preparation:
A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.
Evaluation criteria:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and total erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining 6th animal of each sex is usually evaluated in case an animal dies in its test group spontaneously.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Toxic Symptoms in the Main Experiment

In the main experiment for the highest dose group 24 animals (12 males, 12 females) received i.p. a single dose of 50 mg/kg b.w. A 018 formulated in aqueous DMSO (30%) . The volume administered was 10 mL/kg b.w..

The animals treated with 50 mg/kg b.w. expressed toxic reactions as shown in the table:

Toxic

 

Reactions

hours post-treatment hours post-treatment

male / female

1 h

2-4 h

6 h

24 h

48 h*

reduction of spontaneous activity

12/12

12/12

12/12

12/9

3/2

abdominal position

12/12

12/12

12/12

0/0

0/0

ruffled fur

12/12

12/12

12/12

12/5

2/1

apathy

1/2

0/0

0/0

0/0

0/0

*: data only from 6 animals per sex.

For the mid dose group 12 animals (6 males, 6 females) received i.p. a single dose of   25 mg/kg b.w. A 018 formulated in aqueous DMSO (30%) . The volume administered was 10 mL/kgb.w..

The animals treated with 25 mg/kg b.w. expressed toxic reactions as shown in the table:

 

toxic reactions

hours post-treatment

male / female

1 h

2-4 h

6 h

24 h

reduction of spontaneous activity

6/6

6/6

6/6

4/3

abdominal position

1/2

4/5

3/2

0/0

ruffled fur

5/6

6/6

6/6

4/3

For the low dose group 12 animals (6 males, 6 females) received i.p. a single dose of

12.5 mg/kg b.w. A 018 formulated in aqueous DMSO (30%) . The volume administered was 10 mL/kg b.w..

The animals treated with 12.5 mg/kg b.w. expressed toxic reactions as shown in the table:

 

toxic reactions

hours post-treatment

male / female

1 h

2-4 h

6 h

24 h

reduction of spontaneous activity

2/3

6/6

6/6

4/3

ruffled fur

3/2

3/3

1/2

2/1

Summary of Micronucleus Test Results

test group

dose mg/kg b.w.

sampling time (h)

PCEs with micronuclei (%)

range

PCE per 2000 erythrocytes

 

vehicle

 

0

 

24

 

0.105

 

0 -4

 

1099

 

test item

 

12,5

 

24

 

0.080

 

0 -4

 

1124

 

test item

 

25

 

24

 

0.105

 

0 -4

 

1096

 

test item

 

50

 

24

 

0.080

 

0 -4

 

1082

positive control

 

40

 

24

 

2.270

 

20 -76

 

1035

 

test item

 

50

 

48

 

0.090

 

0 -4

 

1089

 

Biometry

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

 

Vehicle control versus test group

 

Significance

 

p

 

12.5 mg A 018/kg b.w.; 24 h

 

n.t.

 

-

 

25 mg A 018/kg b.w.; 24 h

 

n.t.

 

-

 

50 mg A 018/kg b.w.; 24 h

 

n.t.

 

-

 

40 mg CPA/kg b.w.; 24 h

 

+

 

< 0.0001

 

50 mg A 018/kg b.w.; 48 h

 

n.t.

 

-

 

-     =    not significant

+    =    significant;

n.t =    not tested, as the mean micronucleus frequency was not above the vehicle controlvalue


Applicant's summary and conclusion

Conclusions:
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)) will be used as an aid in evaluating the results. 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.
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Executive summary:

The test item naphthalene-1,5-diol 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 formulated in aqueous DMSO (30%) , which was also used as vehicle control. The volume administered i.p. 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.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

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

The following dose levels of the test item were investigated:

24 h preparation interval: 12.5, 25 and 50 mg/kg b.w.. 48 h preparation interval: 50 mg/kg b.w..

As estimated by pre-experiments 50 mg naphthalene-1,5-diol per kg b.w. was the highest applicable dose without significant effects on the survival rates. At this dose the animals showed clear signs of toxicity. At a higher dose (75 mg/kg) one male and one female animal died.

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 A 018 had no cytotoxic properties in the bone marrow. The analysis of the blood samples of the males (see annex 2) treated with 50 mg test item /kg b.w. showed, that the test item could be quantified in the blood of the treated animals only in the blood samples taken 20 minutes after the treatment (2.5, 1.0 and 0.5 µg/mL). The samples from the later time points did not contain any quantifiable levels of the test item (data of the 1 and 4 h samplings not included). Thus, the bioavailability of the test item could be confirmed.

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 naphthalene-1,5-diol were below or near to the value of the vehicle control group.

40 mg/kg b.w. cyclophosphamide administered i.p. 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 did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.