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

Endpoint summary

Description of key information

- Subacute NOAEL (rat): 250 mg/kg bw/day (OECD 422, GLP)

- Subchronic NOAEL (rat): 62.5 mg/kg bw/day (OECD 408, GLP)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

some minor deviations

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

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

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

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

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

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

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

Frequency of treatment:

once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Control

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: preliminary study

Positive control:

none

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

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

OPHTHALMOSCOPIC EXAMINATION: No

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

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

URINALYSIS: No

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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

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

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

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

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

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

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

Statistics:

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

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

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

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

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

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

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

Endocrine findings:

not examined

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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

Gross pathological findings:

not examined

Description (incidence and severity):

See organ weight findings above

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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

Histopathological findings: neoplastic:

not examined

Details on results:

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

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Critical effects observed:

no

Conclusions:

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

Executive summary:

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

 

General and detailed clinical signs:

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

 

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

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

 

Haematology and clinical biochemistry: no effects

 

Necropsy:

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

 

Histology: no effects

 

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

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 20 MAY 2016 to 19 APRIL 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material:KK-1883
- Expiration date of the batch:October 2017
- Purity test date:Feb 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS:
Purity: 100%

Species:

rat

Strain:

other: Wistar (Cmdb: WI)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: xperimental Medicine Centre at the Medical University in Białystok, Poland
- Age at study initiation: 7 – 8 weeks
- Weight at study initiation: 209g – 279g (males); 147g – 212g (females)
- Housing: The animals were kept in plastic cages covered with wire bar lids. Thedimensions of the cages were 58 x 37 x 21 cm (length x width x height). UV-sterilized, autoclaved, dust-free wood shavings were used as bedding. During the study, 5 animals were kept together in one cage. Each sex was kept separately.
- Diet: “Labofeed H Standard” standard granulated laboratory fodder produced by Zofia Połczyńska Wytwórnia Pasz "Morawski", Kcynia (batch number 1/16) ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 26°C
- Humidity (%): 38 - 100%
- Air changes (per hr): 12 hours light / 12 hours dark
- Photoperiod (hrs dark / hrs light): 13 - 16 times/h

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:Solutions of the test item for each group of animals were prepared once a week. The solutions were kept at room temperature (18 - 23°C).
The test item was diluted with corn oil at the corresponding concentration. The test item and vehicle were given to animals in a constant volume of 0.52 mL/100 g b.w in the doses level: 0 mg/kg b.w.; 62.5 mg/kg b.w.; 250 mg/kg b.w. and 0.51 mL/100 g b.w. in the highest dose (1000 mg/kg b.w.)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The solutions of the test item in the medium (corn oil) for each dose were chemically analysed. Samples of the test item solutions as well as samples of the vehicle were sent for chemical analyses to the Test Site LAUS GmbH, Auf der Schafweide 20, D-67489 Kirrweiler, Germany. The samples were sent in three sessions: at the beginning (June 06, 2016), in the middle (July 25, 2016) and at the end (September 12, 2016) of the study. The % target recovery for the samples measured during the 3 sessons was 100.2-123.8% (Appendix 1).

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 days per week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Main group

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Remarks:

Main group

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Main group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Main group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Satellite group

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Satellite group

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for selecting satellite groups: there were two satellite groups (10 males and 10 females). These were:
- A control group (group 0SAT) treated with corn oil once a day (by gavage);
- Group treated with test item (3SAT) at the highest dose, i.e. 1000 mg/kg b.w. once a day (by
gavage).
- Post-exposure recovery period in satellite groups: The satellite groups received the test item/corn oil every day for 90 days (7 days/week). After that,
they were observed for 14 days to evaluate the reversibility, stability, or delay in the onset of possible harmful effects of the test item.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
The evaluation of general condition of the animals, i.e. the observation of all animals for morbidity and mortality was conducted twice a day or once a day (on days off).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before the test item /vehicle administration, and then once a week

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to the beginning of the experiment (day 0) and then twice a week during the experiment.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food intake/cage was measured once a week during the entire study.
Then, it was converted into average food intake/100 g b.w.


OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmic examinations were conducted on the day before the introduction of the animals to the experiment and one day before euthanasia. An indirect ophthalmoscope was used. In case of
the satellite groups, these examinations were also conducted after the end of the test item/vehicle administration

HAEMATOLOGY: Yes
Circulatory blood examinations,Bone marrow examinations and Coagulological examinations were performed.

CLINICAL CHEMISTRY: Yes
Biochemical and Enzymatic examinations were performed.

URINALYSIS: Yes
To collect urine, the animals were placed in metabolic cages for 18 hours on the last day of the experiment, one animal per cage. Urine collection lasted about 18 hours. The animals were given free access to water. General urinalysis and urine sediment were performed.

NEUROBEHAVIOURAL EXAMINATION: Yes
Open field observations, Evaluation of responses to stimuli, Measurement of the fore- and hindlimb grip strength and Evaluation of locomotor activity were performed.

IMMUNOLOGY:
The immune system was preliminary evaluated on the basis of: blood morphology with a picture of peripheral blood and bone marrow, concentration of albumin as an acute phase negative protein, total protein, and albumin/globulin ratio, urea nitrogen, creatinine, cholesterol, total bilirubin, activity of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, absolute and relative weights of the thymus and spleen, as well as histopathological evaluation of the thymus, spleen, and lymph nodes.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross examinations and organ weights were performed

HISTOPATHOLOGY: Yes
The control groups (0 and 0SAT) and the groups treated with the test item at the highest dose (groups 3 and 3SAT) were examined. The examination was extended to animals of other dosage groups (1 and 2) because of treatment-related changes that were observed in the high dose groups.

