Reaction mass of Cardanol diene and Cardanol monoene and Cardanol triene 

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

90 Days

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Studies on the reproductive, cytological and biochemical toxicity of Ginkgo Biloba in Swiss albino mice

Author:

Abdulaziz A. Al-Yahya, Abdulhakeem A. Al-Majed ∗, A.M. Al-Bekairi, O.A. Al-Shabanah, S. Qureshi

Year:

2006

Bibliographic source:

Journal of Ethnopharmacology 107 (2006) 222–228

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Effects of Ginkgo biloba on biochemical parameters on mice.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Cardanol diene and Cardanol monoene and Cardanol triene 

Cas Number:

37330-39-5

Molecular formula:

C(21)H(31-36)O

IUPAC Name:

Reaction mass of Cardanol diene and Cardanol monoene and Cardanol triene 

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source:Experimental Animal Care Center,Riyadh, Saudi Arabia.- Age at study initiation:6–8 weeks- Weight at study initiation: 25–28 g- Fasting period before study:Not available- Housing:Details not available- Diet (e.g. ad libitum):Purina chowdiet ad libitum- Water (e.g. ad libitum):ad libitum- Acclimation period:Not availableENVIRONMENTAL CONDITIONS- Temperature (°C):Standard conditions- Humidity (%):Standard conditions- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Aqeous suspension of Ginkgo biloba was administered by gastric intubation (oral) daily for a period of 90 days.

Vehicle:

water

Details on oral exposure:

Exposure Period: Aqueous suspension of Ginkgo biloba was administered by gastric intubation (oral) daily for a period of 90 days.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 Days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Dose Concentration: 0,25, 50, 100 mg/kg/bw was selected on the basis of MTD (Maximum tolerated dose viz.1.6 gm/kg) corresponding to 1/64, 1/32, 1/16.

No. of animals per sex per dose:

Total 240 animals;.group 1, control (tap water)-5 male micegroup 2, Ginkgo biloba-25 mg/kg/day-5 male micegroup 3, Ginkgo biloba-50 mg/kg/day-5 male micegroup 4,Ginkgo biloba-100 mg/kg/day-5 male mice

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data available - Time schedule: No data available- Cage side observations checked in table [No.?] were included.: No data availableDETAILED CLINICAL OBSERVATIONS: No data available - Time schedule: No data availableBODY WEIGHT: Yes - Time schedule for examinations: Before the initiation of studyFOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data available- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available FOOD EFFICIENCY: No data available- 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: No data available WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data available - Time schedule for examinations: No data availableOPHTHALMOSCOPIC EXAMINATION: No data available - Time schedule for examinations: No data available- Dose groups that were examined: No data availableHAEMATOLOGY: No data available- Time schedule for collection of blood: No data available- Anaesthetic used for blood collection: No data available - Animals fasted: No data available - How many animals: No data available - Parameters checked in table [No.?] were examined.: No data available CLINICAL CHEMISTRY: No data available- Time schedule for collection of blood: No data available- Animals fasted: No data available- How many animals: No data available- Parameters checked in table [No.?] were examined.: No data availableURINALYSIS: No data available - Time schedule for collection of urine: No data available- Metabolism cages used for collection of urine: No data available - Animals fasted: No data available- Parameters checked in table [No.?] were examined.: No data available NEUROBEHAVIOURAL EXAMINATION: No - Time schedule for examinations: No Data Available. - Dose groups that were examined: No Data Available- Battery of functions tested: sensory activity / grip strength / motor activity / other: No Data AvailableOTHER: Yes Frozen Samples of testes were used for estimation of proteins, ribose nucleic acid (RNA) and deeoxyribose nucleic acid (DNA), Malondialdehyde (MDA) and Non-Protein Sulphahydryl group (NP-SH).

Sacrifice and pathology:

The mice were killed using ether after the last day of sub-chronic treatment.

Other examinations:

-Organ Weight: The organs such as testes, caudae epididymis, seminal vesicles and prostate gland were weighed after sacrifice. -Estimation of total proteins and nucleic acids: Testes were homogenized and the homogenate was suspended in ice-cold trichloroacetic acid (TCA). After centrifugation, the pellet was extracted with ethanol. The levels of DNA were determined by treating the nucleic acid extract with diphenylamine reagent and reading the intensity of blue color at 600 nm. For quantification of RNA, the nucleic acid extract was treated with orcinol and the green color was read at 660 nm. Standard curves were used to determine the amounts of nucleic acids present. -Determination of MDA concentrations: Testes were homogenized in TCA solution and the homogenate suspended in thiobarbituric acid. After centrifugation the optical density of the clear pink supernatant was read at 532 nm. Malondialdehyde bis (dimethyl acetal) was used as an external standard.-Quantification of NP-SH levels: The testes were homogenized in ice-cold 0.02M ethylene-o-amine tetra acetic acid disodium (EDTA) and mixed with TCA. The homogenate was centrifuged at 3000×g. The supernatant was suspended in tris buffer, 5-5-dithiobis-(2 nitrobenzoic acid) (DTNB) and red at 412 nm against reagent blank with no homogenate.

Statistics:

The different studies undertaken were statistically analyzed by analysis of variance (ANOVA).

