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Description of key information

OECD 422:

In a subacute toxicity study Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) was administered to 5 Sprague-Dawley rats/sex/dose via gavage at dose levels of 0, 15, 150, or 1000 mg/kg bw/day).
Systemic changes in the lungs, mesenteric lymph node, stomach and duodenum occurred at the highest dose of 1000 mg/kg bw/day. A NOAEL of 150 mg/kg bw/day has been identified for systemic toxicity. A NOAEL of 1000 mg/kg bw/day has been identified for reproductive parameters.
This repeat dose/reproductive toxicity screening study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study OECD 422 in rats.

OECD 408

Administration of Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) by once daily oral gavage for 90 days did not result in adverse effects in male and female rats at levels of the treatment doeses of 50 and 150 mg/kg bw/day. However, adverse effects were measured at the highest test dose of 500 mg/kg bw/day. Histopathological examinations showed, epithelial hyperplasia of the non-glandular mucosa of the stomach (forestomach, squamous mucosa) at 50, 150 and 500 mg/kg bw/day in males and females with a dose-related increase in severity, up to marked in males and moderate in females.  At 150 and 500 mg/kg bw/day this correlated with macroscopically irregular stomach surfaces.  These findings at 500 mg/kg bw/day were interpreted to be adverse due to the overall severity and concurrent presence of ulceration in some animals. These localised effects were considered to be due to the known irritancy of the test substance. Systemic toxicity was observed in males at 500 mg/kg bw/day in terms of a lower body weight gain which was also considered to be adverse.

Other test substance-related changes in clinical signs, body weight, clinical pathology, organ weights and histopathology were considered non-adverse.

Based on the results generated a No Observed Adverse Effect Level (NOAEL) for the systemic toxicity of Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) of 150 mg/kg bw/day was established. This NOAEL also provides protection against the observed local effects on stomach morphology due to the known irritancy of the test substance.


Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
Remarks:
Study conducted under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Age at study initiation: 8 weeks
- Weight at study initiation: males, 290-335g; females 191-215g
- Housing: Initially in groups of 5 in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbant paper. During mating cages held one male and one female. Males were returned to their original cages and females were housed individually.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
arachis oil
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Up to 54 consecutive days.
Frequency of treatment:
Daily.
Remarks:
Doses / Concentrations:
0, 15, 150, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on results of a preliminary range-finder study.
- Rationale for animal assignment (if not random): Random based on stratified bodyweights.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before and after dosing, and one and five hours after dosing during the working week; before and after dosing, and one hour after dosing during the weekend.


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for males; weeks 1, 2 and 3 and days 1 and 4 post partum for females.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data:


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: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 13, females at day 5 post-partum
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals:5 males and 5 females/dose group


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:Day 13, females at day 5 post-partum
- Animals fasted: No data
- How many animals:5 males and 5 females/dose group

URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine:
- Animals fasted:


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: End of mating phase, 5 males/dose group; Day 4 post-partum, 5 females/dose group
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Mating, pregnancy and parturition, number of pups born, number and sex of pups alive recorded daily, clinical condition of pups, individual pup and litter weights,
Statistics:
ANOVA, incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis, ANOVA and Mann-Whitney 'U' test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no toxicologically significant deaths during the study.

Increased salivation was detected prior to dosing and up to 5 hours after dosing for animals of either sex treated with 1000 mg/kg bw/day from Day 9 onwards. One female treated with 150 mg/kg bw/day developed clinical signs consistent with inappropriate dosing on Day 5 and was subsequently terminated. One female treated with 1000 mg/kg bw/day had given birth to a number of pups of which the majority were found dead. Several clinical signs were observed in this animal, and the animal and litter were terminated.

BODY WEIGHT AND WEIGHT GAIN
A slightly reduced bodyweight gain was observed for 1000 mg/kg bw/day males during the first two weeks of the study. Reduced bodyweight gain was also observed for 1000 mg/kg bw/day females during the later stages of the gestation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No adverse effect on dietary intake or food efficiency were detected.

FOOD EFFICIENCY
No adverse effect on dietary intake or food efficiency were detected.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No intergroup differences were detected.

OPHTHALMOSCOPIC EXAMINATION
N/A

HAEMATOLOGY
Haematological assessments revealed elevated platelet counts in animals of either sex treated with 1000 mg/kg bw/day. Elevated haemoglobin, erythrocyte and haematocrit was also evident for males treated at 1000 mg/kg bw/day. No such effects were detected at 150 and 15 mg/kg bw/day.

CLINICAL CHEMISTRY
An increase in aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels were observed for 1000 mg/kg bw/day animals of either sex, together with elevated inorganic phosphorus, bilirubin and urea, and reduced cholesterol levels.

URINALYSIS
N/A

NEUROBEHAVIOUR
Open field arena observations revealed increased salivation for individual animals of either sex treated with 1000 mg/kg bw/day from Week 3.

ORGAN WEIGHTS
Females treated with 1000 mg/kg bw/day showed elevated liver weights.

GROSS PATHOLOGY
None

HISTOPATHOLOGY: NON-NEOPLASTIC
Groups of alveolar macrophages were seen with a higher incidence for females treated with 1000 mg/kg bw/day.
A higher incidence of sinus histiocytosis and/or foamy histiocytes was observed in relation to treatment with 1000 mg/kg bw/day.
Hyperkeratosis, frequently associated with acanthosis was seen in the forestomachs of animals of either sex treated with 1000 mg/kg bw/day. Focal ulceration of the forestomach epithelium was also seen in one high dose female.
Mucosal hypertrophy was seen in three males at the top dose group. A low incidence of mucosal hypertrophy was observed in females in all dose groups.

OTHER FINDINGS
MATING
No adverse effects on mating or fertility were observed.

OFFSPRING LITTER SIZE AND VIABILITY
No effects detected.

OFFSPRING GROWTH AND DEVELOPMENT
No effects detected.

LITTER OBSERVATIONS
No effects detected.

UTERINE EXAMINATION
No effects detected.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Based on systemic changes in the lungs, mesenteric lymph node, stomach and duodenum.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No treatment effects occurred on reproduction or offspring development.
Critical effects observed:
not specified
Conclusions:
A NOAEL of 150 mg/kg bw/day has been identified for systemic toxicity. A NOAEL of 1000 mg/kg bw/day has been identified for reproductive parameters.
Executive summary:

In a subchronic toxicity study Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) was administered to 5 Sprague-Dawley rats/sex/dose via gavage at dose levels of 0, 15, 150, or 1000 mg/kg bw/day).

 

Systemic changes in the lungs, mesenteric lymph node, stomach and duodenum occurred at the highest dose.The NOAEL is 150 mg/kg bw/day.

 

This repeat dose/reproductive toxicity screening study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study OECD 422 in rats.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19th September 2018 to 19th February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
OECD TG408 dated June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han
Sex:
male/female
Details on test animals or test system 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 164 and 214 g and females were between 133 and 167 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 at least 5 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 48 to 58%.
- 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:
Route of administration:
oral: gavage
Vehicle:
propylene glycol
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 50 mg/kg bw/day (low dose) 150 mg/kg bw/day (intermediate dose) and 500 mg/kg bw/day (high dose) were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

Duration of treatment / exposure:
91 days
Frequency of treatment:
7 days/week
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Actual ingested
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
Actual ingested
Dose / conc.:
500 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 91 inclusive, at the dose levels of 50, 150 and 500 mg/kg bw/day. One group of males and females was only administered with the vehicle, propylene glycol, 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.
Positive control:
None used
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 1.5 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 1.5 hours ± 30 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

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 / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

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 12-13 in the control and substance 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).

- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked:
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)

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)


CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table :


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:


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 1.5 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 1.5 hours ± 30 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 AND WEIGHT GAIN: Yes
Time schedule - Weekly, starting Day 1 (at pre-dose) animals were individually weighed. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION: Yes
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 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 / No / Not specified WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOLOGICAL 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 12-13 in the control and substance 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: each test animals exposed to the test substance

HAEMATOLOGY: Yes
Time schedule - 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).
Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:

HAEMATOLOGY
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)

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
• 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.


IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
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, and bone marrow smears) 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.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Increased salivation was observed in animals treated at 50, 150 and 500 mg/kg bw/day with a dose-related trend being apparent. 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.

Hunched posture and piloerection was incidentally observed for male and females treated at 150 and/or 500 mg/kg bw/day.

Abnormal breathing sounds were also observed for males treated at 150 and 500 mg/kg bw/day and female controls and animals treated at 50 and 500 mg/kg bw/day.

Skin lesions, scabs and fur loss were incidentally noted, which were within the range of background findings expected for rats of this age and strain.

Mortality:
no mortality observed
Description (incidence):
No mortalities were observed in the controls and the 50, 150 and 500 mg/kg bw/day treatment groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test substance-related changes in body weight (gains) were noted in males at 50 mg/kg bw/day and in females up to 150 mg/kg bw/day.

At 150 mg/kg bw/day, slightly lower body weight (0.96x controls on Day 91) and body weight gain (0.92x on Day 91) was observed in males starting in the fourth week of dosing.

At 500 mg/kg bw/day, a moderate decrease in body weight (0.87x controls on Day 91) and body weight gain (0.73x on Day 91) was observed in males starting in the first week of dosing. In females, body weight (0.96x controls on Day 91) and body weight gain (0.93x on Day 91) was slightly lower starting in the third week of dosing.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related changes in food consumption were noted in males and females up to 50 mg/kg bw/day. At 150 and 500 mg/kg bw/day, food consumption was slightly lower in males and females in most dosing weeks. The lower food consumption observed in males at 50 mg/kg bw/day in Week 9 (0.71x controls) was considered to be an incidental finding and not related to treatment with the test substance.
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. The nature and incidence of ophthalmology findings noted during the pre-treatment period and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No test substance-related changes in haematological parameters were noted in males at 50 mg/kg bw/day and in females up to 150 mg/kg bw/day. At 150 mg/kg bw/day, a minimal decrease in red blood cells (0.96x controls) was observed in males.

At 500 mg/kg bw/day, a slight increase in white blood cells (WBC; 1.28x controls), lymphocytes (1.26x controls) and neutrophils (1.42x controls) was observed in males. In addition, red blood cells (0.94x controls) was slightly increased in males. In females neutrophils were slightly higher (2.00x controls) and mean corpuscular haemoglobin concentration (MCHC) were slightly decreased (0.97x controls).

Any other statistically significant changes in haematology parameters were considered to have arisen as a result of slightly high or low control values and/or were in the absence of a dose-related distribution, considered to be unrelated to treatment with the test substance.

No test item-related changes in coagulation parameters were noted in males up to 500 mg/kg bw/day. At all dose levels, a statistically significant dose-related decrease in activated partial thromboplastin time (APTT; 0.82x, 0.74x, and 0.71x controls at 50, 150 and 500 mg/kg bw/day, respectively) was observed in females.

The statistically significant changes in prothrombin time were considered to be unrelated to treatment with the test substance as these occurred in the absence of a dose-related trend and remained within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No marked test substance-related changes in clinical chemistry parameters were noted in males at 50 mg/kg bw/day and in females up to 150 mg/kg bw/day.

At 150 mg/kg bw/day, a slight to moderate increase in ALP activity in males and females (2.68x and 2.35x controls, respectively) and a slight decrease in total protein concentration (0.96x controls) in males were observed.

At 500 mg/kg bw/day, a slight to moderate increase in alanine and aspartate aminotransferase (ALAT and ASAT, respectively) and alkaline phosphatase activities (ALP) were observed in males and females (ALAT: 3.23x and 3.66x, respectively; ASAT: 1.39x (both sexes) and ALP: 3.60x and 3.90x controls, respectively). In addition, slightly lower total protein (0.88x and 0.91x controls) and albumin concentrations (0.94x and 0.93x controls) were observed in males and females, respectively.

