Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Source study has reliability 1. Details on the read across are attached in section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
other: Mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
Males - animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (days 38 and 39 of study). Males were treated for a total of 37 or 38 d.
Females - animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until day 3 post partum (for at least 40 d).
Duration of treatment / exposure:
Males - 37-38 d
Females - 40 d
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
reduced from high dose of 1000 mg/kg bw/day due to toxicity
No. of animals per sex per dose:
5 per sex per group
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C

Examinations

Tissues and cell types examined:
Erythrocytes from bone marrow
Details of tissue and slide preparation:
Extraction of bone marrow
The last two treatments were performed at approximately 24 hour interval. Samples of bone marrow were collected approximately 24 hours following the final treatment and approximately 48 hours following the second last treatment from the same 5 males and 5 females of the main groups randomly selected for clinical pathology investigation. Samples of bone marrow were also collected approximately 24 hours after the single treatment from all animals of Group 7 (Positive Control group). One femur of each animal was rapidly dissected out and cleaned of surrounding tissue. In order to extract the bone marrow, the bone was cut at the proximal end, and irrigated with foetal calf serum using a syringe. The suspension of cells was aspirated, and this procedure was repeated several times.

Preparation of the smears
The suspension thus obtained was centrifuged at 1000 rpm for at least 5 minutes and the supernatant was completely removed. The cells of the sediment were resuspended and transferred onto clean microscope slides as smear preparations. They were air-dried and then fixed with methanol for 10 minutes. Subsequently slides were stained with haematoxylin and eosin solutions. Finally, slides were rinsed in distilled water and allowed to dry.

Scoring of the slides and data analysis
In the first instance, only slides from males were evaluated. In addition, since inter-sex differences of toxicity were observed, also slides from females were evaluated. For each animal, four slides were prepared. These slides were randomised and coded by staff not subsequently involved in the scoring. The adequate quality and a sufficient number of cells were evaluated before scoring. Scoring was performed using a microscope and highpower objective. Immature polychromatic erythrocytes (PCEs) stain a pink-purple colour (since they retain basic ribosomal material for approximately 24 hours after enucleation), and can be distinguished from the pink normochromatic erythrocytes (NCEs). Erythrocytes lack nuclei, making micronuclei obvious when present; Schmid criteria (1976) were used to score micronuclei. Four thousand polychromatic erythrocytes(PCEs) per animal were scored for the presence of micronuclei. At the same time, the number of normal and normochromatic erythrocytes (NCEs) were also recorded. The proportion of immature erythrocytes tog total erythrocytes gives an indication of the toxicity of the treatment; a reduction in the proportion indicates inhibition of cell division. Finally, the incidence of micronucleated PCEs provides an index of induced genetic damage.
Evaluation criteria:
The assay is considered valid if the following criteria are met:
1. incidence of micronucleated PCEs of vehicle control group falls within the historical negative control range.
2. positive control item results falls within the historical control range and are significantly increased, at statistical analysis, when compared with the concurrent negative control
3. 5 animals per sex and per group are available for slide analysis.

Evaluation of results
The test item is considered to induce micronuclei if a statistically significant increase in the micronucleus incidence of polychromatic erythrocytes (at p<0.05) is observed in any treatment group and a dose-effect relationship is demonstrated.
Where statistically significant increases in the incidence of micronucleated PCEs are observed, but all results are inside the distribution of negative control values within this laboratory, then historical control data are used to demonstrate that these increases do not have any biological significance.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was considered to be non-genotoxic in the rat.
Executive summary:

Method

A study was conducted to determine the genotoxic potential of the test substance according to OECD guideline 474.

The micronucleus test was conducted on 5 male and 5 female rats in order to assess the ability of the test substance to induce cytogenetic damage in rat bone marrow, as measured by the induction of micronuclei in polychromatic erythrocytes. Rats were administered test substance orally by gavage. Main group animals were exposed to concentrations of 62.5, 250, 500, 1000 and 1000/500 mg/kg bw/day. Males animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (days 38 and 39 of study). Female animals were dosed once a day, 7 d/week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post-coitum and post-partum periods until day 3 post-partum (for at least 40 d). Mitomycin C was used as the positive control substance.

Results

No relevant inhibitory effect on erythropoietic cell division was observed at any dose level. Therefore, test substance was not considered to be genotoxic in rats.