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Genetic toxicity: in vivo

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Administrative data

Endpoint:

genetic toxicity in vivo, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 December 2019 to 14 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Test material form:

solid: flakes

Test animals

Species:

mouse

Strain:

Swiss

Remarks:

Albino

Details on species / strain selection:

Mouse is one of the recommended species by regulatory agencies for conducting in vivo Micronucleus test among rodents

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: 18 to 23 g
- Assigned to test groups randomly: Through grouping and randomization
- Fasting period before study: NA
- 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. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3 to 22.9°C
- Humidity (%): 44 to 65%
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light

IN-LIFE DATES: From: 26/12/2019 To: 05/03/2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Corn oil is universally accepted vehicle for acute oral dosing
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot no. (if required): A1712001

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 Corn oil was added to the mortar rinsed and transferred to the measuring cylinder. The rinsing procedure was repeated until the test item was transferred completely into the measuring cylinder. The final volume was made up with corn oil 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:

The test item was administered through oral route once a day for 2 consecutive days using gavage cannula.

Frequency of treatment:

once a day for 2 consecutive 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:

Limit Study: 3 groups
10 (5 Males + 5 Females)

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

cyclophosphamide;
- Justification for choice of positive control(s): As per inhouse validation study
- Route of administration: Oral
- Doses / concentrations: 100 mg/kg body weight/10 mg/mL

Examinations

Tissues and cell types examined:

For each animal 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 erythrocytes ratio.

For each animal, a minimum of 4000 Polychromatic Erythrocytes (PCEs) were scored for the incidence of micronucleated immature erythrocytes (MNPCEs)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In pre study, none of the doses produced observable toxic effects (no depression of bone marrow proliferation or other evidence of cytotoxicity were observed up to 2000 mg/kg) and hence; 2000 mg/kg was selected as the limit dose for both the sexes in Limit study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Post 18 to 24 hours (Day 3) of the second day treatment, all surviving animals were sacrificed by cervical dislocation, the femur was isolated from each animal for bone marrow collection

DETAILS OF SLIDE PREPARATION: 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.
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 approximately 5 to 6 minutes. The air dried slides were stained with May-Gruenwald and Giemsa stain for evaluation.




METHOD OF ANALYSIS:

OTHER:

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 erythrocytes 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 erythrocytes 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)

Statistics:

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

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Yes
- Solubility: Suspension in corn oil
- Clinical signs of toxicity in test animals: No clinical signs observed
- Evidence of cytotoxicity in tissue analyzed: No toxicity observed
- Harvest times: 18 to 24 hours of day 2 treatment

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Yes
- Ratio of PCE/NCE (for Micronucleus assay): Yes
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes

Any other information on results incl. tables

INDIVIDUAL ANIMAL MICRONUCLEUS DATA - MAIN STUDY

Sex	Group & Dose (mg/kg)	Animal No.	Total NCEs	Total PCEs	PCE: Total Erythrocytes Ratio	Mean of PCE: Total Erythrocytes Ratio	SD	% Reduction Of PCE: Total Erythrocytes Ratio	Total No. of PCE's Scored	Total Number of MNPCEs	% of MNPCEs	Mean of % of MNPCEs
Male	G1 & 0	Mc5271	255	276	0.52	0.51	0.00	NA	4010	2	0.05	0.05
		Mc5272	254	266	0.51				4021	3	0.07
		Mc5273	253	267	0.51				4151	2	0.05
		Mc5274	256	266	0.51				4008	2	0.05
		Mc5275	255	270	0.51				4215	1	0.02
	G2 & 100 (CPA)	Mc5276	274	234	0.46	0.47*	0.01	7.84	4017	30	0.75	0.69*
		Mc5277	270	240	0.47				4032	26	0.64
		Mc5278	280	245	0.47				4011	25	0.62
		Mc5279	265	240	0.48				4017	30	0.75
		Mc5280	280	246	0.47				4015	27	0.67
	G3 & 2000	Mc5281	285	251	0.47	0.46*	0.01	9.80	4121	4	0.10	0.07
		Mc5282	270	231	0.46				4015	3	0.07
		Mc5283	275	225	0.45				4012	2	0.05
		Mc5284	286	244	0.46				4022	3	0.07
		Mc5285	280	240	0.46				4125	3	0.07

SD: Standard deviation, CPA: Cyclophosphamide Monohydrate, *: Statistically significant

INDIVIDUAL ANIMAL MICRONUCLEUS DATA - MAIN STUDY

Sex	Group & Dose (mg/kg)	Animal No.	Total NCEs	Total PCEs	PCE: Total erythrocytes ratio	Mean of PCE: Total erythrocytes ratio	SD	% Reduction of PCE: Total erythrocytes ratio	Total No. of PCE's scored	Total Number of MNPCEs	% of MNPCEs	Mean of % of MNPCEs
Female	G1 & 0	Mc5286	260	266	0.51	0.50	0.01	NA	4125	2	0.05	0.05
		Mc5287	265	264	0.50				4085	1	0.02
		Mc5288	255	258	0.50				4151	2	0.05
		Mc5289	258	263	0.50				4063	3	0.07
		Mc5290	265	258	0.49				4017	2	0.05
	G2 & 100 (CPA)	Mc5291	290	243	0.46	0.47*	0.01	6.00	4021	25	0.62	0.65*
		Mc5292	287	259	0.47				4125	30	0.73
		Mc5293	279	248	0.47				4063	27	0.66
		Mc5294	280	266	0.49				4011	25	0.62
		Mc5295	289	257	0.47				4032	25	0.62
	G3 & 2000	Mc5296	270	230	0.46	0.45*	0.01	10.00	4015	2	0.10	0.08
		Mc5297	275	235	0.46				4101	3	0.07
		Mc5298	278	223	0.45				4022	4	0.10
		Mc5299	288	223	0.44				4021	3	0.07
		Mc5300	287	248	0.46				4031	2	0.05

SD: Standard deviation, CPA: Cyclophosphamide Monohydrate, *: Statistically significant

Applicant's summary and conclusion

Conclusions:

Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, Licocare RBW 300 FL TP is non-genotoxic at the limit dose of 2000 mg/kg.

Executive summary:

Test item,Licocare RBW 300 FLwas evaluated in the MammalianErythrocyte Micronucleus Test.

This study was conducted to determine the genotoxic potential ofLicocare RBW 300 FL 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 300 FL 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 main 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 a limit dose of 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 pre study, no clinical signs,no body weight changes, no gross pathological findingsand no mortality were observed in any of the animals dosed withLicocare RBW 300 FL TPin any of the dose levels.

In pre study, there was statistical significant decrease in thePCE: total erythrocytesratio at 500, 1000 and 2000 mg/kg in females and in males at 2000 mg/kgwhen compared to vehicle control group. The reduction in PCE: total erythrocytes was well below 50% (3.92% in males and 9.80% in females) and the test item was therefore considered not to be cytotoxic to the bone marrow. 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 main study.

In the main 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 statistical significant decrease in proportions of PCE: total erythrocytes ratio in animals dosed at 2000 mg/kg of test item and also in positive control group in both the sexes when compared to vehicle control group. The average percentage of MNPCEs was 0.05 in both the sexes 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.