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Endpoint summary

Description of key information

In order to assess the acute toxicity of the test substance registered a repeated oral dose 28-day toxicity study in rat with Dodicor V 5654, according to OECD 407,  is available.

No test item related changes were noted up to 1000 mg/kg in both the sex: (male/female) based on: (test mat.) body weight and weight gain ; clinical biochemistry ; clinical signs ; food consumption and compound intake ; gross pathology ; haematology ; histopathology: non-neoplastic ; mortality ; ophthalmological examination ; organ weights and organ / body weight ratios ; urinalysis. In addition, no delayed effects were noted in the animals post recovery period of 14 days.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 February 2020 to 28 May 2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

03 October 2008

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Clariant Ibérica Producción, S.A.
Batch No.: ESD0033040
- Expiration date of the batch: August 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: The stability of the test item Dodicor V 5654 in dose formulations were established under Bioneeds Study No.: BIO-ANM 1468. As per the stability results, the test item formulations at the concentrations of 5 mg/mL and 250 mg/mL were stable up to 48 hours at room temperature.
- Solubility and stability of the test substance in the solvent/vehicle: Corn oil was used as vehicle for the preparation of formulations as evidenced by the in-house miscibility test results. The details of the vehicle used for formulation was recorded in the raw data. Corn oil is the universally accepted vehicle in preclinical formulations.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Test material was diluted to required concentration with the vehicle, corn oil and administered to animals.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the recommended species by regulatory agencies for conducting preclinical toxicological studies among rodent species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Females nulliparous and non-pregnant: [yes/no]: Yes
- Age at study initiation: 7 Weeks
- Weight at study initiation: Males: 131.89 g to 149.94 g and Females: 120.49g to 139.83g
- Housing: Maximum of two animals of same sex were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 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. Clean sterilized paddy husk was provided as bedding material.
Polycarbonate rat tunnels were provided to the individually housed rat as an enrichment object
- 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 acclimatization and 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: 15 February 2020 to 19 February 2020

DETAILS OF FOOD AND WATER QUALITY: Certificate of Analysis of feed and water attached as annexures in the study report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8ºC to 23.0ºC
- Humidity (%): 42% to 65%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 15 February 2020 To: 21 April 2020

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered through oral route using stainless steel (gavage) cannula.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared and used within the stability period. Required quantity of test item was weighed into a clean glass beaker and there by adding little volume of vehicle in to beaker, mixed well with clean glass rod and transferred to a measuring cylinder. This procedure was repeated until to ensure entire quantity of test item formulation was transferred to measuring cylinder. Finally, the volume was adjusted to required mark in measuring cylinder with vehicle to get a desired concentration of dose formulations.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was used as vehicle for the preparation of formulations as evidenced by the in-house miscibility test results. The details of the vehicle used for formulation was recorded in the raw data. Corn oil is the universally accepted vehicle in preclinical formulations.
- Concentration in vehicle:
G1 (Vehicle Control): 0 mg/mL; G2 (Low Dose): 10 mg/mL;
G3 (Mid Dose): 30 mg/mL; G4 (High Dose); 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot no.: O21904007 and L12017004

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analysis for homogeneity and concentration verification was done by Analytical Chemistry Department of Bioneeds India Private Limited, as per validated analytical methods detailed in the Study No. BIO-ANM 1468.
Formulation analysis for homogeneity and concentration verification was performed for all the dose formulations prior to first treatment and during week 4 of the study.
For homogeneity and concentration verification analysis, the prepared formulations were sampled in duplicate sets (5 mL each from top, middle and bottom layers) from all the dose groups prior to first treatment and during week 4 of the treatment period. From vehicle control, samples were drawn from middle layer only in duplicate sets (5 mL each) during scheduled sampling.
The collected samples were transferred to Analytical Chemistry Department of Bioneeds India Private Limited, for homogeneity and dose concentration analysis. One set of aliquot of each formulation was analysed. The second aliquot was discarded after the confirmation of results to be within the limits of acceptable range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10.0%.

Duration of treatment / exposure:

28 consecutive days

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

G1/G1R (Vehicle Control)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

G2 (Low Dose)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

G3 (Mid Dose)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

G4/G4R (High Dose)

No. of animals per sex per dose:

5 males and 5 females per sex per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): The animals for the experiment were weighed and arranged in ascending order of their body weights. These body weight stratified rats were distributed to all the experimental groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the experiment did not exceed ±20% (Male: -10.29% to +15.23% and Female: -9.43% to +7.63%) of the mean body weights of each sex. The grouping was done one day prior to the initiation of treatment. Body weight of the animals were analyzed statistically for mean body weight to rule out the statistical significant difference between groups within each sex.

