Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11th June 2019 to 5th November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
OECD TG408 dated June 2018
Deviations:
no
Remarks:
No deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in test report): Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade)
- Physical state: liquid
- Other: Straw coloured to dark brown liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: From Charles River Laboratories Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: At the initiation of dosing animals were approximately 6 to 8 weeks old
- Weight at study initiation: At the initiation of dosing males were between 134 and 174 g and females were between 108 and 143 g.
- Fasting period before study:
- Housing: On arrival and following randomization, animals were group housed (up to 5 animals of the same sex and same dosing group together) in Macrolon plastic cages (Type IV, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany).
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare Corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage enrichment, bedding material, food and water.

- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
During motor activity measurements, animals did not have access to food for a maximum of 2 hours.

- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
During motor activity measurements, animals did not have access to water for a maximum of 2 hours.

- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for 13 days before the commencement of dosing.

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target temperatures of 18 to 24°C with the actual daily mean temperature during the study period being 20 to 22°C.
- Humidity (%): Relative target humidity of 40 to 70% with the actual daily mean relative humidity being 50 to 73%.
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): A 12 hour light/12 hour dark cycle was maintained, except during designated procedures.

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Dried and deacidified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity/stability analyses of dosing solutions were performed at each dose level prior to the initiation of the study.

The concentrations analyzed in the formulations of the 100 mg/kg bw/day (low dose) 300 mg/kg bw/day (intermediate dose) and 1000 mg/kg bw/day (high dose) were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).
Duration of treatment / exposure:
90 days
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
actual ingested
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Actual ingested
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Actual ingested
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Actual ingested
No. of animals per sex per dose:
10 males and 10 nulliparous and non-pregnant females

Control animals:
yes, concurrent vehicle
Details on study design:
A total of 40 male and 40 nulliparous and non-pregnant Wistar Han rats were allocated to four groups (10 per sex per group) Three groups were administered with the test substance Distilled Grade, by oral gavage daily from day 1 to day 90 inclusive, at the dose levels of 100, 300 and 1000 mg/kg bw/day. One group of males and females was only administered with the vehicle, dried and deacidified corn oil, at the same constant dose volume of 5 mL/kg.
Individual volumes were adjusted through the study according to the most recently recorded body weight.

Rationale for dose selection: These doses were selected based on the results of a 14 day range finding test for the OECD TG408 study.
Positive control:
None used

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage side observations were made at least once daily, 0 to 30 minutes post-dose during the dosing period. Animals were not removed from the cage during the observations, unless it was necessary for identification or confirmation of possible findings
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed once daily, beginning during the first administration of the test substance and lasting throughout the dosing period. During the dosing period, these observations were performed 1.0 hours ±15 minutes after dosing (based on the peak effect of occurrence of clinical signs observed in the dose range finder). The observations included those for salivation, hunched posture, piloerection (erect fur), abnormal breathing sounds, skin lesions, scabs and fur loss.

ARENA OBSERVATIONS: Yes
Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout the dosing period. These observations were conducted 1.0 hours ± 15 minutes after dosing.

MORTALITY: Yes
Time schedule - At least twice daily throughout the study animals were observed for general health/mortality and moribundity. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly, starting Day 1 (at pre-dose) animals were individually weighed. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Time schedule: Weekly, starting Day 1, throughout the dosing period food consumption was quantitatively measured except on the day of scheduled euthanasia.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol 5 mg/ml solution, THEA Pharma, Wetteren, Belgium) during pretreatment in all animals, and at the end of the dosing period in Week 13 in the control and the high dose (1000 mg/kg bw/day) exposed animals. The observations included assessment of corneal oedema, opacity and vascularisation, pupil dilation and dyscovia and effects on the vitreous fluid.
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from all surviving animals prior to necropsy, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands).

