Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled, oligomerisation products with 1-chloro-2,3-epoxypropane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

some minor deviations

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled, oligomerisation products with 1-chloro-2,3-epoxypropane

Cas Number:

68413-24-1

Molecular formula:

C24H36O2

IUPAC Name:

Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled, oligomerisation products with 1-chloro-2,3-epoxypropane

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): CARDOLITE NC-513
- Physical state: liquid
- Batch no.: GH-0129
- Expiry date: 13.07.2014
- Storage condition of test material: room temperature 20 ± 5 °C, keep away from humidity
- Other: the test item was previously considered as 'Cashew, nutshell liq., oligomeric reaction products with 1-chloro-2,3-epoxypropane' (EC number 500-210-7).

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sandhofer Weg 7 97633 Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 259 g – 296 g (males); 173 g – 196 g (females) (at acclimatisation)
- Fasting period before study: no information
- Housing: Clean conventional housing
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6-9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 10 cycles/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle: the test item mixed with the required quantity of vehicle.
Fresh test item dosage forms were prepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Amount of vehicle (if gavage): 5 mL/kg/day (dosage-volume)

PREPARATION OF DOSING SOLUTIONS:
For preparation of the application solutions it was diluted with corn oil as the substance is insoluble in water. The application suspensions was prepared daily according to the following protocol:
1. Fill required volume to produce the high dose application solution of the test item into an appropriate vial.
2. Add corn oil up to the required volume to produce the application suspension as stated in the laboratory work sheets.
3. Stir until suspension is a consistent solution.
4. Prepare a serial dilution (1 in 4) to produce the further application solutions.
5. Stir immediately before the application solution will be drawn into the application syringe.

VEHICLE
- Justification for use and choice of vehicle (if other than water): see above (water insolubility)
- Amount of vehicle (if gavage): 4 ml per kg body weight

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

males: 48 -61 days
females: >= 46 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Control

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: preliminary study

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day
- Cage side observations: Viability and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at least once weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After the pre-mating phase for 5 female animals of each dose group; on the day of scheduled necropsy for 5 male animals of each dose group.
- Anaesthetic used for blood collection: ether
- Animals fasted: no information
- How many animals: see above
- Parameters examined:
Leucocytes
Erythrocytes
Hemoglobin
Haematokrit
Mean cell volume
Mean cell hemoglobin concentration (MCHC)
Mean cell hemoglobin (MCH)
Thrombocytes (PLAT)
Reticulocytes
Neutrophils
Eosinophils
Basophils (B) G/L
Lymphocytes (L) G/L
Monocytes (M) G/L

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: no information
- How many animals: see above
- Parameters examined:
Bile acids
Alkaline phosphatase
Aspartate aminotransferase
Alanine aminotransferase
Cholesterol
Urea
Sodium
Potassium
Chloride
Calcium
Glucose
Albumin
Total protein
Globulin
Albumin/globulin ratio
Creatinine

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: grip strength, beam walking test

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Sacrifice
Animals were euthanised when found to be moribund or when the adequate number of litters according to guideline OECD 422 was achieved. All animals were sacrificed humanely by asphyxiation in a CO2 atmosphere.

Organ weights
The body weight of all animals killed at the end of the treatment period was recorded before sacrifice, and the organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Macroscopic post-mortem examination
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

Preservation of tissues
For all study animals, the tissues specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except for the eyes and Harderian glands which were fixed in Davidson's fixative, and the testes and epididymides which were preserved in Bouin's fluid).

Microscopic examination
All tissues required for microscopic examination were embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS).
A microscopic examination was performed on:
- all tissues listed below for animals of the control and high-dose groups (groups 1 and 4) killed at the end of the treatment period,
- all macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3) killed on completion of the treatment period.

Tissues (preserved, organ weights (for ** only))
Organs
Macroscopic lesions
Adrenals **
Brain** (not preserved)
Cecum
Cerebrum
Cerebellum
Colon
Duodenum
Epididymides **
Esophagus
Heart **
Ileum
Jejunum
Kidneys **
Liver **
Lungs
Lymph nodes
Ovaries **
Peripheral nerve
Pons
Prostate
Rectum
Spinal cord
Spleen **
Bone marrow
Sternum
Stomach
Testes **
Thymus **
Thyroids
Trachea
Urinary bladder
Uterus
Vagina

Statistics:

Descriptive statistics
The arithmetic mean and standard deviation were calculated for all grouped numerical data originating from monitoring the body weight, food- and water consumption, organ weights (gross pathology) and litter size and weight (for details see appendix). Where appropriate, detailed column statistics were applied (minimum / maximum data, 25% quantiles, standard error, upper and lower confidence interval 95%).