The following organs and tissues were examined: brain with the cerebellum, pituitary gland, eye with the optic nerve, spinal cord, skeletal muscle with the peripheral nerve, mandibular salivary gland with the lymph nodes, trachea, esophagus, thyroid with the parathyroids, stomach, small and large intestines (duodenum, jejunum, ileum, cecum, colon, and rectum), liver, spleen, pancreas, lungs with the bronchi, heart, aorta, thymus, kidneys, adrenals, urinary bladder, ovaries, uterus with the cervix, testicles, epididymides, accessory sex glands (prostate with the seminal vesicles and coagulating glands), skin, mammary gland and gross lesions.

The immune system was preliminary evaluated on the basis of: blood morphology with a picture of peripheral blood and bone marrow, concentration of albumin as an acute phase negative protein, total protein, and albumin/globulin ratio, urea nitrogen, creatinine, cholesterol, total bilirubin, activity ofaspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, absolute and relative
weights of the thymus and spleen, as well as histopathological evaluation of the thymus, spleen, and lymph nodes.

Statistics:

The results obtained in the treated groups (groups 1, 2, and 3) were compared to the ones obtained in the control group (group 0). The results obtained in the treated satellite group (group 3SAT) were compared to the ones obtained in the control satellite group (group 0SAT).

The clinical results (body weights, locomotor activity, the numbers of fecal boluses and urine pools, fore- and hindlimb grip strength, latency of pain responses) were statistically analyzed using the oneway analysis of variance, the Dunnet’s test (group 0, 1, 2, 3) and Student’s t-test (group 0 SAT, 3
SAT) (p ≤ 0.05).

Food intake is summarized in tables. No statistical analyses were conducted, because the amount of
data was insufficient (two cages for males and two cages for females/group). The results of the clinical-chemical examinations in groups 1, 2, 3 were statistically analyzed using the one-way analysis of variance and Dunnet’s test (p ≤ 0.05). The results of the clinical-chemical examinations in group 3SAT was statistically analyzed using the one-way analysis of variance and Student’s t test (p ≤ 0.05). Some results of the urinalysis such as bilirubin and urine sediment were statistically analyzed using nonparametric Statistics test - Kruskal-Wallis test (group 1, 2, 3) and U Mann-Whitney test (group 3SAT), (p ≤ 0.05).
Absolute and relative weights of internal organs were statistically analysed using the one-way analysis of variance, the Dunnet’s test (group 0, 1, 2, 3) and Student’s t-test (group 0 SAT, 3 SAT) (p ≤ 0.05). The statistical analyses were conducted using Statistica 10, and Microsoft Excel 2007.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The results of the clinical observations from groups 0, 1, 2, and 3 are shown in Table 1. Clinical signs were observed in 14 animals during the entire experiment: in 1 male and 1 female of group 0, 5 males and 1 female of group 1, 2 males and 1 female of group 2, 3 females of group 3. During the entire experiment, there were no differences in appearance and behavior between the treated and the control groups. Thinning on the forelimbs was observed in one female from group 2
(occurred in the 10th week and remained till the 13th week of experiment). Thinning on the head was observed in five males from group 1 and in one male from group 2 (occurred in the 6th week and remained till the end of the experiment). Alopecia on the forelimbs was observed in one female from group 0 and in one female from group 2 (occurred in the 5th week and remained till the end of experiment). Scabs on the head were observed in one male from group 1 (occurred in the 9th week and remained till the 10th week of experiment). Porphyrin deposition around the eye was observed in one male from group 0 (occurred in the 1st week and remained till the end of the experiment) an in one female from group 3 (occurred in the 12th week and remained till the end of the experiment). Wavering gait while walking, distinct decrease in locomotor activity, bristled coat, respiratory murmurs, difficult respiration and saliva flowing from the snout were stated in one male from group 2 (change occurred in the last day of experiment). The animal was lying on the side and it allowed picking it up and taking away.

The results of the clinical observations from the satellite groups, i.e. 0SAT and 3SAT are shown in
Table 2. Clinical signs were observed in 17 animals during the entire experiment: in 4 males and 1 female of group 0SAT, 4 males and 8 females of group 3SAT.
During the entire experiment, there were no differences in appearance and behaviour between the
treated and the control groups. Thinning on the back was observed in two females from group 0SAT (occurred in the13th week and remained till the end of the experiment) and in two males from group 3SAT (occurred in the 8th week and remained till the end of the experiment). Thinning on the head was observed in four males from group 0SAT (change occurred in the 7th week and remained till the end of the experiment). Scabs on the head were observed in three males from group 0SAT (change occurred in the 8th week and remained till the 11th week of the experiment). Scabs on the nose was observed in one female from group 0SAT (change occurred in the 3rd and 5th week and remained till the 6th week of the experiment). Porphyrin deposition around the eye was observed in one female from group 3SAT (change occurred in the 2nd week and remained till the 7th week of the experiment).

Mortality:

no mortality observed

Description (incidence):

There were no mortalities (Table 1).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight losses were stated in one female of group 1 and two females of group 3 (Table 1).
Average body weights of the animals from groups 0, 1, 2, and 3, measured at weekly intervals, are
shown in Table 3 (males), and Table 4 (females).

During the study, there were no statistically significant differences in average body weights of males
and females between the treated and the control groups. The only exception was a statistically
significant decrease in the average body weight of males from group 3 on the 13 week of the
experiment compared to the control group.