For haematology analysis 0.5 ml samples were taken into K3-EDTA anticoagulant tubes; (Greiner Bio-One GmbH, Kremsmünster, Austria). The following haematology parameters were measured:
• Total leukocyte count (White blood cells)
• Erythrocyte count (Red blood cells)
• Haemoglobin
• Haemocrit
• Mean corpuscular volume (MCV)
• Mean corpuscular haemoglobin (MCH)
• Mean corpuscular haemoglobin concentration (MCHC)
• Platelet count
• Reticulocyte count (absolute)
• Neutrophils (absolute)
• Lymphocytes (absolute)
• Monocytes (absolute)
• Eosinophils (absolute)
• Basophils (absolute)
• Red Blood Cell Distribution Width (RDW)
• Large unstained cells (LUC) (absolute)

For the measurement of coagulation parameters, Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT), 0.45 ml target blood volumes were taken into Citrate anticoagulant tubes (Greiner Bio-One GmbH, Kremsmünster, Austria)

SERUM CHEMISTRY: Yes
For serum chemistry determinations 0.5 ml plasma samples were taken into Li-Heparin anticoagulant tubes; (Greiner Bio-One GmbH, Kremsmünster, Austria). The following serum chemistry parameters were measured:
• Alanine aminotransferase (ALAT)
• Aspartate aminotransferase (ASAT)
• Alkaline Phosphatase (ALP)
• Total protein
• Albumin
• Total Bilirubin
• Cholesterol (including HDL and LDL Cholesterol)
• Creatinine
• Glucose
• Urea
• Calcium
• Chloride
• Inorganic Phosphate
• Potassium
• Sodium

THYROID HORMONE ANALYSIS: Yes
One sample with a target volume of 0.5 mL blood was collected in Li-Heparin tubes and processed to plasma. Another sample, with a target volume of 1 mL (blood) was collected in serum tubes and processed to serum. These plasma and serum samples were initially stored at ≤-75°C for subsequent analysis of triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
The following Functional Battery Observations (FOB) were recorded in the test organisms:

OPEN FIELD OBSERVATIONS (EVALUATED OVER A 2-MINUTE OBSERVATION PERIOD)
Once before the first administration of the test substance and at weekly intervals during the exposure period animals were observed for clinical signs outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded.

FUNCTIONAL TEST OBSERVATIONS
Functional tests were performed on the first 5 animals per sex per group during Week 12-13. These tests were performed before dosing, after completion of clinical observations (including arena observation, if applicable).
The following tests were performed:

• Hearing ability (Score 0 = normal/present, score 1 = abnormal/absent).
• Pupillary reflex (Score 0 = normal/present, score 1 = abnormal/absent).
• Static righting reflex (Score 0 = normal/present, score 1 = abnormal/absent).
• Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).

OTHER:
LOCOMOTOR ACTIVITY
Locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). was evaluated once during the treatment period (in Week 12-13). Tests were performed on the first 5 animals per sex per group. These tests were performed after clinical observations (including arena observation, if applicable). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
Sacrifice and pathology:
Animals surviving until scheduled euthanasia had a terminal body weight recorded and were deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy, though water was available.

ORGAN WEIGHT ASSESSMENT: Yes
Identified organs (brain, cervix, epididymis, heart, kidney, liver, ovary, spleen, testis, thymus, uterus and the adrenal, pituitary, prostate, seminal vesicle and thyroid glands) were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. Organ weight as a percent of body weight (using the terminal body weight) was calculated.

GROSS PATHOLOGY: Yes
All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

HISTOPATHOLOGY: Yes
Representative samples of these tissues were collected from all animals at necropsy including: animal identification, artery, aorta, body cavity, nasopharynx, bone (femur, marrow and sternum), brain [cerebellum, mid-brain, cortex] (8 levels), cervix, epididymis oesophagus, eye, glands (adrenal, clitoral, harderian, lacrimal, mammary, parathyroid, pituitary, preputial, prostate, salivary, seminal vesicle , (para) -thyroid), gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (caecum, colon and rectum), larynx, liver, lung, lymph node (mandibular and mesenteric), muscle (skeletal), nerve (optic and sciatic), ovary, pancreas, skin, small intestine (duodenum, ileum and jejunum), spinal cord, spleen, stomach, testis, thymus, tongue, trachea, urinary bladder, uterus and vagina.

Most of these tissues were preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands). Certain tissues (epididymis, eye, harderian gland, optic nerve and testis were preserved in Modified Davidson’s fixative prepared at Charles River Den Bosch using formaldehyde 24%,ethanol, acetic acid - glacial (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA).