Any other statistically significant changes in clinical chemistry parameters were considered to have arisen as a result of slightly high or low control values and/or appeared in the absence of a dose-related distribution. These were therefore considered to be unrelated to treatment with the test substance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all the animals examined in Week 13. In females at 50, 150 and 500 mg/kg bw/day, an increase in total movements (1.37x, 1.20x and 1.42x controls, respectively) and ambulations (1.56x, 1.33x and 1.64x controls, respectively) were observed. Motor activity was similar between males in the control and treatment groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. In absence of a clear dose-related trend and corroborative findings, these effects were considered to not be test substance-related.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No test-substance related changes in organ weights were observed in either males or females at 50 and 150 mg/kg bw/day. However, test substance-related increases in liver weights (relative to body weight) were noted in the 500 mg/kg bw/day group males and females. The higher liver weights were statistically significant only when expressed relative to body weight in males (10%) and females (13%) and were without histological correlation.

Some other organ weight differences were statistically significant at 500 mg/kg bw/day when compared to the control group, but were considered to be the result of a test substance-related effect on final body weight (-14% in males and -6% in females at 500 mg/kg bw/day) and were without any histological correlation. These included lower absolute heart and kidney weight in females (-8 and -9% respectively), and higher, relative to body weight values, for brain (12%), epididymis (18%), spleen (16%), and testis (20%) in males.

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 a) the adrenal gland, brain, pituitary gland, spleen, thymus gland, thyroid gland and thymus in animals of both sexes; b) the epididymis, prostate gland, seminal vesicle and testis in males and c) the ovaries and uterus in females.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related irregular surface morphology was observed in the non-glandular mucosa of the stomachs in males and females starting at 150 mg/kg bw/day. At 150 mg/kg bw/day 50% of males and females showed an irregular stomach surface whilst at 500 mg/kg bw/day the incidence was 90% in males and 100% in females.

Dark discoloration or dark focus in the mesenteric lymph node was observed in 40% and 10% respectively of males at 500 mg/kg bw/day.

The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related macroscopic findings after treatment were noted in the forestomach of males and females at all dose levels (starting at 50 mg/kg bw/day), the thyroid of males at all dose levels (starting at 50 mg/kg bw/day) and the mesenteric lymph node of males at 500 mg/kg bw/day.

In the stomach, test substance-related changes were present in males and females in the non-glandular region (i.e. squamous mucosa, forestomach) starting at 50 mg/kg bw/day. The primary change was diffuse epithelial hyperplasia (with hyperkeratosis) with a dose-related increase in severity, up to marked in males and moderate in females at 500 mg/kg bw/day. Secondary changes included minimal or mild mixed cell infiltrates of the non-glandular mucosa at 150 and 500 mg/kg bw/day, minimal or mild ulceration of the non-glandular mucosa in a few males and females at 500 mg/kg bw/day only, and submucosal oedema in females at each dose level. Based on the severity of the effects observed only those at 500 mg/kg bw/day were considered to be adverse. Functionally, the non-glandular stomach serves as a temporary storage organ and, due to prolonged exposure, is potentially a major site for interaction with the test substance. As humans lack a non-glandular stomach, the relevance of these test substance-related changes in this tissue for humans remains uncertain. However, it is reasonable to assume that the squamous mucosa of the oesophagus (in species without the non-glandular stomach) will react in a similar way to this test substance as the non-glandular stomach epithelium.

Males, starting at the lowest dose of 50 mg/kg bw/day, showed an increased incidence (compared to concurrent control males) of diffuse, bilateral, follicular cell hypertrophy. In most cases these changes were minimal in severity, but a few individuals showed a mild degree of severity, primarily in the highest dose of 500 mg/kg bw/day. In females a low incidence and minimal severity of follicular cell hypertrophy was noted in treated animals. It should be recognised that follicular cell hypertrophy of the thyroid gland at a low level is common background finding in rats of the strain and age used in the study. The changes observed, even in the males at the highest dose of 500 mg/kg bw/day, were without any degenerative changes (i.e. they were not characterised by progressive deterioration and loss of function in the organs and tissues).

