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Toxicological information

Genetic toxicity: in vivo

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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
Study period:
August 30, 2004 to September 27, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, well performed and reported according to good scientific standards
Cross-reference
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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): EUF (TPI 1618)
- Substance type: Formaldehyde releaser
- Physical state: clear colourless liquid
- Analytical purity: Reaction product of urea, etyleneglycol and paraformaldehyde. Therefore purity is not exactly known
- Composition of test material, percentage of components: Reaction product of urea, etyleneglycol and paraformaldehyde. Releasable formaldehyde: 46.1%
- Lot/batch No.: LJ 526/37
- Expiration date of the lot/batch: January 2006
- Storage condition of test material: at room temperature, closed container

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: 8-12 weeks at delivery
- Weight at study initiation: males:31.0 – 36.6 g; females: 42.7 – 30.2 g
- Assigned to test groups randomly: yes, under following basis: unambiguously identification
- Fasting period before study: 18 hours
- Housing: individually in Macrolon Type II cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+-2
- Humidity (%): 55+-15
- Air changes (per hr): 10-20
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From September 08, 2004 to September 21, 2004

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: absence of water to maintain a stable solution
- Concentration of test material in vehicle: 3, 6 and 12%
- Amount of vehicle (if gavage): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in corn oil at a 3, 6 and 12% suspension to obtain the required concentrations for the dosing solutions.
Cyclophosphamide was dissolved in water

Duration of treatment / exposure:
Negative control group: 5 m/5 f; sampling time 24 and 48 h
Positive control group: 5 m/5 f, sampling time 24 h
Low dose group: 5 m/5 f, sampling time 24 h
Mid dose group: 5 m/5 f, sampling time 24 h
High dose group: 5 m/5 f, sampling time 24 and 48 h
Frequency of treatment:
All animals recieved a single oral treatment by gavage
Post exposure period:
24 h after treatment (all exposure groups and control groups)
48 h after treatment (highest dose group and negative control group)
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 300, 600 and 1200 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 males/5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): to secure the sensitivity of the test system
- Route of administration: oral gavage
- Doses / concentrations: 60 mg/kg bw

Examinations

Tissues and cell types examined:
Tissue: Bone marrow
Type of cells: Erythrocytes in bone marrow
Parameter: -Number of micronucleated cells per 2000 polychromatic erythrocytes
-Number of polychromatic erythrocytes per 200 blood cells
-Ratio polychromatic/normochromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Doses were selected after a preliminary dose reange finding

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
negative control: 5 males + 5 females per sampling time (24 and 48 h)
positive control: 5 males + 5 females per sampling time (24 h)
Low dose group: 5 m/5 f, sampling time 24 h
Mid dose group: 5 m/5 f, sampling time 24 h
High dose group: 5 m/5 f, sampling time 24 and 48 h

DETAILS OF SLIDE PREPARATION:
Bone marrow smears were prepared and stained according to Pappenheim

METHOD OF ANALYSIS:
Microscopic evaluation
Evaluation criteria:
A genotoxic effect is claimed if a dose-related increase in the number of polychromatic micronucleated erythrocytes or a statistically significant increase in the number of micronucleated cells in a single dose group at a sampling time is observed, but biological relevance of the results is considered first.
Statistics:
U-test according to Mann-Whitney

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: A female animal died at 2000 mg/kg bw, wheras no male animal died. At 1000 mg/kg bw. there were no signs in males or females, however, at 650 mg/kg bw, one male showed clinical signs but recovered within 3 hours.

- Rationale for exposure: In the light of these findings, 300, 600 and 1200 mg/kg bw were chosen for the main study.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): 48 h after EUF application, male and female animals of the high dose groups demonstrated a slight decrease rather than an increase in the mean frequency of micronucleus formation. The observed reduction in micronucleus formation could be due to slight inhibition of blood formation, indicated by a reduction in the ratio of PCE/200 RBC in the bone marrow of EUF exposed animals
- Appropriateness of dose levels and route: approriate, as clinical signs were observed
- Statistical evaluation: no statistically significant effects

Any other information on results incl. tables

Table 1: Induction of Micronuclei (Number per 2000 PCE, Mean ± SD

Groups/Dosages

Males

Females

Negative controls (24 h)

4.6 ± 2.7

3.2 ± 0.8

Positive controls (24 h)

122.2 ± 14.7 **P<0.01

84.6 ± 31.0 **P<0.01

300 mg EUF/kg bw (24 h)

4.6 ± 1.6

4.6 ± 1.9

600 mg EUF/kg bw (24 h)

4.6 ± 1.5

3.4 ± 1.9

1200 mg EUF/kg bw (24 h)

4.8 ± 2.8

4.0 ± 2.0

Negative contols (48 h)

5.4 ± 2.9

3.4 ± 2.6

1200 mg EUF/kg bw (48 h)

4.2 ± 2.9

2.6 ± 2.4

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
EUF (TPI 1618) is not considered to be clastogenic in vivo in the mouse micronucleus test in the bone marrow cells after a single oral administration
Executive summary:

The study represents a micronucleus assay in bone marrow cells of the mouse with EUF (TPI1618) given once, orally, up to the maximum tolerable dose. The test was performed according to OECD 474 to NMRI mice. Doses used were 300, 600 and 1200 mg/kg bw. Evaluation times were after 24 hours for positive and negative control and for the 300 and 600 mg/kg dose levels. The vehicle control and high dose group (1200 mg/kg bw.) was evaluated 24 and 48 hours post administration.

There was a slight tendency towards inhibition of blood formation especially 48 h after application in the high dose as shown by slightly reduced numbers of polychromatic erythrocytes and PCE/NCE ratios, indicating that some EUF or EUF hydrolytes reached the bone marrow compartment. A lack of DNA reactivity was already observed for some other formaldehyde releasing biocides (Richardson et al., 1983; Charles et al., 2005) and was assigned to formaldehyde detoxification mechanisms existing in vivo which prevent generation of formaldehyde levels capable of causing geno-toxicity at sites others than the direct application site.