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Diss Factsheets

Administrative data

genetic toxicity in vivo, other
Mammalian Erythrocyte Micronucleus Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 January 2020 to 28 April 2020
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Adopted on 29th July 2016.
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test

Test material

Constituent 1
Test material form:
solid: flakes
Specific details on test material used for the study:
- Source and lot/batch No.of test material: Clariant Plastics and Coatings (Deutschland) GmbH
- Expiration date :16.01.2022
- lot/batch: DEF2105728
- Purity test date:9 9.63%

- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the solvent/vehicle: 0.5% carboxymethyl cellulose
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

Test animals

Details on species / strain selection:
Mouse is one of the recommended species by regulatory agencies for conducting in vivo Micronucleus test among rodents
Details on test animals or test system and environmental conditions:
- Source: In-house bred animals
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: Male - 18.54 to 20.71 g, Female - 18.31 to 19.93 g (Pre study)
Male - 18.29 to 20.85 g, Female - 18.03 to 22.76 g (Main study)

- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Maximum of five animals of same sex and group were housed together in a standard polypropylene cage (L 280 × B 220 × H 140 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Sterilized paddy husk was used as a bedding material.
- Diet (e.g. ad libitum): Altromin Maintenance Diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period
- Acclimation period: Healthy adult young animals were acclimatized for six days in pre study and five days in limit study

- Temperature (°C): 18.5 to 22.8°C
- Humidity (%): 47 to 69%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

IN-LIFE DATES: From: 10 January 2020 To: 18 March 2020

Administration / exposure

Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used: 0.5 % w/v CMC (carboxymethyl cellulose);
- Justification for choice of solvent/vehicle: universally accepted vehicle for acute oral dosing
- Concentration of test material in vehicle: 2000 mg/kg body weight
- Lot/batch no. (if required): 210515
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Required quantity of test item was weighed as per the dose. The weighed test item was transferred to a clean mortar and ground using pestle by adding a small quantity of vehicle to get a uniform suspension. The content was transferred to a measuring cylinder. Again, a small quantity of 0.5% carboxymethyl cellulose was added to the mortar rinsed and transferred to the measuring cylinder. The rinsing procedure was repeated until the test item is transferred completely into the measuring cylinder. The final volume was made up with 0.5% carboxymethyl cellulose to get the desired concentration as per the dose requirement. Formulation of the test item was prepared freshly every day before dosing
Duration of treatment / exposure:
24 hours interval
Frequency of treatment:
Once in a day for two days
Post exposure period:
18 to 24 hours
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
none; cyclophosphamide;
- Justification for choice of positive control(s): As per inhouse validation study and as per the OECD 474
- Route of administration: Oral
- Doses / concentrations: 100 mg/kg


Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: As per the OECD Guideline No. 474, 2000 mg/kg body weight was selected as highest dose

DETAILS OF SLIDE PREPARATION: The femur was isolated from each animal for bone marrow collection. Bone marrow cells were obtained by cut opening the epiphyses of femur bone immediately following sacrifice. The marrow was flushed out in to a centrifuge tube using the Fetal Bovine Serum (FBS). The femur bone marrow cells were centrifuged at about 2700 rpm for 10 minutes. Prior to smear preparation, the supernatant was discarded and the cell pellet is then resuspended in approximately 50 µL of Fetal Bovine Serum (FBS).
Minimum of three slides were prepared for pre study and main study per animal. Smears before staining was fixed by immersing the slides in methanol for 5 minutes.The air dried slides were stained with May-Gruenwald and Giemsa stain for evaluation.