In males at 500 mg/kg bw/day, minimal to mild erythrophagocytosis was noted in the mesenteric lymph nodes which correlated to macroscopic dark discoloration/foci in 5/10 animals. Minimal erythrophagocytosis noted in 1/10 females at 500 mg/kg bw/day was considered to be within the range of background for this common histological finding in lymph nodes.

There were no other test substance-related histologic 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 histologic 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 50, 150 and 500 mg/kg bw/day
Other effects:
effects observed, treatment-related
Description (incidence and severity):
THYROID HORMONE ANALYSIS
Analysis of the samples from animals at necropsy showed that mean total T3 levels in both males and females did not show clear dose–response relationships. At the lowest test dose of 50 mg/kg bw/day, both the total T3 and total T4 levels remained within the same range as the concurrent levels in control animals. Male rats treated at the highest test dose of 500 mg/kg bw/day showed a minimal decrease in mean total T3 levels (i.e. 0.88x those in the controls). A similar pattern of a small decrease in mean T3 levels in females exposed to 500 mg/kg bw/day was observed (this being 0.93x of those in controls). These changes were not statistically significant.

For T4 mean levels, males showed a general decrease with increasing dose, whereas in females the mean T4 levels showed a gradual increase with increasing dose. Male rats treated at the highest test dose of 500 mg/kg bw/day showed a minimal decrease in mean total T4 levels (0.84x those in the controls). In contrast, female rats showed small increases in mean T4 levels at the higher test doses (these being 1.30x and 1.34x those in the controls in the 150 and 500 mg/kg bw/day doses respectively). Based on the variation in the direction of the change, the changes of T4 concentrations in male and females were not considered to be toxicologically relevant.

In males the mean serum levels of the thyroid stimulating hormone (TSH) were consistent with those in the controls at the lowest test dose of 50 mg/kg bw/day, but increased at the 150 and 500 mg/kg bw/day doses (by 4.2x and 4.6x respectively). However, at the two highest doses there was considerable variability in the responses between animals with coefficients of variation of 60% and 95% respectively. The TSH levels in individual males ranged from 0.080 to 1.09 µLU/ml at 150 mg/kg bw/day and from 0.119 to 1.96 µLU/ml at 500 mg/kg bw/day (compared to control values of 0.044 to 0.329 µLU/ml). However, the variability in these rats is consistent with the mean historical TSH value for control male rats in the test laboratory at the end of OECD TG408 studies which is 0.224 µLU/ml with 95% Confidence Limits of 0.047 to 0.728 µLU/ml. In the study only 3 animals in the 150 mg/kg bw/day and 4 animals in the 500 mg/kg bw/day exceeded the 95%ile limit for the historical control TSH values.

For female rats there was a discernible dose-response relationship for mean TSH levels with increases of 2.2x and 3.3x relative to those in controls at the two higher test doses of 150 and 500 mg/kg bw/day. Marked variability in responses were also evident at those two doses with coefficients of variation of 81% and 96% respectively. The TSH levels in individual females ranged from 0.027 to 0.485 µLU/ml at 150 mg/kg bw/day and from 0.076 to 0.871 µLU/ml at 500 mg/kg bw/day (compared to control values of 0.015 to 0.115 µLU/ml). However, the variability in these rats is consistent with the mean historical TSH value for control female rats in the test laboratory at the end of OCD TG408 studies which is 0.074 µLU/ml with 95% Confidence Limits of 0.012 to 0.211 µLU/ml. In the study 5 animals in the 150 mg/kg bw/day, but only 3 animals in the 500 mg/kg bw/day exceeded the 95%ile limit for the historical control TSH values.