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity.
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were subjected to detailed clinical examinations before initiation of the treatment and at weekly intervals thereafter during the experiment.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weights were recorded on the Day 1, before test item administration and at weekly intervals thereafter. Fasting body weight of all the animals were recorded at terminal sacrifice.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examination was performed for all the animals before start of the treatment and during week 4 of treatment for G1 and G4 group animals and during week 6 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1/G1R (Vehicle control) and G4/G4R (High Dose)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Main groups on day 29 and from recovery group animals on day 43.
- Anaesthetic used for blood collection: Yes (30% Isoflurane and 70% propylene glycol)
- Animals fasted: Yes
- How many animals: 60 Animals
- Parameters checked in table [No.10] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Main groups on day 29 and from recovery group animals on day 43.
- Animals fasted: Yes
- How many animals: 60 Animals
- Parameters checked in table [No.11] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Main groups on day 29 and from recovery group animals on day 43.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.12] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations :Week 4 for vehicle control and high dose main group animals (G1 and G4) and during Week 6 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1/G1R (Vehicle control) and G4/G4R (High Dose).
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Not Applicable

Statistics:

The data was subjected to statistical analysis. The computer printout of the data (in the form of appendix) was verified with the original raw data. After verification, the data was subjected to statistical analysis using SPSS software version 22. Body weight, body weight gain (percent change in body weight with respect to Day 1 body weight), feed consumption, organ weights and ratios, haematological and clinical chemistry estimations and urine analysis parameters (urobilinogen, pH, specific gravity and urine volume), FOB parameters (rearing, urination, defecation, excessive grooming, body temperature, grip strength, motor activity and hind limb foot splay) were subjected to statistical analysis. One-way ANOVA followed by Dunnett’s post test was done for different treatment groups comparing with the control group data. Comparison of means between recovery groups was done using student ‘t’ test. All analysis and comparisons were evaluated at the 95% level of confidence (p<0.05). Statistically significant changes obtained from the aforementioned tests were designated by the superscripts in the summary table throughout the report as stated below:
*: Statistically significant (p<0.05).

Clinical signs:

not specified

Description (incidence and severity):

No clinical signs of toxicity were noted in any of the treated group animals in either sex.

Mortality:

no mortality observed

Description (incidence):

No mortality/morbidity were noted in any of the treated group animals in either sex.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related changes in mean body weight were noted in all the tested dose groups. However, statistically significant lower body weight gain (percent change in body weight with respect to Day 1) were observed during days 1-15 (G3M), 1-22 (G3M, G4M). In the absence of associated variation in feed consumption, the noted variations are considered incidental.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

Not applicable

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related changes in feed consumption were noted in any of the tested group animals in either sex. However, statistically significant lower feed consumption was noted during week 2 (G4F, G4RM), week 3 (G4RM), week 4 (G4RM and G4RF). The lower feed consumption noted did not have any associated changes in mean body weight and hence considered incidental.

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

Not applicable

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ocular changes were observed during ophthalmoscopic examination in groups examined.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse test item related changes were observed in haematology parameters. However, the following statistically significant changes were observed when compared to respective vehicle control groups.
In main group, statistically significant increase in absolute and percent monocytes (G2M), absolute monocytes (G4M), absolute neutrophils (G4M), percent eosinophils (G3F), APTT (G3M, G4M); decrease in APTT (G2F, G3F) was noted. These changes are considered as incidental in the absence of dose responsiveness.
In recovery groups, statistically significant increase in percent eosinophils (G4RM) and decrease in absolute neutrophils (G4RM) was noted. All these changes are considered as incidental and not related to test item in the absence of such changes at the end of treatment period and also, all the changes could be due to random biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse test item related changes were observed in clinical chemistry parameters when compared to control group animals. However, the following statistically significant variations were noted.
In main group, decrease in total cholesterol (G3M); increase in total cholesterol (G4M), potassium (G3F) which is considered incidental in the absence of dose responsiveness.
In recovery groups, decrease in ALP (G4RM) and increase in calcium (G4RM) was noted. All these changes are considered as incidental in the absence of such changes at the end of treatment period or in females.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item treatment related changes in urinalysis parameters were noted.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test item related changes were observed in neurological/functional examination test in any of the test item treated groups.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse test item related changes in organ weights (absolute and relative) were noted. However, the following statistically significant changes were noted in organ weights and its ratios.

