Formaldehyde, telomer with 1,3-benzenedimethanamine, 1,3-benzenediol and ethenylbenzene

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Formaldehyde, telomer with 1,3-benzenedimethanamine, 1,3-benzenediol and ethenylbenzene

EC Number:

615-240-7

Cas Number:

710292-85-6

Molecular formula:

Not available for this UVCB

IUPAC Name:

Formaldehyde, telomer with 1,3-benzenedimethanamine, 1,3-benzenediol and ethenylbenzene

Test material form:

other: waxy solid

Details on test material:

Batch 5, 100% purity, Stable at room temperature, Expiry date: 30 June 2006

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

NMRI BR mice (SPF) were used as test system. The animals were provided by Charles River, Sulzfeld, Germany. In the main test 5 male mice were treated per sampling time in each treatment group. Young adult animals were selected (6-7 weeks old). The body weights of the mice at the start of the treatment were within 20% of the sex mean. The mice were identified by a unique number on the tail written with a marker pen. On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health. Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
The animals were housed in an air-conditioned room with approximately 15 air changes per hour and a controlled environment with a temperature of 21 ± 3°C (actual range 18.6 — 21.5°C) and a relative humidity of 30 — 70% (actual range 34 — 84%). Due to cleaning procedures or performance of functional observations in the room, temporary deviations from the maximum level for humidity (with max. 14%) occurred. Based on laboratory historical data these deviations are considered not to affect the study integrity. The room was illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.
Group housing of 5 animals per sex per cage in labelled polycarbonate cages (type MII height:14 cm) containing Woody Clean bedding (Woody-Clean type 3/4; Technilab-BMI BV, Someren, The Netherlands). Paper bedding was provided as nest material (Technilab-BMI BV). Certificates of analysis were examined and then retained in the NOTOX archives.

Free access to standard pelleted laboratory animal diet (Altromin (code VRF 1), Lage, Germany) Certificates of analysis were examined and then retained in the NOTOX archives.
Free access to tap-water. Certificates of analysis (performed quarterly) were examined and then retained in the NOTOX archives.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Beckopox VEH 2626-Polymer was suspended in corn oil (Roth, Karlsruhe, Germany), Beckopox VEH 2626-Polymer concentrations were blended and treated with ultra-sonic waves to obtain a homogeneous suspension. Beckopox VEH 2626-Polymer concentrations were dosed within
2.5 hours after preparation.

Details on exposure:

The mice received an intraperitoneal injection of a maximum tolerated (high), an intermediate and a low dose of Beckopox VEH 2626-Polymer. The route and frequency of administration and the volume administered of the negative and the positive control was the same as those of the test article. The systemic toxic signs were recorded at least once a day. The animals were weighed just prior to dosing. Selection of an adequate dose range for the micronucleus main test was based on a dose range finding study. Three dose groups, two comprising 1 male and 1 female, and one comprising 3 males and 3 females received a single dose of Beckopox VEH 2626-Polymer. The group comprising 3 males and 3 females were dosed with the highest concentration that was used for the main study. The study duration per dosing was three days. During this period mortality and physical condition were recorded at least once a day. In a dose range finding study ten animals (group A and B: 1 male and 1 female,
group C: 3 males and 3 females) were dosed intraperitoneally with 2000, 1500, and 750 mg/kg body weight (groups A, B and C, respectively).

In the main micronucleus study, five male mice were used per sampling time in each treatment group. The animals were dosed once and sampled at either 24 or 48 hours after test material administration.

Duration of treatment / exposure:

24 or 48 h

Frequency of treatment:

once

Post exposure period:

24 or 48 h

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
190 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
375 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
750 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Positive control(s):

The positive control used in the micronucleus test was cyclophosphamide (CP;
CAS no. 50-18-0; Endoxan, Asta-Werke, Germany) dissolved in physiological saline (Ziekenhuis Apotheek Noordoost-Brabant, Den Bosch, The Netherlands) dosed as a single intraperitoneal injection of 50 mg/kg body weight

Examinations

Tissues and cell types examined:

Erythrocytes from bone marrow

Details of tissue and slide preparation:

Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing, The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of fetal calf serum (Invitrogen). The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide, which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether (Merck, Darmstadt, Germany) and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. Two slides were prepared per animal. The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA¬tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in MicroMount (Klinipath) and mounted with a coverslip.
All slides were randomly coded before examination. At first the slides were screened at a magnification of 100x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000x, The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated. Observations/measurements in the study were recorded electronically using the following programme: REES Monitoring system version 1.5 (REES Scientific, Trenton, NJ, USA).

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range (mean ± three times the standard deviation): Males: 1.3%0 ± 4.3%0 indicated are means for n=251).
A test substance is considered positive in the micronucleus test if:
It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time).
A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant
(p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes.
The increase or decrease in the ratio polychromatic to normochromatic erythrocytes of the treatment groups compared to the ratio polychromatic to normochromatic erythrocytes of the negative control group is statistically evaluated using Wilcoxon Rank Sum Test (two sided test at p < 0.05).

Statistics:

Averages and standard deviations were calculated for polychromatic and normochromatic erythrocytes.
The increase or decrease in the ratio polychromatic to normochromatic erythrocytes of the treatment groups compared to the ratio polychromatic to normochromatic erythrocytes of the negative control group is statistically evaluated using Wilcoxon Rank Sum Test (two sided test at p < 0.05).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Toxicity was demonstrated in the high dose and the low dose. No changes in the PCE/NCE ratio were noted for the intermediate dose group.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Mice in all dose ranges displayed clinical signs of lethargy, rough coat and hunched posture.

Table 3 .      Mean number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes and ratio of polychromatic/normochromatic erythrocytes
	MALES
Group Treatment	Dose (mg/kg body weight)	Sampling time (hours)	Number of micronucleatedpolychromatic erythrocytes per 2000 polychromatic erythrocytes (Mean ± SD)	Ratio polychromatic/ normochromatic erythrocytes (Mean ± SD)
Solvent control= corn oil	0	24	0.2 ± 0.4	1.07 ± 0.07
Beckopox VEH 2626-Polymer	750	24	1.0 ± 1.4	0.69 ± 0.11*
Beckopox VEH 2626-Polymer	750	48	2.2 ± 1.9	0.52 ± 0.21*
Beckopox VEH 2626-Polymer	375	24	1.2 ± 1.1	0.93 ± 0.24
Beckopox VEH 2626-Polymer	190	24	0.0 ± 0.0	0.85 ± 0.14*
Positive control = CP	50	48	41.2 ± 15.2*	0.35 ± 0.22*

CP = cyclophosphamide

* Significantly different from corresponding control group (Wilcoxon Rank Sum Test, p < 0.05)

Applicant's summary and conclusion

Conclusions:

The substance, when administered by intraperitoneal injection to mice at doses from 190 to 750 mg/kg bw, did not cause an increase in micronucleated polychromatic erythrocytes in bone marrow. The substance is negative for clastogenic effects in vivo, under conditions of this study.