Body weight losses were stated in one female of group 0SAT, four males of group 3SAT and eight
females of group 3SAT (Table 2).
Average body weights of the animals from the satellite groups, i.e. 0SAT and 3SAT, measured at
weekly intervals, are shown in Table 5 (males) and Table 6 (females).

During the 90-day study and the 14-day additional observation period, there were no statistically
significant differences in average body weights of females between the treated and the control satellite
groups. The only exception was a statistically significant decrease in the average body weight of
males from group 3SAT on the 12 and 15 week of the experiment compared to the control satellite
group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Average food intake/100 g b.w. by the animals from groups 0, 1, 2, and 3 is shown in Table 7 (males)
and Table 8 (females).

Average food intake by the animals from the treated groups, i.e. 1, 2, and 3 and the control group,
i.e. 0 was similar.

Average food intake/100 g b.w. by the satellite groups, i.e. 0SAT and 3SAT is shown in Table 9
(males) and Table 10 (females).

During the 90-day study and the 14-day additional observation period, average food intake by the
animals from the treated satellite group, i.e. 3SAT and the control satellite group, i.e. 0SAT was
similar.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmic examinations did not reveal any pathological changes in any test groups.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Circulatory blood examinations
The results of the hematological examinations of circulatory blood in groups 0, 1, 2, and 3 are shown
in Table 35 (males) and Table 36 (females).

No statistically significant changes were found in males. As for females from group 2 and 3, there was
a decrease in MCHC value.

The results of the hematological examinations of circulatory blood collected from the satellite groups
(0SAT and 3SAT) are presented in Table 37 (males) and Table 38 (females).No statistically significant changes were found in males.
As for females, statistically significant decrease in the MCHC value was stated.

The results of the leukocytogram estimation in groups 0, 1, 2, and 3 are shown in Table 39 (males)
and Table 40 (females). No statistically significant changes were found in males and females. The only exception was an
increase in eosinocytes number in females of group 2.

The results of the leukocytogram estimation in both satellite groups, i.e. 0SAT and 3SAT are
presented in Table 41 (males) and Table 42 (females). No statistically significant changes were found in males and females from group 3SAT. The only
exception was an increase in other cells number in males of group 3SAT.

Bone marrow examinations
The results of the examinations of the erythrocyte system of bone marrow in groups 0, 1, 2, and 3 are
shown in Table 43 (males) and Table 44 (females). No statistically significant changes were found in males and females.

The results of the examination of the erythrocyte system of bone marrow in the satellite groups,
i.e. 0SAT and 3SAT are presented in Table 45 (males) and Table 46 (females).
No statistically significant changes were found in males and females.

The results of the examinations of the leukocyte system of bone marrow in groups 0, 1, 2, and 3 are
shown in Table 47 (males) and Table 48 (females).
No statistically significant changes were found in males and females.

The results of the examinations of the leukocyte system of bone marrow in the satellite groups,
i.e. 0SAT and 3SAT are presented in Table 49 (males) and Table 50 (females).
No statistically significant changes were found in males and females.

The numbers of different cells in bone marrow in groups 0, 1, 2, and 3 are shown in Table 51 (males)
and Table 52 (females). No statistically significant changes were found in males and females.
The numbers of different cells in bone marrow in the satellite groups, i.e. 0SAT and 3SAT are
illustrated in Table 53 (males) and Table 54 (females). No statistically significant changes were found in males and females.

Coagulation examinations
The results of the determination of PT and APTT in groups 0, 1, 2, and 3 are shown in Table 35
(males) and Table 36 (females). No statistically significant changes were found in males and females.

The results of the determination of PT and APTT in the satellite groups, i.e. 0SAT and 3SAT are
illustrated in Table 37 (males) and Table 38 (females). No statistically significant changes were found in males from group 3 SAT.
As for females from group 3SAT a decrease in PT was stated.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The results of the biochemical examinations of blood serum in groups 0, 1, 2, and 3 are shown in
Table 55 (males) and Table 56 (females).
There was a statistically significant increase in the concentration of urea nitrogen in males from group
2 and 3. There was also a statistically significant increase in the concentration of albumin, A/G ratio
and decrease of concentration of globulin in males from group 3.
As for females from group 1, the concentration of chlorides was higher than in the control group.
In females from group 2 and 3, the concentration of calcium was higher than in the control group.
Females from group 3 had additionally higher concentration of albumin than in the control group, A/G
ratio, cholesterol, and urea nitrogen.

The results of the biochemical examinations of blood serum in the satellite groups, i.e. 0SAT and
3SAT are given in Table 57 (males) and Table 58 (females).
There was an increase in the concentration of cholesterol and calcium in males from group 3SAT.
As for females from group 3SAT, there was an increase in the concentration of urea nitrogen and
calcium.

Enzymatic analysis
The results of the enzymatic examinations of blood serum in groups 0, 1, 2, and 3 are shown
in Table 59 (males) and Table 60 (females).
No statistically significant changes were found in males and females. The only exception was an
increase in AP activity in males from group 3.

Table 61 (males) and Table 62 (females) summarize the results of the enzymatic examinations of
blood serum in the satellite groups, i.e. 0SAT and 3SAT.
No statistically significant changes were found in males and females from group 3SAT.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

General urinalyses: The results of the general urinalysis in groups 0, 1, 2, and 3 are shown in Table 63 (males) and Table 64 (females).
No statistically significant changes were found in males and females. The only exception was a
decrease in ketone bodies in males from group 2 and 3.