Increase in relative weight of liver (G4M) and absolute weight of brain (G2F, G3F) was noted. Increase in brain weight is considered incidental in the absence of dose responsiveness. Increase in liver weight is also considered incidental without any associated macroscopic and microscopic changes in liver.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathological changes were noted at any of the tested dose group animals in main groups and recovery group.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related, microscopic findings in the study.
Few microscopic findings observed in this study such as ultimobranchial cyst in thyroid, epithelial cysts in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats of this age.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Not applicable

Other effects:

no effects observed

Description (incidence and severity):

Not applicable

Details on results:

Not applicable

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Key result

Critical effects observed:

no

Conclusions:

Based on the results discussed, the No Observed Adverse Effect Level (NOAEL) of the test item, Dodicor V 5654 was found to be 1000 mg/kg body weight/day, which is the highest dose administered for a period of 28 consecutive days repeatedly by oral (gavage) route to Sprague Dawley rats under the experimental conditions and the doses employed. In addition, no delayed effects were noted in the animals post recovery period of 14 days.

Executive summary:

The objective of this study was to assess the toxic potential of test item Dodicor V 5654 when administered for a period of 28 consecutive days repeatedly by oral (gavage) route to Sprague Dawley rats. This study provides information on major toxic effects, target organs, possibility of cumulative effects and reversibility of effects after 14 daysofrecovery period and an estimate of the No Observed Adverse Effect Level (NOAEL).

A total of 60 Sprague Dawley rats (30males +30 females) were distributed to 4 main (G1, G2, G3 & G4) and 2 recovery groups (G1R & G4R). Each group consisted of 5 rats/sex. The rats allocated to groups G2, G3 and G4/G4R were administered with test item, Dodicor V 5654 at the doses of 100, 300 and 1000 mg/kg body weight/day respectively and G1/G1R group animals received vehicle (corn oil) alone. The vehicle/test item formulations were administered by oral (gavage) route at an equivolume of 10 mL/kg body weight.

The test item formulations were stable at the concentrations of 5 mg/mL and 250mg/mL for 48 hours at room temperature. The dose concentration of the test item formulations were analyzed prior to the day of first administration and during week 4 of the dosing period for dose concentration determination. The results were found within the acceptable range of ±15% recovery to the nominal concentration for Dodicor V 5654 with <10% RSD (relative standard deviation).

All the rats were observed once daily for clinical signs and twice daily for mortality and morbidity, weekly for detailed clinical examination, body weight and feed consumption; ophthalmoscopic examination was performed during acclimatization period for all the rats and during week 4 of dosing period for G1 and G4 groups and during week 6 for G1R and G4R groups. Functional observational battery was performed during week 4 of dosing period for G1 and G4 groups and during week 6 for G1R and G4R groups.

At the end of treatmentand recovery period, i.e., Day 29 and Day 43, respectively, blood and urine samples were collected and analyzed from overnight fasted rats. Subsequently, theratswere sacrificed using CO₂asphyxiation followed by exsanguination and subjected to gross pathological examination. The study plan specified organs/tissueswere collected, weighed and preserved for histopathological examination. Histopathology examination was performed on organs of vehicle control and high dose group (G1 and G4).

No mortality and clinical signs were observed throughout the dosing and recovery period. Notest item-related changes in body weight, percent change in body weight and feed consumption were observed throughout the experimental period. No test item-related neurological/functional abnormalities and no ocular changes were observed during examination. 

No toxicologically significant changes noted in haematology, coagulation, clinical chemistry and urinalysis parameters.There were no test item-related effects inabsolute and relative organ weights, gross and histopathology parameters in both the sexes.

Based on the results discussed, the No Observed Adverse Effect Level (NOAEL) of the test item, Dodicor V 5654 was found to be 1000 mg/kg body weight/day, which is the highest dose administered for a period of 28 consecutive days repeatedly by oral (gavage) route to Sprague Dawley rats under the experimental conditions and the doses employed. In addition, no delayed effects were noted in the animals post recovery period of 14 days.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subacute

Species:

rat

Quality of whole database:

reliability 1, GLP conform, OECD Guideline study

Justification for classification or non-classification

Based on the results from the available repeated dose toxicity data it is concluded that the registered substance is not subject to classification and labelling according to the criteria of the EU Dangerous Substances Directive (67/548/EEC) (DSD) and of the EU Classification, Labelling and Packaging Regulation (1972/2008/EC) (CLP).