ESTRUS STAGE DETERMINATION
No test substance-related changes in estrous stages were noted across the dose groups. Most females were in di-estrous or estrous, with exception of one female at 150 mg/kg bw/day (No. 68) and 500 mg/kg bw/day (No. 77), which were in met-estrous. NO EFFECTS OBSERVED
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Remarks:
Local effects
Effect level:
150 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:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
organ weights and organ / body weight ratios
Key result
Dose descriptor:
LOAEL
Remarks:
Local effects
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no
Conclusions:
Administration of Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) by once daily oral gavage for 90 days did not result in adverse effects in rats at levels of 50 and 150 mg/kg bw/day. Histopathological examinations showed, epithelial hyperplasia of the non-glandular mucosa of the stomach (forestomach, squamous mucosa) at 50, 150 and 500 mg/kg bw/day in males and females with a dose-related increase in severity, up to marked in males and moderate in females. At 150 and 500 mg/kg bw/day this correlated with macroscopically irregular stomach surfaces. These findings at 500 mg/kg bw/day were interpreted to be adverse due to the overall severity and concurrent presence of ulceration in some animals. Furthermore, a lower body weight gain was observed in males at 500 mg/kg bw/day which was also considered to be adverse.

Other test substance-related changes in clinical signs, body weight, clinical pathology, organ weights and histopathology were considered non-adverse.

In the study, no toxicologically relevant treatment related changes in T3 or T4 were evident in either males or females. An increase of TSH was observed in males at 150 and 500 mg/kg bw/day and females at 500 mg/kg bw/day. However, there was considerable variability in the responses observed within the groups and these were largely within the upper 95%ile of the historical control data. This change was associated with the limited extent of diverse follicular cell hypertrophy in the thyroid gland of males and females. These changes were, in most instances, minimal in severity, with the exception of a few males in the higher test doses (where changes of mild severity were observed) and were not degenerative in nature. Follicular cell hypertrophy of the thyroid gland at a low level is common background finding in rats of the strain and age used in the study. No statistically significant effects on the absolute or relative weights of organs potentially sensitive to endocrine disruptors were evident at any of the test doses.

Based on the results generated a No Observed Adverse Effect Level (NOAEL) for the systemic toxicity of Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) of 150 mg/kg bw/day was established. This NOAEL also provides protection against the observed local effects on stomach morphology due to the known irritancy of the test substance.
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, 50, 150 and 500 mg/kg bw/day. The test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.


Clinical signs which were observed incidentally during the dosing period consisted of abnormal breathing sounds and hunched posture in males and females at 500 mg/kg bw/day. In addition, piloerection was observed incidentally in males and females at 150 and 500 mg/kg bw/day. At the incidence observed, these findings were considered to be not adverse.


A moderate decrease in body weight and body weight gain was observed in males and slightly lower body weight and body weight gain in females at 500 mg/kg bw/day. At 150 mg/kg bw/day, body weight and body weight gain were also slightly lower in males. The body weight effects in males at 500 mg/kg bw/day were, at the severity observed, considered to be adverse while the effects in females at 500 mg/kg bw/day and males at 150 mg/kg bw/day were only slight in nature and considered to be not adverse. The body weight effects correlated to the lower food consumption observed at 150 and 500 mg/kg bw/day.


Haematology and coagulation parameters showed slight increases in white blood cells (WBC), lymphocytes and neutrophils in males at 500 mg/kg bw/day. Neutrophils were also slightly higher in females at 500 mg/kg bw/day. In addition, red blood cells in males and mean corpuscular haemoglobin concentration (MCHC) in females were slightly decreased and mean corpuscular volume (MCV) were slightly increased in males at 500 mg/kg bw/day. Furthermore, a slight decrease in activated partial thromboplastin time (APTT) was observed in females at all dose groups with a dose-related response. At the severity observed and in absence of any histopathological correlations, these findings were considered to be not adverse. At 150 mg/kg bw/day, a minimal decrease in red blood cells was observed in males. As this slight change remained within the range considered normal for rats of this age and strain, it was considered to not be toxicologically relevant.


