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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Due to cleaning procedures or performance of functional observations in the room, temporary deviations from the level of humidity (with max 20%) occurred. Based on lab historical data these deviations are considered not to affect the study integrity.
GLP compliance:
yes
Type of assay:
other: mammalian germ cell cytogenic assay: micronucleus

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Clear colourless liquid, purity 96.5%.

Test animals

Species:
mouse
Strain:
NMRI
Remarks:
NMRI BR (SPF)
Details on species / strain selection:
The mice selected (NMRI BR (SPF) are recommended by international guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Females were nulliparous and non-pregnant. The animals were provided by Charles River, Sulzfeld, Germany.

Young adult animals were selected (6-8 weeks old). The body weights of the mice at the start of the treatment were within 20% of the sex mean.

Conditions:
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21 +/- 3 oC and a relative humidity of 30-70%. The room was illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.

Accomodation:
Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing Woody Clean bedding (Woody-Clean type 3/4: Tecnilab-BMIBV, Someren, The Netherlands). Paper bedding was provided as nest material (B.M.I. Helmond, The Netherlands). Acclimatization period was at least 5 days before start of treatment under laboratory conditions.

Diet:
Free access to standard pelletized laboratory animal diet (code VRF 1, Altromin, Lage, Germany).

Water:
Free access to tap water.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The mice received an oral intubation of a maximum tolerated (high), an intermediate and a low dose of Pentamethyl-trioxepane. The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.

Feed was withheld 3-4 h prior to dosing.

The dosing volume was 10 ml/kg body weight. The route and frequency of administration and the volume administered of the negative and positive control was the same as those of the test article.

The systemic toxic signs were recorded at least once a day. The time of death was recorded as precisely as possible. The animals were weighed just prior to dosing.
Duration of treatment / exposure:
single treatment via oral intubation, exposure period/sampling time 24 hrs, with an additional one at 48 hrs for high dose (1000 mg/kg bw male, 750 mg/kg bw female)
Frequency of treatment:
once
Post exposure period:
24hrs and 48 hrs for high dose groups (1000 mg/kg bw male, 750 mg/kg bw female)
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
male
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
male
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
male
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
female
Dose / conc.:
375 mg/kg bw/day (actual dose received)
Remarks:
female
Dose / conc.:
190 mg/kg bw/day (actual dose received)
Remarks:
female
No. of animals per sex per dose:
five
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CP; CAS 50-18-0; Endoxan, Asta-Werke, Germany) dissolved in physiological saline (B. Braun, Melsungen, Germany) dosed as a single oral intubation of 50 mg/kg body weight.

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
Bone marrow samples: femurs were removed from the animals and flushed with 2 ml fetal calf serum. The cell suspension is collected and centrifuged at 1000 rpm for 5 min. supernatant was removed with a Pasteur pipette. One drop of serum was left. One drop of cell suspension is placed on a slide. 2 slides were prepared per animal. Slides were stained using the “Wright-stain procedure” in an “Ames” HEMA-tek slide stainer. Slides were scored at 1000x magnification.
Evaluation criteria:
The polychromatic/normochromatic ratio was determined by counting and differentiating the 1st 1000 erythorcytes at the same time. 2000 micronucleated polychromatic erythrocytes were examined.
Statistics:
The polychromatic/normochromatic ratio was determined. Averages and standard deviations were calculated. A wilcoxon rank sum test; two sided test at p < 0.05 was used to test if there was a significant increase in micronucleated polychromatic erythrocytes.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Cyclophosphamide induced a significant increase in the number of micronucleated polychromatic erythrocytes and a decrease in the polychromatic to normochromatic erythrocytes ratio (= toxic effects on erythropoiesis). The animals treated with pentamethyl-trioxepane and the negative control showed no increase in the number of micronucleated polychromatic erythrocytes and no decrease in the polychromatic to normochromatic erythrocytes ratio.

During the first hours the highest dose groups showed ataxia and increased activity. Within 18 hours the animals recovered.

Applicant's summary and conclusion

Conclusions:
pentamethyl-trioxepane is not mutagenic in the micronucleus test under the experimental conditions described in this report.
Executive summary:

pentamethyl-trioxepane is not mutagenic in the micronucleus test under the experimental conditions described in this report.