The results of the general urinalysis in groups 0SAT and 3SAT are shown in Table 65 (males)
and Table 66 (females).
There was a decrease in the concentration of ketone bodies and an increase in leukocytes number in males from group 3SAT. As for females from group 3SAT, there was an increase in the concentration of protein.

Urine of all animals was yellow.

Urine sediment examinations: The results of the urine sediment examinations in groups 0, 1, 2, and 3 are shown in Table 67 (males) and Table 68 (females).
No statistically significant changes were found in males and females.

The results of the urine sediment examinations in groups 0SAT and 3SAT are shown in Table 69
(males) and Table 70 (females).
No statistically significant changes were found in males and females.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Open field observations
The results of the open field observations of the animals from groups 0, 1, 2, and 3, are presented in Table 11 (males) and Table 12 (females).
In case of groups 0, 1, 2, and 3, involuntary clonic and tonic movements, changes in gait, or stereotypical behavior were not observed.
A slight decrease in arousal was noticed in two males and one female from group 0, one female from group 1, two males from group 2, and two females from group 3. A moderate decrease in arousal was noticed in one male from group 2. A slight increase in arousal was noticed in three males and two females from group 0, five males and three females from group 1, two males and four females from group 2, and five males and two females from group 3.
There were no statistically significant differences in the number of fecal boluses left by males and
females between the treated groups, i.e. 1, 2, and 3, and the control group. There were no statistically significant differences in the number of urine pools left by males and females between all treated groups, i.e. 1, 2, and 3, and the control group. There were no statistically significant differences in horizontal locomotor activities of males and females between the treated and the control groups.
There were no statistically significant differences in vertical locomotor activities of females between the treated and the control groups.
There were no statistically significant differences in vertical locomotor activities of males between two treated groups, i.e. 1 and 3, and the control group. A decrease in vertical locomotor activities of males from group 3 was statistically significant.

The results of the open field observations of the animals from the satellite groups, made at the end of the treatment (week 13 of the experiment - measurement 1) are shown in Table 13 (males) and Table 14 (females). In case of the satellite groups (males and females), involuntary clonic and tonic movements, chnges in gait, or stereotypical behavior were not observed. A slight decrease in arousal was noticed in two females from group 0SAT. A slight increase in arousal was noticed in four males from group 0SAT, four males and three females from group 3SAT. A strong
increase in arousal was noticed in one male from group 3SAT. There were no statistically significant differences in the numbers of fecal boluses left by males and females between groups 3SAT and 0SAT.
There were no statistically significant differences in the numbers of urine pools left by males between groups 3SAT and 0SAT. A decrease in the number of urine pools left by females from group 3SAT was statistically significant. There were no statistically significant differences in locomotor activity (horizontal and vertical) of males and females between groups 3SAT and 0SAT.

The results of the open field observations of the satellite groups, made at the end of the additional observation (measurement 2) are shown in Table 15 (males) and Table 16 (females). During the open field observations of males and females, involuntary clonic and tonic movements, changes in gait, or stereotypical behavior were not noticed. A slight decrease in arousal was noticed in three males from group 0SAT and three males from group 3SAT. A slight increase in arousal was noticed in one male and one female from group 0SAT, two males and three females from group 3SAT. There were no statistically significant differences in the numbers of fecal boluses and urine pools left by males and females between groups 3SAT and 0SAT. There were no statistically significant differences in locomotor activity (horizontal and vertical) of males between groups 3SAT and 0SAT. There were no statistically significant differences in horizontal locomotor activity of females between groups 3SAT and 0SAT. An increase in vertical locomotor activities of females from group 3SAT was statistically significant.

Evaluation of sensorimotor responses to stimuli: Sensorimotor responses to stimuli are shown in Table 17 (males) and Table 18 (females). The study aimed at observing reactions of the treated animals (males and females) and the ones from group 0 (responses to objects, being touched, and sound, and the pinna reflex) did not reveal any changes. However, no reactions in one male from group 2 were stated. As for the latency of pain responses of males and females, there were no statistically significant differences between the treated groups, i.e. 1, 2, and 3, and the control group. Responses of the satellite groups to stimuli, measured at the end of the treatment (measurement 1) are illustrated in Table 19 (males) and Table 20 (females).
There were no negative effects on responses to objects, being touched, and sound, and the pinna reflex of males and females from group 3SAT when compared to group 0SAT. The latency of pain responses of males and females from groups 3SAT and 0SAT was similar.
Responses of the satellite groups to stimuli, measured at the end of the additional observation period (measurement 2) are given in Table 21 (males) and Table 22 (females). There were no negative effects on responses to objects, being touched, and sound, and the pinna
reflex of males and females from group 3SAT when compared to group 0SAT. Pain reaction latency time of females of group 3SAT was level with control group, whereas pain reaction latency time of males was statistically significantly shorter.

Measurement of the fore- and hindlimb grip strength: The results of the measurement of the fore- and hindlimb grip strength are given in Table 23 (males) and Table 24 (females).
There were no statistically significant differences in the fore- and hindlimb grip strength of males and females between the treated and the control groups.
As for the satellite groups, the results of the fore- and hindlimb grip strength measurement made at the end of the treatment (measurement 1) are presented in Table 25 (males) and Table 26 (females). There were no statistically significant differences in the fore- and hindlimb grip strength of males and females between groups 3SAT and 0SAT. The results of the fore- and hindlimb grip strength measurement made at the end of the additional observation period (measurement 2) are illustrated in Table 27 (males) and Table 28 (females). There were no statistically significant differences in the fore- and hindlimb grip strength of males and females between groups 3SAT and 0SAT.