Clinical chemistry analysis showed, slight to moderate increases in alanine and aspartate aminotransferase (ALAT and ASAT) and alkaline phosphatase (ALP) activities in males and females at 500 mg/kg bw/day, which correlated with higher liver weights. At 150 mg/kg bw/day, ALP activity was also slightly to moderately increased in males and females, but lacked any histopathological correlation, and was therefore considered to be not adverse. Additionally, slightly lower total protein and albumin concentrations were observed in males and females at 150 and/or 500 mg/kg bw/day. As the changes in total protein and albumin concentration remained within the range considered normal for rats of this age and strain and no histopathological correlations were observed, these findings were considered to not be toxicologically relevant.


The functional observations showed an increase in total movements and ambulations was observed in females at 50, 150 and 500 mg/kg bw/day. In absence of a clear dose-related trend and corroborative findings, these effects were considered to not be test substance-related.


Histopathological examinations showed, epithelial hyperplasia of the non-glandular mucosa of the stomach (forestomach, squamous mucosa) at 50, 150 and 500 mg/kg bw/day in males and females with a dose-related increase in severity, up to marked in males and moderate in females. At 150 and 500 mg/kg bw/day this correlated with macroscopically irregular stomach surfaces. These findings at 500 mg/kg bw/day were interpreted to be adverse due to the overall severity and concurrent presence of ulceration in some animals. At 150 mg/kg bw/day, epithelial hyperplasia up to moderate severity was noted, without degenerative changes or ulceration and with only minimal mixed cell infiltrates and no to slight body weight changes. Therefore, the effects on the stomach at 50 and 150 mg/kg bw/day were interpreted to be not adverse. Functionally, the non-glandular stomach serves as a temporary storage organ and, due to prolonged exposure, is potentially a major site for interaction with the test item. As humans lack a non-glandular stomach, the relevance of these test item-related changes in this tissue for humans remains uncertain. However, it is reasonable to assume that the squamous mucosa of the oesophagus (in species without the non-glandular stomach) will react in a similar way to this test substance as the non-glandular stomach epithelium.


In the mesenteric lymph nodes, erythrophagocytosis (up to a mild degree of severity) was noted in males at 500 mg/kg bw/day. This is a common, non-adverse background change but the incidence at 500 mg/kg bw/day (along with macroscopic correlating dark discoloration and foci) is suggestive of a test substance-related effect and may be secondary to the changes in stomach morphology.


In the liver, minor increases in liver weight were noted in males and females at 500 mg/kg bw/day, which may correlate with the increases in liver enzymes (alanine and aspartate aminotransferase (ALAT, and ASAT) and ALP) that were noted in these animals. In absence of a histopathological correlation, these findings were considered to be not adverse.


Males, starting at the lowest dose of 50 mg/kg bw/day, showed an increased incidence (compared to concurrent control males) of diffuse, bilateral, follicular cell hypertrophy. In most cases these changes were minimal in severity, but a few individuals showed a mild degree of severity, primarily in the highest dose of 500 mg/kg bw/day. In females a low incidence and minimal severity of follicular cell hypertrophy was noted in treated animals. It should be recognised that follicular cell hypertrophy of the thyroid gland at a low level is common background finding in rats of the strain and age used in the study. The changes observed, even in the males at the highest dose of 500 mg/kg bw/day, were without any degenerative changes (i.e. they were not characterised by progressive deterioration and loss of function in the organs and tissues). The test report concluded that “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 it was not taken into account when determining the NOAEL.”.


No mortality occurred and no test substance-related changes were noted in the remaining parameters investigated in this study (i.e. ophthalmoscopy).


Overall, no marked effects on endocrine-disruption related apical morphological and histological endpoints in males and females (thyroid weight and histopathology and indicators of thyroid hormonal activity (T3, T4 and TSH) were observed in the study at any of the exposure doses of 50, 150 and 500 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

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 toxicity: via oral route - systemic effects (target organ) digestive: duodenum; digestive: stomach; glandular: other; respiratory: lung

Justification for classification or non-classification

In an OECD 422 repeat dose study via oral route, the lowest no adverse effect level (NOAEL) associated with a reproducible effect is 150 mg/kg bw/day.

In an OE CD 408 repeat dose study via oral route, the lowest no adverse effect level (NOAEL) associated with a reproducible effect is 150 mg/kg bw/day.