The identified tissue issues (except animal identification, nasopharynx, femur, clitoral gland, lacrimal gland, preputial gland, larynx and tongue) were embedded in paraffin (Klinipath, Duiven, The Netherlands), sectioned, mounted on glass slides, and stained with hematoxylin and eosin (Klinipath, Duiven, The Netherlands). All tissues were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

Other examinations:
ESTROUS STAGE DETERMINATION
On the day of necropsy vaginal lavage was performed for all females to determine the stage of the estrous. The estrous stage in females was evaluated by examining the vaginal cytology of samples obtained by vaginal lavage
Statistics:
All statistical tests were performed using appropriate computing devices or programs. Data were analysed between the control and treatment groups (low, intermediate and high doses) as appropriate depending on data availability and the normality of data distribution using relevant parametric or non-parametric approaches. Significant differences between control animals and those treated with the test substance were expressed at the 5% of 1% level. Each mean for an endpoint was given with the standard deviation (S.D) and the number of animals used to calculate the mean.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No toxicologically relevant clinical signs were noted during the clinical observations.

Increased salivation was observed in the majority of animals treated at 300 and 1000 mg/kg bw/day. This clinical sign is considered to be a physiological response related to the taste of the test substance rather than a sign of systemic toxicity
Mortality:
no mortality observed
Description (incidence):
No mortalities were observed in the controls and the 100, 300 and 1000 mg/kg bw/day treatment groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of males treated up to 300 mg/kg bw/day and females treated up to 1000 mg/kg bw/day remained in the same range as controls over the study period.

A slightly lower body weight gain was noted for males treated at 1000 mg/kg bw/day from Day 15 onwards (except between Days 64-71 and 85-91), this resulted in a slightly lower body weight up to -7.7% compared to controls. Based on the magnitude of the change it was not considered to be toxicologically relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related changes in food consumption were recorded for males treated up to 1000 mg/kg bw/day and females treated up to 300 mg/kg bw/day.

Increased food consumption (up to +15.6% compared to controls) was observed for females treated at 1000 mg/kg bw/day from Day 8 onwards. Based on the direction and magnitude of the change this was not considered to be toxicologically relevant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
A subjective appraisal of water consumption was maintained during the study, but no quantitative investigation was conducted as no effect was suspected

Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to treatment with the test substance.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were noted in haematological parameters.

Slightly increased platelet counts (1.16x of controls) and slightly decreased haemoglobin concentrations (0.95x of controls) were noted in males and females treated at 1000 mg/kg bw/day, respectively. Based on the minimal magnitude of the change and/or the absence of changes in related parameters they were not considered to be toxicologically relevant.

Other statistically significant changes in hematology parameters were considered to be unrelated to treatment with the test substance based on the magnitude of the change and/or the fact that these occurred in the absence of a dose-related trend.

No toxicologically relevant changes were noted in coagulation parameters. A shorter activated partial thromboplastin time (APTT) was noted for males (0.84x of controls) and females (0.86x of controls) treated at 1000 mg/kg bw/day. This was considered to be of no toxicological relevance as the opposite effect (i.e. a prolongation) would be expected in case of target organ toxicity.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes in liver parameters distinguished treated animals from the controls at the end of the treatment period and were considered test substance related (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):

• Increased aspartate aminotransferase (AST) activity in males and females treated at 1000 mg/kg bw/day (1.59x and 1.25x, respectively). Values for both sexes were slightly outside the range of historical control values.
• Increased alanine aminotransferase (ALT) activity in males and females treated at 1000 mg/kg bw/day (4.29x and 4.05x, respectively).Values for both sexes were outside the range of historical control values.
• Increased alkaline phosphatase (ALP) activity in males and females treated at 1000 mg/kg bw/day (2.46x and 2.39x, respectively). Values for both sexes were slightly outside the range of historical control values.
• Increased concentrations of total bilirubin (TBIL) in males and females treated at 1000 mg/kg bw/day (1.48x and 1.24x, respectively). Values of males were slightly outside the range of historical control values.