Measurement of locomotor activities: Table 29 (males) and Table 30 (females) illustrate locomotor activity. There were no differences in horizontal and vertical locomotor activities of males between the treated and the control groups (0-30 minutes). As far as individual stages of the experiment are concerned, i.e. 0-10 minutes, 10-20 minutes, and 20-30 minutes, there were no differences in horizontal and vertical locomotor activities of males between the treated and the control groups. There were no differences in horizontal and vertical locomotor activities of females between the treated and the control groups (0-30 minutes). As far as individual stages of the experiment are concerned, i.e. 0-10 minutes, 10-20 minutes, and 20-30 minutes, there were no differences in horizontal and vertical locomotor activities of females between the treated and the control groups.

Table 31 (males) and Table 32 (females) present the results of the locomotor activity measurement in the satellite groups made at the end of the treatment (measurement 1). There were no differences in locomotor activity (horizontal and vertical) of males and females between groups 3SAT and 0SAT (0-30 minutes). During particular time-intervals: 0-10 min., 10-20 min., 20-30 min., locomotor activity (vertical and horizontal) of treated animals was level with control group with the exception for group 3SAT of males in which statistically significant lower vertical locomotor activity was observed during time-interval of 10-20 min.

Table 33 (males) and Table 34 (females) present the results of the locomotor activity measurement in the satellite groups made at the end of the additional observation period (measurement 2). There were no statistically significant differences in locomotor activity (horizontal and vertical) of males and females between groups 3SAT and 0SAT (0-30 minutes). During particular time-intervals: 0-10 min., 10-20 min., 20-30 min., locomotor activity (vertical and horizontal) of treated animals was at the same level as control group with the exception of femalesfrom group 3SAT in which statistically significant lower vertical locomotor activity was observed during time-interval of 20-30 min.

Immunological findings:

effects observed, treatment-related

Description (incidence and severity):

Evaluation of the immune system:
The clinical-chemical parameters which were used for evaluation of the immune system were as
follows: hematological (Tables 35-54), albumin, total protein, and albumin/globulin ratio, creatinine, urea nitrogen, cholesterol, total bilirubin (Tables 55-58), aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase (Tables 59-62).

Analyses of these parameters reveal some statistically significant changes:
- decrease of MCHC value in females from group 2, 3, and 3SAT
- increase in eosinocytes number in females from group 2
- increase in other cells number in males from group 3SAT
- increase in the concentration of urea nitrogen in males from group 2 and 3
- increase in the concentration of albumin, A/G ratio, AP activity, and a decrease of concentration of globulin in males from group 3
- increase in concentrations of albumin, A/G ratio, cholesterol, and urea nitrogen in females from group 3
- increase in the concentration of cholesterol in males from group 3SAT
- increase in the concentration of urea nitrogen in females from group 3SAT.
Absolute and relative weights of the thymus and spleen (Tables 72-79), as well as histopathological evaluation of the thymus, spleen, and lymph nodes (Table 80-81) were used for evaluation of the immune system.

Single histopathological lesions in the thymus (hyperemia, edema, lymphocytic infiltrations), spleen, (hyperemia, depletion of the white pulp, hemosiderin deposits), and mandibular lymph nodes (hyperemia, ecchymoses, edema, lymphocytic infiltrations, hyperplasia of lymphocytic tissue) in animals exposed to the test item and in control animals were stated. The statistical analysis of the weight of the thymus and the spleen showed some statistically significant changes in animals from group 1 (increased absolute and relative weight of the thymus in females), animals from group 2 (decreased absolute weight of the thymus as well as increased relative weight of the spleen in males), animals from group 3 (decreased absolute and relative weight of the thymus as well as increased relative weight of the spleen in mails) and animals from group 3SAT (increased relative weight of the spleen in mails and decreased absolute and relative weight of the thymus in females).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Weights of organs:

Absolute weights of organs of males and females from groups 0, 1, 2, and 3 are summarized in Table 72 (males) and Table 73 (females). Relative weights of organs of males and females from groups 0, 1, 2, and 3 are summarized in Table 74 (males) and Table 75 (females).

There were no statistically significant changes in absolute and relative weights of all the examined organs of males from group 1. In females from group 1 there was statistically significant increase in the absolute and relative weight of the thymus.
There were no statistically significant changes in absolute and relative weights of most of the examined organs of males from group 2, except a decrease in absolute weight of the thymus and an increase in relative weight of the spleen. In females from group 2 there were no statistically significant changes in absolute and relative weights of all the examined organs.
In males from group 3 there was a statistically significant decrease in absolute and relative weight of the thymus and an increase in absolute and relative weight of the heart and also an increase of relative weights of the brain, liver, spleen, kidneys, adrenals and testicles. As for females from group 3, there were statistically significant increases in absolute and relative weights of the heart and liver and also an increase in relative weights of kidneys and ovaries.

Absolute weights of organs of males and females from the satellite groups, i.e. 0SAT and 3SAT are summarized in Table 76 (males) and Table 77 (females).
Relative weights of organs of males and females from the satellite groups, i.e. 0SAT and 3SAT are
summarized in Table 78 (males) and Table 79 (females).

In males from group 3 SAT there were statistically significant increases in the absolute weight of adrenals and in relative weights of the heart, spleen, kidneys and adrenals.
As for females from group 3 SAT, there was a statistically significant decrease in absolute and relative weight of thymus and an increase in the relative weights of brain, heart, liver and kidneys.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross examinations: Table 71 contains a list of gross changes observed in group 2. In groups 0, 1 and 3 no gross lesions were observed.

