Registration Dossier

Administrative data

Description of key information

NOAEL was observed to be in the concentration of proteins to 100 mg/kg bw/day where decrease in RNA and DNA concentrations and increase in MDA concentrations are observed. Also, a NOAEL 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

Key value for chemical safety assessment

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:
90 Days
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
no guideline available
Principles of method if other than guideline:
Effects of Ginkgo biloba on biochemical parameters on mice.
GLP compliance:
not specified
Species:
mouse
Strain:
Swiss
Sex:
male
Details on test animals 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
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
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
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).
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.
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
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
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.
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.
Critical effects observed:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
K2 level publication

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose oral-

Based on the various studies available with Klimish rating 2 for the read across substances for CAS: 37330-39-5 based on the category approach of organic functional group along with similar mechanistic approach and having structural similarities defined by QSAR toolbox. This data is combined with the prediction done using the QSAR toolbox for the target chemical based on similar category approach, the results is summarized as follows.

Sr. No

End point

Value

Species

Route

Effects

Remarks

1.

NOAEL

100 mg/kg bw/day

Mice

Oral

No adverse effects observed.

publication data

2.

NOAEL

40mg/kg bw/d

Rat

Oral

No adverse effects observed.

Study report RA-27193-86-8

3

NOAEL

100mg/kg bw/day (nominal)

Rat

Oral

No change in body weight were observed in treated and control dose group.

Publication data

 

Based on the studies summarized in the above table with various routes it can be observed that NOAEL values varies from 40 - 161.657424927 mg/Kg bw/ d. The no effects observed on the these doses was listed as follows

-Overall effects-no adverse effects observed on body weight,food consumption etc.

-No effects on clinical signs, body weight,histopathology.

Thus based on above discussion it can be concluded that substance CAS: 37330-39-5 is expected to show the similar toxicological effect based on the effects observed on the other category members. Since the no effective dose value (NOAEL) is higher than 40 mg/kg bw/day thus based on this value it can be concluded that substance CAS: 37330-39-5 is considered to be not toxic via repeated dose toxicity oral route.Also there is no known evidence of adverse effect on Human of CAS: 37330-39-5 as well as mechanistic triggers does not classify this substance as toxic substance.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The NOAEL (no observed adversed effects level) value of Cardanol in was observed at a dose level of 161.65 mg/kg bw /day.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
This end point was considered for waiver considering that the vapour pressure of Cardanol is very low i.e. 0.0000195 Pascal.The inhalation route is the least likely route of exposure for test substance. Thus, repeated dose toxicity by the inhaltion route shall be all the more rare and hence this end point was considered for waiver

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
This end point was considered for waiver considering that the vapour pressure of Cardanol is very low that is 1.27E-006 mm Hg at 25 deg c.The inhalation route is the least likely route of exposure for test substance. Thus, repeated dose toxicity by the inhaltion route shall be all the more rare and hence this end point was considered for waiver

Justification for classification or non-classification

The substance cardanol is unlikely to show repeated dose toxicity effect for oral,dermal and inhalation route and thus will not be considered for further classification.