Other test substance-related changes in clinical biochemistry parameters that distinguished treated animals from the controls were lower creatinine concentrations in females treated at 300 and 1000 mg/kg bw/day, higher cholesterol concentrations in females treated at 1000 mg/kg bw/day, lower concentrations of calcium in males treated at 1000 mg/kg bw/day, and higher concentrations of LDL, sodium and inorganic phosphatase in males treated at 1000 mg/kg bw/day. These changes were not considered to be toxicologically relevant based on the magnitude of the change.

Other statistically significant changes in clinical chemistry parameters were considered to be unrelated to treatment with the test substance as these occurred in the absence of a dose-related trend.

Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Grip strength, hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.

Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Any statistically significant changes in functional observations were considered to be unrelated to treatment with the test substance as these occurred in the absence of a dose-related trend
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related higher liver weights were noted in males and females at the end of treatment. In males, higher liver weights were noted at 300 and 1000 mg/kg bw/day (relative to body weight only). In females, higher liver weights were noted at 300 mg/kg bw/day (relative to body weight only) and at 1000 mg/kg bw /day (absolute and relative to body weight).

Any other differences, including those that reached statistical significance were considered not to be test substance-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values. These related to potential effects on the heart and thymus in males.

Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance-related macroscopic changes were observed in treated animals.

Slight variability was noted in the incidence of dark foci in the glandular mucosa of the stomach in test substance-treated males compared to the control males. However, this was of low incidence generally and without a dose relationship and was not suggestive of a test substance-related effect.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related microscopic findings after treatment were noted in the liver, thyroid glands, mesenteric lymph node, and/or stomach of males (at 1000 mg/kg bw/day) and females (starting at 300 mg/kg bw/day), and the spleen of females at 1000 mg/kg bw/day.

In males at 1000 mg/kg bw/day, minimal hepatocellular hypertrophy (centrilobular) of the liver was noted in 40% of animals, and increased incidence of multinucleated hepatocytes (moderate severity) and karyomegaly (mild severity) was noted in 10% of animals. In females at 300 and 1000 mg/kg bw/day, minimal hepatocellular hypertrophy (centrilobular) was noted in 10% and 80% respectively; and increased multinucleated hepatocytes (up to moderate severity) was noted in 40% and 90%, respectively.

In males and females at 1000 mg/kg bw/day, there was an increased incidence and severity (up to mild) of diffuse follicular hypertrophy in the thyroid, compared to the control animals.

In males and females at 1000 mg/kg bw/day, there was an increased incidence of erythrophagocytosis in the sinuses of the mesenteric lymph nodes (up to a mild degree).

In males at 1000 mg/kg bw/day, erosion/ulcers of the stomach were noted in the glandular mucosa (up to a mild degree of severity) and hyperplasia of the non-glandular mucosa (up to a mild degree of severity) were noted. A single incidence of minimal focal erosion/ulcer of the glandular mucosa of the stomach in the pyloric region, and a single incidence of minimal hyperplasia of the non-glandular mucosa in males at 100 mg/kg bw/day was interpreted as not test substance-related due to the low incidence, lack of similar effect at the next highest dose (300 mg/kg bw/day), and to the possible spontaneous nature of this finding. In females at 300 and 1000 mg/kg bw/day, hyperplasia of the non-glandular mucosa (minimal) was noted.

In females at the highest dose of 1000 mg/kg/day, a slight decrease in the severity of pigment was noted in the spleen (minimal to mild) compared to the control group (mild to moderate).

There were no other test substance-related histological changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test substance-related alteration in the prevalence, severity, or histological character of those incidental tissue alterations.

Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplastic histopathological effects were evident in the controls and treatment groups of 100, 300 and 1000 mg/kg bw/day.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
THYROID HORMONE ANALYSIS
Statistically significant changes in thyroid hormone parameters (T3 changes in males at 300 mg/kg bw/day) were considered to be unrelated to treatment with the test substance based on the magnitude of the change and as these occurred in the absence of a dose-related trend.

An increased mean concentration of Thyroid-Stimulating Hormone (TSH) was noted in males treated at 300 and 1000 mg/kg bw/day (4.17x and 3.18x of controls, respectively) and females treated at 1000 mg/kg bw/day (3.45x of controls). However, there was significant variability in the levels of TSH measured in individual animals at these treatment doses. Mean values of females treated at 1000 mg/kg bw/day were slightly outside the range of historical control data.