Group 2
- Yellow pedunculated nodule with a diameter of 4mm in peritoneal cavity in 1 rat (male)
- Edema of the left side of the neck and mandibular salivary gland area with abscess in muscle tissue in 1 rat (male)
- Right polycystic kidney in 1 rat (female)

There were no gross lesions observed in the satellite groups, i.e. 0SAT and 3SAT.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were also cases of individual lesions in other organs (brain, pituitary gland, thyroid, mandibular lymph nodes, thymus, lungs, heart, liver, spleen, pancreas, intestines, kidneys, urinary bladder, adrenal glands, prostate, peritoneal cavity and connective and muscle tissue of the neck). Among the observed histopathological lesions in test animals were blood circulation disorders, inflammatory lesions, cases of regressive changes as well as cases of proliferation or hyperplasia in some tissues.
The lesions were sporadic and scattered among the treated and control groups. None of them showed severity caused by increasing doses of the test item. With the exception of the pathological lesions in the mandibular salivary gland all cases of histopathological lesions in organs and tissues of treated animals were considered as not associated with the test item. Further details in Tables 80-81.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The histopathological evaluation showed the presence of significant lesions in the mandibular salivary gland. In this organ the partial degeneration of the secretory cells of the acini, degeneration and exfoliation of the epithelium of ducts, focal metaplasia of the epithelium of ducts, hyperemia and lymphocytic infiltrations occurred. The lesions were considered as related to the test item. Their severity progressed along with the increase of the test item dose. None of them occurred in control animals as well as animals treated in the low dose: 62,5 mg/kg b.w./day. Noticeable was a slight impact of the test item at the mid dose: 250 mg/kg b.w./day as well as a progressive impact at the high dose: 1000 mg/kg b.w./day Due to number of affected animals (in the Group 3 and 3 SAT almost all males were affected), it is concluded, that males are more sensitive. The degeneration of the epithelium of ducts with exfoliation seems to be persistent. The same lesions found also in the animals after the 14-day additional observation period. Other lesions such as focal metaplasia of the epithelium of ducts, hyperemia and lymphocytic infiltrations are persistent in the mandibular salivary gland of males from group 3 SAT only. It may also confirm that males are more sensitive than females.
In females such lesions seems to be reversible due to their absence in the Group 3 SAT. Further details in Tables 80-81.

Dose descriptor:

NOAEL

Effect level:

62.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Conclusions:

A repeated dose 90-day oral toxicity study of CARDOLITE® NC-513 Cashew Nutshell Liquid, polymer with epichlorohydrin in male and female Wistar rats was conducted. The doses of the test item were 62.5, 250 and
1000 mg/kg b.w./day. On the basis of the results of the clinical, behavioral, clinical-chemical, and postmortem examinations which were parts of this study, it may be concluded that the NOAEL for male and females is 62.5 mg/kg b.w./day.

Executive summary:

In a subchronic toxicity study (S-52/16), CARDOLITE® NC-513 (100%) was administered to 3 groups of Wistar (Cmdb: WI) rats (10 animals/sex/group) by gavage in corn oil at dose levels of 0, 62.5, 250, and 1000 mg/kg bw/day 7 days per week for 90 days.

 

There were no mortalities during the study. The clinical examinations did not reveal any test item-related effects. There were no significant differences in physical appearance and behaviour between the treated and control groups. The ophthalmic examinations did not show any changes in the eyes of the control and the treated animals. The administration of the test item did not influence body weights at 62.5 and 250 mg/kg bw/day. A statistically significant decrease was noted in the average body weight of males at 1000 mg/kg bw/day (week 13) and in the 1000 mg/kg bw/day satellite group  (week 12 and week 15 week) compared to the control group and was considered test-item related. The administration of the test item did not influence food consumption.

 

The behavioral studies showed that the nervous system functioned properly. The test substance had an impact on the treated animals at the dose of 1000 mg/kg bw/day (albumin, globulin, A/G ratio, BUN, cholesterol, calcium, AP) as well as at the dose 250 mg/kg bw/day (BUN, calcium). Statistically significant changes in BUN and Ca concentration in SAT group means that the test item demonstrated a persistent dosage related response in the case of these two parameters. On the basis of the results of the clinical-chemical and post-mortem examinations, it was concluded that the test item had no harmful effects on the immune system of the treated animals.

 

The gross examinations of tissues and organs of animals in the control and treated groups showed macroscopic changes with the exception of two males and one female at 250 mg/kg bw/day.  However, none of these changes were perceived as a test item-related effect. Changes in weights of the brain, heart, liver, spleen, kidneys, adrenals, testicles, ovaries and thymus of animals exposed to the test item were statistically significant. These changes occurred mainly in the animals exposed to the highest dose of the test item. The occurrence of statistically significant differences in weights of the organs may indicate the impact of the test item at the highest dose (1000 mg/kg b.w./day) on the treated males (the weight of the brain, thymus, heart, liver, spleen, kidneys, adrenals, testicles) and females (the weight of the heart, liver, kidneys, ovaries). Some of these changes seem to be persistent because they were also found after the period of 14-day additional observation in males (the weight of the heart, spleen, kidneys, adrenals) and females (the weight of the heart, liver, kidneys) from the satellite 1000 mg/kg bw/day group. In addition the increased relative weight of the brain and the decreased weight of the thymus (absolute and relative) in females from the satellite 1000 mg/kg bw/day group may be associated with the test item as a delayed effect.