METHOD OF ANALYSIS: Slide evaluation

Evaluation criteria:
In the dose range finding study for each animal, minimum of 500 erythrocytes (which include mature and immature erythrocytes) were scored to determine the Polychromatic Erythrocytes (PCE) and Normochromatic Erythrocytes (NCE) ratio, in order to determine PCE: total RBC ratio. This result was used to determine the cytotoxicity of the test item.
In the limit study, all the slides including those of positive and vehicle controls were coded before microscopic evaluation to avoid group bias during evaluation.
For each animal, a minimum of 500 erythrocytes (which included mature and immature erythrocytes) were scored from first slide of the animal to determine PCE: total RBC ratio along with the incidence of micronucleus. The subsequent slides were scored only for the number of PCEs and incidence of micronucleated PCEs. For each animal, a minimum of 4000 Polychromatic Erythrocytes (PCEs) were scored for the incidence of micronucleated immature erythrocytes (MNPCEs).
Body weight of both pre study and limit study was analyzed by SPSS, version No. 22 at a 95% level (p≤0.05) of significance. Inter group comparison of Body weight of Day 1, 2 and 3 were done. Slides from limit study were decoded after analysis, the number of PCE (Polychromatic erythrocytes), RBC (Red blood corpuscles), MNPCE (Micronucleated Polychromatic erythrocytes) and PCE/ total erythrocytes ratio (Polychromatic erythrocytes/ total erythrocytes) and frequency of MNPCE was calculated. The data of positive control and the treatment groups were compared with that of the vehicle control for the incidence of MNPCEs and the proportion of PCEs among total RBCs by SPSS at a 95% level (p≤0.05) of significance. All analysis and comparisons were evaluated at the 95% level of confidence (p<0.05).
Statistically significant changes obtained were designated by the superscripts in the summary table throughout the report as stated below:
*: Statistically significant (p<0.05).

Results and discussion

Test results
Key result
no effects
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
- Dose range: Yes
- Solubility: Soluble in 0.5% W/V CMC
- Clinical signs of toxicity in test animals: No
- Evidence of cytotoxicity in tissue analyzed: No
- Rationale for exposure: No

- Induction of micronuclei (for Micronucleus assay): No
- Ratio of PCE/NCE (for Micronucleus assay): Yes
- Appropriateness of dose levels and route: Yes
- Statistical evaluation:Yes

Applicant's summary and conclusion

Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, Licocare RBW 106 TP is non-genotoxic at the limit dose of 2000 mg/kg.
Executive summary:

The test itemLicocare RBW 106 TPwas evaluated in the MammalianErythrocyte Micronucleus Test .

 This study was conducted to determine the genotoxic potential ofLicocare RBW 106 TP inthe micronucleus test using bone marrow cells of Swiss Albino mice. The pre study consisted of four groups, vehicle control, 500, 1000 and 2000 mg/kg.Licocare RBW 106 TPwasadministered at a dose volume of 10 mL/kg. In the pre study, each group of mice consisted of 3 males and 3 females. The limit study consisted of 3 groups of mice and each group consisted of 5 males and 5 females. G1 group animals were administered with vehicle, G2 group animals were administered with cyclophosphamide monohydrate at 100 mg/kg and animals of G3 group were administered with 2000 mg/kg for two consecutive days by oral route using gavage cannula. Post 18 to 24 hours of last dosing, all mice were sacrificed by cervical dislocation and bone marrow cells were collected. The slides of bone marrow cells were stained with May-Gruenwald and Giemsa stain and observed for incidences of micronucleated polychromatic erythrocytes (MNPCE).In the pre study, no clinical sign,no body weight changes, no gross pathological findingsand no mortality were observed in any of the animals dosed with  Licocare RBW 106 TPin any of the dose levels.

Percent reduction in P/E: total RBC ratio was1.92, 3.85 and 3.85 in males and 1.92, 1.92 and 3.85 in females dosed at 500, 1000 and 2000 mg/kg respectively when compared to respective vehicle control.There was no statistical change in theP/E: total RBC ratio at 500, 1000 and 2000 mg/kg in both the sexes.

In pre study, none of the doses produced observable toxic effects (no depression of bone marrow proliferation or other evidence of cytotoxicity) hence,2000 mg/kg was selected as the limit dose for both the sexes in limit study.

In the limit study, there was no clinical signs, no body weight changes, no gross pathological findings and no mortality were observed in any of the animals dosed with test item at 2000 mg/kg and also in positive control dosed animals. There was no statistical significant change in proportions of PCE: total RBC ratio in animals dosed at 2000 mg/kg of test item and there was statistically significant decrease in proportions of PCE: total RBC ratio in positive control group in both the sexes when compared to vehicle control groups respectively. The average percentage of MNPCEs was 0.05 in males and 0.04 in females dosed with vehicle. There was no statistically significant increase in thepercentageof micronucleated PCEs (per 4000 PCEs scored) at 2000 mg/kg in comparison with vehicle control.

The positive control,cyclophosphamide monohydrate at 100 mg/kgexhibited statistically significant increase in the percentage of MNPCE, when compared to vehicle control. Thisdemonstrated the sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate.