Urea, reaction products with diethylene glycol, formaldehyde and glyoxal

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Fixapret PC, vor Magnesiumchlorid-Zugabe

IUPAC Name:

Fixapret PC, vor Magnesiumchlorid-Zugabe

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Fixapret PC, vor Magnesiumchlorid-Zugabe
- Physical state: liquid
- Analytical purity: Reaction product of urea, formaldehyde, glyoxal and diethylene glycol, content: 62.1 % in water
- Impurities (identity and concentrations): 37.9 % water (solvent)
- Lot/batch No.: 153
- 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; avoid temperatures < 5°C >30°C

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: mean: 28.8g
- Assigned to test groups randomly: [yes: The animals were assigned to the test groups according to a randomization plan prepared
with an appropriate computer program.]
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): Fully air-conditioned rooms with central air conditioning
- 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 test substance is an aqueous solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed and topped up with the vehicle deionized water to achieve the following concentrations: 80.5 mg/mL, 161 mg/mL and 322 mg/mL test substance refering to 50 mg/mL, 100 mg/mL and 200 mg/mL active ingredients, respectively.

Duration of treatment / exposure:

The animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance and the vehicle. The positive controls, both dissolved in deionized water were administered to male animals once orally (CPP) or intraperitoneally (VCR) each in a volume
of 10 mL/kg body weight.

Frequency of treatment:

once

Post exposure period:

The animals were sacrificed 24 hours (all test substance concentrations, vehicle, both positive controls) and 48 hours (highest test substance
concentration, vehicle) after the treatment, respectively.

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg
Basis:
other: active ingredient

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPP); vincristine sulfate (VCR)
- Justification for choice of positive control(s): The stability of CPP and VCR is well-defined under the selected conditions, since both substances are well-established reference clastogens and aneugens, respectively.
- Route of administration: Orally (CPP), Intraperitoneally (VCR)
- Doses / concentrations: 20 mg (CPP), 0.15 mg (VCR)

Examinations

Tissues and cell types examined:

polychromatic erythrocytes (PCE) and normochromatic erythrocytes (= normocytes / NCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, at 3 220 mg/kg body weight (corresponds to 2 000 mg/kg body weight of the active ingredients) recommended as the highest dose according to the OECD Guideline all animals (male and female) survived without any signs of toxicity. Besides, there were no distinct differences in clinical observations between males and females. Thus, only male animals were used for the
cytogenetic investigations as requested by the current OECD Guideline 474.
Based on the data of the pretest a dose of 3 220 mg/kg body weight (corresponds to 2 000 mg/kg body weight of the active ingredients) was administered as the highest dose in the present cytogenetic study. 1 610 mg/kg (corresponds to 1 000 mg/kg body weight of the
active ingredients) and 805 mg/kg body weight (corresponds to 500 mg/kg body weight of the active ingredients) were administered as further
doses.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the study, the animals were weighed and the substance to be administered or the amount of volume was related to the specific weight of the individual animals.
Male animals per sacrifice interval were given Fixapret PC, vor Magnesiumchlorid-Zugabe dissolved in deionized water at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight active ingredients. Treatment consisted of a single administration. The volume
administered was 10 mL/kg body weight. The animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the
test substance and the vehicle. The positive controls, both dissolved in deionized water were administered to male animals once orally (CPP) or intraperitoneally (VCR) each in a volume of 10 mL/kg body weight. The animals were sacrificed 24 hours (all test substance
concentrations, vehicle, both positive controls) and 48 hours (highest test substance concentration, vehicle) after the treatment, respectively.

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by Schmid and Salamone et al.
- The animals were anesthetized with isoflurane and afterwards sacrificed by cervical dislocation. Then the two femora were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (FCS with EDTA) which was pre-heated up to 37°C (about 2 mL/femur).

Removal of nucleated cells
The procedure applied is based on the method described by Romagna et al.
Preparing the cellulose columns
- A 13-mm filter disk with a pore size of 8 μm was placed at the bottom of a 5-mL plastic syringe and a 10-mL plastic syringe.
- Equal parts by weight of microcrystalline cellulose, Sigmacell® type 50 and α-cellulose fibers, were mixed thoroughly.
- 1st cellulose column: About 500 mg of this cellulose mixture was added to the column of the 5-mL plastic syringe. To achieve optimal packing, the syringe was tapped on a hard surface until the 2 mL calibration mark of the syringe was reached.
- 2nd cellulose column: About 1000 mg of the cellulose mixture was added to the column of the 10-mL plastic syringe. To achieve optimal packing, the syringe was tapped on a hard surface until the 3 mL calibration mark of the syringe was reached.
Cell separation
- The bone marrow suspension was mixed gently before transferring to the 1st cellulose column. As soon as the suspension was fully soaked into the column 4 mL HBSS (Hanks Balanced Salt Solution with Ca and Mg) was added. This mixture was eluted for about 10 - 20 minutes.
- The eluate containing the erythrocytes was mixed gently before transferring to the 2nd cellulose column. As soon as the suspension was fully soaked into the column 4 mL HBSS (Hanks Balanced Salt Solution with Ca and Mg) was added. This mixture was eluted for about 10 - 20 minutes. The eluate containing the erythrocytes was centrifuged at 300 x g for 5 minutes.
- The cell suspension was resuspended with 1 mL PBS (Phosphate Buffered Solution with Ca and Mg) and was stored on ice until preparation of cytospin slides (at least two slides per animal).
- Slides equipped with cell funnels was clamped into the rotor of the cytospin (Cellspin I-12, Tharmac GmbH, Waldsolms, Germany). Then 200 μL cell suspension was added in each cell funnel and was centrifuged at 1 400 rpm (approx. 220 x g) for 7 minutes.
- After drying overnight the slides were stained.

Staining of the slides
- The slides were stained with pure May-Grünwald solution for about 4 minutes. Then staining with a mixture of May-Grünwald solution and deionized water (ratio 1:1) for about 4 minutes was followed.
- After having briefly been rinsed in deionized water twice the slides were stained with Giemsa solution (7.5% [v/v] in deionized water) for about 15 minutes.
- Finally the slides was rinsed twice in deionized water and were dried overnight.
The preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
MICROSCOPIC EVALUATION
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10 000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The
following parameters were recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value. A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice interval.
- Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the bone
marrow, means the target determined for genotoxic effects.
- Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4) [d = diameter of micronucleus, D = cell diameter]
The size of micronuclei may indicate the possible mode of action of the test substance ( 10), i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4).
Slides were coded before microscopic analysis.
Since the absolute values shown were rounded, but further calculation was based on unrounded values, there may be deviations in the relative values given.

Acceptance criteria
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data for PCEs.
- The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Evaluation criteria:

A finding is considered positive if the following criteria are met:
- Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative
frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.

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:

MICROSCOPIC EVALUATION
All concentrations given in this paragraph refer to the content of active ingredients.
The single oral administration of the vehicle deionized water in a volume of 10 mL/kg body weight led to 1.6‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.7‰ after the 48-hour sacrifice interval, respectively.
After the single administration of the highest dose of 2 000 mg/kg body weight, 1.7‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.9‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of 1.3‰ (1 000 mg/kg group) and 1.8‰ (500 mg/kg group) were detected at a sacrifice interval of 24 hours in each case.
The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (21.9‰) in the number of polychromatic erythrocytes containing exclusively small micronuclei, as expected.
Vincristine sulfate, a spindle poison, produced a statistically significant increase (25.2‰) in the number of polychromatic erythrocytes containing micronuclei. A significant portion increase, 4.2‰ was attributable to large micronuclei.
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups at any of the sacrifice intervals.
No inhibition of erythropoiesis induced by the treatment of mice with Fixapret PC, vor Magnesiumchlorid-Zugabe was detected.
CLINICAL EXAMINATIONS
The single oral administration of the vehicle in a volume of 10 mL/kg body weight was tolerated by all animals without any clinical observations.
Besides the administration of the test substance did not lead to any clinical signs of toxicity.
Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine sulfate in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

Any other information on results incl. tables

According to the results of the present study, there are thus no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (500 mg/kg, 1 000 mg/kg and 2 000 mg/kg active ingredients) or between the two sacrifice intervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range. Nor were large micronuclei (d ≥ D/4)

observed either in the vehicle control group or in the three dose groups treated with Fixapret PC, vor Magnesiumchlorid-Zugabe.

In this study, after single oral administration of the vehicle deionized water the ratio of PCEs/NCEs in the vehicle control animals at both sacrifice intervals was within the normal range for the animal strain selected. Besides the number of cells containing micronuclei in

these vehicle control animals was within the range of the historical vehicle control data for PCEs.

In addition, both positive control substances, cyclophosphamide and vincristine sulfate, induced a statistically significant increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.