Results and discussion

Results of examinations

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In male mice, the subchronic treatment resulted in significant increase in reproductive organs such as caudae epididymis and prostate.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Prolonged treatment with Ginkgo biloba failed to induce any significant changes in the protein contents of the testicular cells. Although, the concentrations of RNA and DNA were decreased significantly at the higher dose (100 mg/kg/day). At the same dose the levels of MDA were significantly increased.The concentrations of NP-SHwere found to decrease. The depletion was statistically significant at 25 mg/kg/day and at 50 mg/kg/day.

Effect levels

1

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Significant increase in weights of Caudae epididymis and Prostate gland.

Remarks on result:

other: no other details available

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No significant changes in the protein contents of the testicular cells.

Remarks on result:

other: no other details available

3

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Concentrations of RNA and DNA significantle decreased while concentration of MDA significantly decreased at the same dose level.

4

Key result

Dose descriptor:

LOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: The levels of NP-SH was found to decrease significantly at 25 mg/kg/day and at 50 mg/kg/day.

Target system / organ toxicity

Critical effects observed:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

NOAEL was observed to be in the concentration of proteins while a LOAEL is attributed to 100 mg/kg bw/day where decrease in RNA and DNA concentrations and increase in MDA concentrations are observed. Also, a LOAEL was attributed to 25 mg/kg bw/day wherein decrease in the levels of Non-Protein Sulfahydryl group was observed in male Swiss albino mice

Executive summary:

A repeated dose (90 Days) Oral Toxicity Study was conducted on Swiss Albino mice to determine any reproductive, cytological and biochemical toxicity of Ginkgo biloba. A total of 240 healthy (120 male and 120 female) mice were used in the study. The study duration was 90 days and the test chemical was uniformly mixed with vehicle (tap water) and administered to mice at the graduated dose levels of 25, 50, and 100 mg/kg/day for low dose (G2), mid dose (G3) and high dose (G4) group rats, respectively. The rats in the vehicle control group (G1) groups received vehicle (tap water) alone. The dose volume administered to all the animals was 1.6 gm/kg. The dose of Ginkgo biloba was determined by (i) maximum tolerated dose (MTD) and (ii) human therapeutic dose with reference to the surface area rule. Two individual studies were conducted to identify and analyze the reproductive toxicity of Ginkgo biloba in both male and female mice. In first study, 5 male mice each were inducted in control group and all other dose groups. The rationale behind this study was to analyze the effect of Ginkgo biloba on weight of organs such as prostate, testes and Caudae epididymis and also to assess the effect of Ginkgo biloba on the biochemical parameters in swiss albino mice. In this study, the aqueous suspension of Ginkgo biloba was administered by gastric intubation (oral gavage) for a period of 90 days. After the dosing, the mice were sacrificed and necropsied after the last day of subchronic treatment and weighed for essential reproductive organs namely testes, caudae epididymis, seminal vesicles and prostate gland. After sacrifice and necropsy, it was observed that, the subchronic treatment caused a significant increase in the mean weight of caudae epididymis at 50 and 100 mg/kg/day and prostate at 100 mg/kg/day. Further, effects of Ginkgo biloba on levels and concentration of biochemical parameters such as total proteins, RNA and DNA, levels of Malondialdehyde and Non-Protein Sulfhydryl group were assesed. For estimation of total proteins the testes of male mice was homogenized and was suspended in ice-cold trichloroacetic acid (TCA). After centrifugation, the pellet was extracted in ethanol. The levels of DNA were determined by treating the nucleic acid extract with diphenylamine reagent and reading theintensity of blue colour at 600 nm. For quantification of RNA , the nucleic acid extract was treated with orcinol nd the green colour was read at 660 nm. Total protein estimation was performed by using Modified Lowry estimation. Standard curves were usd to determine the amounts of nucleic acid and proteins present. From all the analysis it was found out that a significant decrease in concentrations of RNA and DNA was observed at 100 mg/kg bw/day. Also, no significant changes in protein concentration was observed at 100 mg/kg bw/day. For determining the MDA concentrations, testes were homogenized in TCA solutionand the homogenate was suspended in thiobarbituric acid. After centrifugation, the optical density of the clear pink supernatant was read at 532 nm. Malondialdehyde bis(dimethyl acetal) was used as an external standard. After this experiment, a significant increase in the concentrations of MDA was observed at 100 mg/kg bw/day dose range. An experiment was also set-up for the quantification of Non-Protein Sulfahydryl (NP-SH) group where the testes were homogenized in ice-cold 0.02 M ethylene-o-amine tetra acetic acid disodium (EDTA) and mixed with TCA. The homogenate was centrifuged at 3000 x g. The supernatant was suspended in tris buffer, 5 -5' dithiobis-(2 nitrobenzoic acid) (DTNB) and was read at 412 nm against reagent blank with no homogenate. From this experiment, it was observed that there was a significant depletion in the levels of NP-SH at 25 mg/kg bw/day and at 50 mg/kg bw/day. From all the results it was concluded that, a NOAEL is seen in the concentration of proteins while a LOAEL is attributed to 100 mg/kg bw/day where decrease in RNA and DNA concentrations and increase in MDA concentrations are observed. Also, a LOAEL was attributed to 25 mg/kg bw/day wherein decrease in the levels of Non-Protein Sulfahydryl group was observed in male Swiss albino mice.