ESTRUS STAGE DETERMINATION
No test substance-related changes in estrous stages were noted across the dose groups.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Local effects
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Remarks:
Systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Remarks:
Local effects
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:

Administration of Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade) by once daily oral gavage resulted in no adverse effects in male and female rats at dose levels of 100 and 300 mg/kg bw/day. Adverse findings were noted on liver weights and liver histopathology (in terms of hepatocellular hypertrophy and an increased incidence of multinucleated hepatocytes) at the highest dose of 1000 mg/kg bw/day. Clinical biochemistry measurements revealed concurrent increases in liver enzymes including a >4-fold increase in alanine aminotransferase activity (ALAT) in males and females treated at 1000 mg/kg bw/day, in combination with lesser increases in aspartate aminotransferase (ASAT) and alkaline phosphatase (ALP) activity and total bilirubin concentration, which suggests hepatocellular damage.

Adverse local effects were also noted on the morphology of the forestomach of males at the highest test dose of 1000 mg/kg bw/day. These were considered to be due to the known irritancy of the test substance.

Based on these results, the No Observed-Adverse-Effect Level (NOAEL) was considered to be 300 mg/kg bw/day. In this study, a marked increase of TSH was observed in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg/day. This change correlated with follicular cell hypertrophy in the thyroid at 1000 mg/kg bw/day which were without degenerative changes. The changes were considered to be test substance-related. However, under the conditions of this study no adverse effect was observed that could be linked to the increase in TSH and thyroid follicular cell hypertrophy and, therefore, this increase was not taken into account when determining the NOAEL.
Executive summary:

Groups of 10 male and 10 nulliparous and non-pregnant Wistar Han rats were treated with Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) for 13 weeks by daily oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. The test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

 

At end of treatment, higher liver weights were noted for males treated at 300 and 1000 mg/kg bw/day and females treated at 300 (relative to body weight only) and 1000 mg/kg bw/day (absolute and relative to body weight). This increased liver weight correlated to hepatocellular hypertrophy and an increased incidence of multinucleated hepatocytes, which were seen microscopically in males and females at 1000 mg/kg bw/day and males at 300 mg/kg bw/day. Additionally, clinical biochemistry measurements revealed concurrent changes in liver enzymes. A >4-fold increase in alanine aminotransferase activity (ALAT) was noted in males and females treated at 1000 mg/kg bw/day, in combination with lesser increases in aspartate aminotransferase (ASAT) and alkaline phosphatase (ALP) activity and total bilirubin concentration, which suggests hepatocellular damage. Therefore, the changes in the liver at 1000 mg/kg bw/day are considered adverse. The liver changes at 300 mg/kg bw/day were, at the severity observed, not considered to be adverse.

 

During microscopic examination, erosion/ulcers were noted in the stomach of males at 1000 mg/kg/day. Due to the degenerative nature of this finding, the effect was considered to be adverse.

An increased concentration of thyroid stimulating hormones was measured in males treated at 300 and 1000 mg/kg bw/day and females treated at 1000 mg/kg bw/day, this correlated with thyroid follicular cell hypertrophy.

 

In the study, a marked increase of TSH was observed in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day. This change correlated with follicular cell hypertrophy in the thyroid at 1000 mg/kg bw/day which were without degenerative changes. The changes were considered to be test substance-related. However, under the conditions of this study no adverse effect was observed that could be linked to the increase in TSH and thyroid follicular cell hypertrophy and, therefore, this increase was not taken into account when determining the NOAEL.

 

Other test substance-related findings at 1000 mg/kg bw /day that were noted consisted of erythrophagocytosis in the mesenteric lymph node, decreased severity of pigment in the female spleens, and epithelial hyperplasia of the non-glandular stomach. These findings were interpreted to be non-adverse based on the severity, lack of degeneration or necrosis, and no indication of altered function or possible sequelae in other organ systems.

 

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality/moribundity, clinical appearance, body weight, food consumption, ophthalmology, functional observations, haematology, coagulation and gross necropsy examination).

 

No adverse effects on endocrine-disruption related apical morphological and histological endpoints in males and females (thyroid weight and histopathology and indicators of thyroid hormonal activity) were observed in the study at any of the exposure doses of 100, 300 and 1000 mg/kg bw/day.