 

The histopathological evaluation showed the presence of significant lesions in the mandibular salivary gland. There were also cases of individual lesions in the brain, pituitary gland, thyroid, mandibular

lymph nodes, thymus, lungs, heart, liver, spleen, pancreas, intestines (duodenum, jejunum, ileum, cecum, colon and rectum), kidneys, urinary bladder, adrenal glands, prostate, peritoneal cavity and

connective and muscle tissue of the neck. Among the observed histopathological lesions in tested animals were blood circulatory disorders like: hyperemia – in the brain, pituitary gland, mandibular lymph nodes, mandibular salivary gland, thyroid, thymus, lungs, heart, spleen, liver, pancreas, cecum, kidneys, adrenal glands, prostate; erythrocytorrhagia – in the brain, lungs, heart and kidneys, ecchymoses in the pituitary gland, mandibular lymph nodes, liver and adrenals; pulmonary edema, edema of the connective and muscle tissue of the neck and edema of the submucosa of cecum.

 

Another stated lesions were inflammatory lesions, i.e. lymphocytic or purulent infiltrations in the mandibular salivary gland, reactive hyperplasia of lymphocytic tissue of the mandibular lymph nodes

or mandibular lymph nodes hyperplasia, inflammatory infiltration in the heart, thymus and thyroid, lymphocytic infiltrations in the duodenum, jejunum, ileum, cecum, colon and rectum, lungs, prostate,

urinary bladder, focal infiltrations of foam cells, bronchitis, pulmonary congestion and foci of emphysema in the lungs, foci of proliferation of Browicz-Kupffers cells in the liver, abscess with

extensive purulent inflammation of the connective and muscle tissue of the left side of the neck. Regressive changes were also found, e.g. degeneration and exfoliation of the epithelium of ducts of the mandibular salivary gland with or without focal metaplasia, partial degeneration of the secretory cells of the acini of the mandibular salivary gland, emphysema of the lungs, small necrotic foci with

hemorrhage in the left lobe of the liver - subcapsular and encysted with fibrous capsule, degeneration of the epithelium of renal tubules, exfoliation of renal tubules, hyaline casts, retention cysts, atrophy of single glomeruli, foci of glomerulosclerosis in the kidneys as well as hemosiderin deposits or depletion of the white pulp of the spleen. Among other stated lesions there where cases of hyperplasia of lymphatic follicle of intestines (duodenum, jejunum, ileum, cecum, colon and rectum), hyperplasia of the parathyroid, proliferation of the cortex of adrenals, hyperplasia of the islets of Langerhans in the pancreas, cases of lymphocytic micronodules in adipose tissue of the pancreas area, hyperplasia of glomeruli in the kidneys, small lipoma in the peritoneal cavity and microcysts in the pituitary gland.

With the exception of the pathological lesions in the mandibular salivary gland all cases of histopathological lesions in organs and tissues of treated animals should not be associated with the

test item.

 

The significant lesions in the mandibular salivary gland were considered as related to the test item, because none of them occurred in control animals. Moreover, their severity progressed along with the

increase of the test item dose (noticeable is a slight impact of the test item at the mid dose: 250 mg/kg bw/day as well as a progressive impact of the highest dose:1000 mg/kg bw/day). Males were more sensitive than females to this lesion (in the main and satellite groups at 1000 mg/kg bw/day, almost all males were affected), especially if we consider the cases of the degeneration of the epithelium of ducts, focal metaplasia, hyperemia and lymphocytic infiltrations. The degeneration of the epithelium of ducts with exfoliation seems to be persistent. The same lesions were found also in animals after the period of 14-day additional observation. Other lesions such as focal metaplasia of the epithelium of ducts, hyperemia and lymphocytic infiltrations are persistent in the mandibular salivary gland of males from the 1000 mg/kg bw/day satellite group only. This may also confirm that males are more sensitive than females. In females, such lesions appeared to be reversible due to their absence in the 1000 mg/kg bw/day satellite group. The NOAEL for males and females is 62.6 mg/kg bw/day.

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408) in rats. It should be noted that no historical control data for any parameters were included in the report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

62.5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

There are 2 key studies that have a Klimisch socre of 1 so the quality of the database is high. The subchronic OECD 408 study in rats was chosen for the hazard assement as the NOAEL is lower.

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

There were 2 studies for the repeated dose toxicity endpoint:

(i) Combined Oral Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in rats

(ii) Repeated dose 90 days oral toxicity study in rats.

 

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

 

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

 

General and detailed clinical signs:

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

 

Body weight, food and water consumption: Regarding the body weight and the body weight gain, no significant differences were

observed between all test item treated animals (male and female) and their respective vehicle control animals. Occasional differences observed for the females were assumed to be of natural origin based on the pregnancy status of the animals.

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

 

Haematology and clinical biochemistry: no effects

 

Necropsy:

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

 

Histology: no effects

 

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

 

Repeated Dose 90 days Oral Toxicity in Rats

 

In a subchronic toxicity study (S-52/16), CARDOLITE® NC-513 (100%) was administered to 3 groups of Wistar (Cmdb: WI) rats (10 animals/sex/group) by gavage in corn oil at dose levels of 0, 62.5, 250, and 1000 mg/kg bw/day 7 days per week for 90 days.

 

No historical control data for any parameters were included in the report. There were no mortalities during the study. The clinical examinations did not reveal any test item-related effects. There were no significant differences in physical appearance and behaviour between the treated and control groups. The ophthalmic examinations did not show any changes in the eyes of the control and the treated animals. The administration of the test item did not influence body weights at 62.5 and 250 mg/kg bw/day. A statistically significant decrease was noted in the average body weight of males at 1000 mg/kg bw/day (week 13) and in the 1000 mg/kg bw/day satellite group  (week 12 and week 15 week) compared to the control group and was considered test-item related. The administration of the test item did not influence food consumption.