Inductive statistics
If appropriate, the respective test item groups were compared to the vehicle group by assessing statistical significance using a two-tailed unpaired Student´s t-test. For all calculations, the significance level was set to 0,05.
For some analysis parameters that returned statistical significances in the t-test, further inductive statistics were applied as outlined in the schematic decision tree displayed in the appendix. Most statistical hypotheses in this study were best characterised as “many to one”– a vehicle control vs. three treatment groups, respectively. Therefore the adequate analysis method was a One-Way ANOVA (Analysis of variance), followed by a post hoc Dunnett´s t-test. In case a sufficient number of values per group were available a Bartlett´s test for equal variances was applied on the data. For all calculations, the significance level was set to 0,05. These further inductive statistics were then performed using Graph Pad Prism for Mac, Version 5.01. Statistical data and analyses were documented in the appendix.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mild discomfort throughout the whole application period was observed for the animals treated with the high and the medium dose of the test item (wiping of nose and mouth through the cage bedding, salivation after application, bleeding of mucous membranes at nose and mouth, respirators sounds). Moreover, some male animals of the high dose group became lethargic after application of the test item on individual days. A biological and particularly toxicological relevance of these observations could not be excluded.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Regarding the body weight and the body weight gain, no significant differences were observed between all test item treated animals (male and female) and their respective vehicle control animals. Occasional differences observed for the females were assumed to be of natural origin based on the pregnancy status of the animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item related tendencies regarding food and water consumption of all test item treated animals (male and female) could be observed throughout the whole study phase when compared to their respective vehicle control animals. All fluctuations observed were most likely of natural origin.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No test item related tendencies regarding food and water consumption of all test item treated animals (male and female) could be observed throughout the whole study phase when compared to their respective vehicle control animals. All fluctuations observed were most likely of natural origin.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No relevant test item induced effects on any haematology or clinical biochemistry parameter
could be detected

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No relevant test item induced effects on any haematology or clinical biochemistry parameter
could be detected

Endocrine findings:

not examined

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Description (incidence and severity):

No alterations regarding general behaviour of the rats were observed during in-life phase

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw (males and females): Statistically significant increase of the liver weight (male; absolute and relative), a statistically significant decrease of the prostate weight (absolute and relative), a statistically significant increase of the brain weight (female; absolute), and a statistically significant weight increase of the right ovary (absolute and relative). Moreover, with increasing dose levels the amount of male animals having slightly developed mammary glands declined. Besides some further individual findings, heterogeneously distributed over all dose groups, no further apparent observations were made that could be related to the administration of the test item.

Gross pathological findings:

not examined

Description (incidence and severity):

See organ weight findings above

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The type, incidence and severity of all microscopic findings observed did not indicate a relationship to the treatment with the test item. The repeated oral administration of the test item did not produce any evidence of pathomorphological findings that are considered to be due to a toxic effect of the test item.

Histopathological findings: neoplastic:

not examined

Details on results:

ORGAN WEIGHTS
For females of the high dose group (1000 mg/kg bw) ovary weight was increased.

Effect levels

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In a GLP-study according to OECD Test Guideline 422 the test item CARDOLITE NC-513, when administered by gavage to Wistar rats at the dose-levels of 62.5, 250 or 1000 mg/kg/day for at least 46 days, was clinically well tolerated. No overt signs of toxicity were observed in hematological, blood biochemical or urinalysis parameters. There were findings in prostate, ovary and liver weight at the high dose group (1000 mg/kg bw). No macroscopic or histopathological findings revealed a treatment-related effect. Under the experimental conditions of the study, the No Observed Effect Level (NOAEL) was established at 250 mg/kg/day in Wistar rats.

Executive summary:

In a GLP-study according to OECD Tes Guideline 422 the toxic effects of the test item CARDOLITE NC-513 at doses of 62.5, 250 and 1000 mg/kg body weight on the subacute toxicity and the development and reproduction of Wistar rats after oral administration were under examination.The substance was at least applied for 46 days daily to rats of both sexes. The following observations were made.

 

General and detailed clinical signs:

Mild discomfort throughout the whole application period was observed for the animals treated with the high and the medium dose of the test item (wiping of nose and mouth through the cage bedding, salivation after application, bleeding of mucous membranes at nose and mouth, respirators sounds). Moreover, some male animals of the high dose group became lethargic after application of the test item on individual days. A biological and particularly toxicological relevance of these observations could not be excluded.

 

Body weight, food and water consumption: Regarding the body weight and the body weight gain, no significant differences were observed between all test item treated animals (male and female) and their respective vehicle control animals. Occasional differences observed for the females were assumed to be of natural origin based on the pregnancy status of the animals.

No test item related tendencies regarding food and water consumption of all test item treated animals (male and female) could be observed throughout the whole study phase when compared to their respective vehicle control animals. All fluctuations observed were most likely of natural origin.

 

Haematology and clinical biochemistry: no effects

 

Necropsy:

The determination of organ weights, as part of the necropsy, showed a statistically significant increase of the liver weight (male; absolute and relative), a statistically significant decrease of the prostate weight (absolute and relative), a statistically significant increase of the brain weight (female; absolute), and a statistically significant weight increase of the right ovary (absolute and relative) within the animals treated with the high dose of the test item. Moreover, with increasing dose levels the amount of male animals having slightly developed mammary glands declined. Besides some further individual findings, heterogeneously distributed over all dose groups, no further apparent observations were made that could be related to the administration of the test item.

 

Histology: no effects

 

In conclusion: A daily oral administration of the test item to female Wistar rats at dose levels of 62,5 mg, 250 mg and 1000 mg/kg body weight over a time period of 46 to 79 days resulted in minor systemic effects in the high dose group. With respect to the findings in prostate and liver weight (in males) and ovary weight (in females) the NOAEL regarding the subchronic toxicity was set to 250 mg/kg body weight.