Registration Dossier

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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

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

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan italy
- Age at study initiation: 7 to 8 weeks (day of allocation)
- Weight at study initiation: 218.3-232.2 g for males, 183.1-207.3 g for females
- Fasting period before study: no
- Housing: 5 animal/sex/cage
- Diet (e.g. ad libitum): ad libitum except for clinical pathology investigation
- Water (e.g. ad libitum):ad libitum except for clinical pathology investigation
- Acclimation period: 12 days

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

IN-LIFE DATES: From: 13 January (allocation) to 14 March 2015 (last necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Polyethylene glycol (PEG) 400 in softened water (by reverse osmosis) - 1:1 v/v.

The vehicle for the positive control item will be sterile water for injection.
Details on exposure:
The animals received the test item, dissolved in Polyethylene glycol (PEG) 400 in softened water (by reverse osmosis) - 1:1 v/v, at a constant volume of 10 mL/kg body weight

The positive control item was dissoolved in the vehicle at a concentration of 0.2 mg/ml
Duration of treatment / exposure:
Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for approximately 6 weeks. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Positive Control group (Group 7)
Animals received a single dose approximately 24 hours before sacrifice.
The Mitomycin-C (positive control) was administered once by intraperitoneal injection at the dose volume of 10 mL/kg body weight. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal
Frequency of treatment:
once a day
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 62.5, 250, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males
Control animals:
yes, concurrent vehicle
Positive control(s):
No of animals: 5 males
The Mitomycin-C (positive control) was administered once by intraperitoneal injection at the dose volume of 10 mL/kg body weight. The dose was
administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Examinations

Tissues and cell types examined:
Bone marrow from one femur of males only
Details of tissue and slide preparation:
Extraction of bone marrow
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 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 males of the 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 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 overnight 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
For each animal, at least three slides were prepared. These slides were randomised and coded by staff not subsequently involved in the scoring. Provided that the slides are of an adequate quality and a sufficient number of cells can be scored, it may only be necessary to score one of the series. Scoring was performed using a microscope and high-power objective. Immature polychromatic erythrocytes (PCE) 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 (NCE). Erythrocytes lack nuclei, making micronuclei obvious when present; the criteria of Schmid (1976) were used to score micronuclei. At least two thousand polychromatic erythrocytes per animal were scored for the presence of micronuclei (unless there is a marked depression in PCE numbers). At the same time the number of normochromatic erythrocytes was recorded, as well as the number of micronucleated NCE. The proportion of immature erythrocytes among 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 PCE provides an index of induced genetic damage.
Evaluation criteria:
The assay is considered valid if the following criteria are met:
i The incidence of micronucleated PCE in the vehicle control group falls within the historical vehicle control range.
ii Five males per group are available for slide analysis.
iii The concurrent positive control results fall within the historical control range and are ssignificantly increased at statistical analysis, when compared with the concurrent negative control.

Evaluation of results
The test item was considered to induce micronuclei if a statistically significant (p<0.05) and biologically meaningful increase in micronucleus incidence
was observed in any treatment group. A dose-effect relationship should be observed.
The evaluation of data from groups in which there was extensive lethality of the test item treatment followed RTC SOPs. Similarly, where erythropoiesis was depressed by the test item treatment and few PCE are available for scoring, evaluation followed RTC SOPs. Where increases in the incidence of micronucleated PCE were observed which were statistically significant, but fell within the range of vehicle control values within this laboratory, then concurrent and historical control data were used to demonstrate that these increases did not have biological significance.
Statistics:
Only counts from polychromatic cells were subjected to statistical analysis.
Using the original observations (and not the micronucleus frequencies per
1000 cells), a modified chi-squared calculation was employed to compare
treated and control groups. The degree of heterogeneity within each group
was first calculated and, where significant, it was taken into account in
the comparison between groups. If there was no significant within-group
heterogeneity, the chi-squared test was used to compare treated groups with
the controls. If there was significant within-group heterogeneity, then that
group was compared with the controls using a variance ratio (F) value
calculated from the between-group and within-group chi-squared values

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
No signs of genotoxicity were detected by measuring the micronuclei in polychromatic erytrocytes.
Executive summary:

The ability of the test item to induce cytogenetic damage and/or disruption of the mitotic apparatus in rat bone marrow was investigated measuring the induction of micronuclei in polychromatic erythrocytes. The test was only performed in males, as no toxicological relevant difference in target organ toxicity was noted.

Samples of bone marrow were collected approximately 24 hours following the final treatment and approximately 48 hours following the second last treatment from 5 males of the main groups randomly selected and from all animals of the positive control group consisting of 5 males. One femur of each animal was removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared to make smears on slides. These slides were air-dried, fixed with methanol and then stained with haematoxylin and eosin solutions and mounted with Eukitt. Three slides were made from each animal.

The slides were randomly coded by a person not involved in the subsequent microscope scoring and examined under low power to select one or more slides from each animal according to staining and quality of smears. Four thousand polichromatic erythrocytes (PCEs) per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time, the numbers of normal and micronucleated normochromatic erythrocytes (NCEs) were also recorded

Findings noted during treatment (discoloured urine/bedding) were considered a proof of absorption.

The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.

Following treatment with the test item, no relevant increase in the number of micronucleated PCEs was observed at any dose level.

A marked increase in the frequency of micronucleated PCEs was observed in the positive control group.

A summary of the results obtained is presented in the following table:

Dose level (mg/kg/day)

Incidence in micronucleated PCEs

PCE/s(PCEs+NCEs) %

over the mean control value

 

mean

SE

Range

 

0.00

1.1

0.5

0.3-3.0

100

62.5

1.1

0.2

0.8-1.8

95

250

1.5

0.1

1.0-1.8

101

1000

1.4

0.3

0.0-1.8

92

Mitomycin-C

2.00 mg/kg

 

11.3

 

1.4

 

7.8-16.0

 

88

 

The incidence of micronucleated PCEs of the negative control group fell within the historical control range (95% confidence limit). Statistically significant increases in the incidence of micronucleated PCEs over the negative control values were seen in the positive control group. The induced response was compatible with the historical control range, demonstrating the laboratory proficiency in the conduct of the test. Five animals per groups were available for micronucleus slide analysis. Based on the stated criteria, the assay was therefore accepted as valid.

On the basis of the results obtained, it is concluded that the test item does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.