 

The behavioral studies showed that the nervous system functioned properly. The test substance had an impact on the treated animals at the dose of 1000 mg/kg bw/day (albumin, globulin, A/G ratio, BUN, cholesterol, calcium, AP) as well as at the dose 250 mg/kg bw/day (BUN, calcium). Statistically significant changes in BUN and Ca concentration in SAT group means that the test item demonstrated a persistent dosage related response in the case of these two parameters. On the basis of the results of the clinical-chemical and post-mortem examinations, it was concluded that the test item had no harmful effects on the immune system of the treated animals.

 

The gross examinations of tissues and organs of animals in the control and treated groups showed macroscopic changes with the exception of two males and one female at 250 mg/kg bw/day.  However, none of these changes were perceived as a test item-related effect. Changes in weights of the brain, heart, liver, spleen, kidneys, adrenals, testicles, ovaries and thymus of animals exposed to the test item were statistically significant. These changes occurred mainly in the animals exposed to the highest dose of the test item. The occurrence of statistically significant differences in weights of the organs may indicate the impact of the test item at the highest dose (1000 mg/kg b.w./day) on the treated males (the weight of the brain, thymus, heart, liver, spleen, kidneys, adrenals, testicles) and females (the weight of the heart, liver, kidneys, ovaries). Some of these changes seem to be persistent because they were also found after the period of 14-day additional observation in males (the weight of the heart, spleen, kidneys, adrenals) and females (the weight of the heart, liver, kidneys) from the satellite 1000 mg/kg bw/day group. In addition the increased relative weight of the brain and the decreased weight of the thymus (absolute and relative) in females from the satellite 1000 mg/kg bw/day group may be associated with the test item as a delayed effect.

 

The histopathological evaluation showed the presence of significant lesions in the mandibular salivary gland. There were also cases of individual lesions in the brain, pituitary gland, thyroid, mandibular lymph nodes, thymus, lungs, heart, liver, spleen, pancreas, intestines (duodenum, jejunum, ileum, cecum, colon and rectum), kidneys, urinary bladder, adrenal glands, prostate, peritoneal cavity and connective and muscle tissue of the neck. Among the observed histopathological lesions in tested animals were blood circulatory disorders like: hyperemia – in the brain, pituitary gland, mandibular lymph nodes, mandibular salivary gland, thyroid, thymus, lungs, heart, spleen, liver, pancreas, cecum, kidneys, adrenal glands, prostate; erythrocytorrhagia – in the brain, lungs, heart and kidneys, ecchymoses in the pituitary gland, mandibular lymph nodes, liver and adrenals; pulmonary edema, edema of the connective and muscle tissue of the neck and edema of the submucosa of cecum.

 

Another stated lesions were inflammatory lesions, i.e. lymphocytic or purulent infiltrations in the mandibular salivary gland, reactive hyperplasia of lymphocytic tissue of the mandibular lymph nodes or mandibular lymph nodes hyperplasia, inflammatory infiltration in the heart, thymus and thyroid, lymphocytic infiltrations in the duodenum, jejunum, ileum, cecum, colon and rectum, lungs, prostate, urinary bladder, focal infiltrations of foam cells, bronchitis, pulmonary congestion and foci of emphysema in the lungs, foci of proliferation of Browicz-Kupffers cells in the liver, abscess with extensive purulent inflammation of the connective and muscle tissue of the left side of the neck. Regressive changes were also found, e.g. degeneration and exfoliation of the epithelium of ducts of the mandibular salivary gland with or without focal metaplasia, partial degeneration of the secretory cells of the acini of the mandibular salivary gland, emphysema of the lungs, small necrotic foci with hemorrhage in the left lobe of the liver - subcapsular and encysted with fibrous capsule, degeneration of the epithelium of renal tubules, exfoliation of renal tubules, hyaline casts, retention cysts, atrophy of single glomeruli, foci of glomerulosclerosis in the kidneys as well as hemosiderin deposits or depletion of the white pulp of the spleen. Among other stated lesions there where cases of hyperplasia of lymphatic follicle of intestines (duodenum, jejunum, ileum, cecum, colon and rectum), hyperplasia of the parathyroid, proliferation of the cortex of adrenals, hyperplasia of the islets of Langerhans in the pancreas, cases of lymphocytic micronodules in adipose tissue of the pancreas area, hyperplasia of glomeruli in the kidneys, small lipoma in the peritoneal cavity and microcysts in the pituitary gland. With the exception of the pathological lesions in the mandibular salivary gland all cases of histopathological lesions in organs and tissues of treated animals should not be associated with the

test item.

 

The significant lesions in the mandibular salivary gland were considered as related to the test item, because none of them occurred in control animals. Moreover, their severity progressed along with the increase of the test item dose (noticeable is a slight impact of the test item at the mid dose: 250 mg/kg bw/day as well as a progressive impact of the highest dose:1000 mg/kg bw/day). Males were more sensitive than females to this lesion (in the main and satellite groups at 1000 mg/kg bw/day, almost all males were affected), especially if we consider the cases of the degeneration of the epithelium of ducts, focal metaplasia, hyperemia and lymphocytic infiltrations. The degeneration of the epithelium of ducts with exfoliation seems to be persistent. The same lesions were found also in animals after the period of 14-day additional observation. Other lesions such as focal metaplasia of the epithelium of ducts, hyperemia and lymphocytic infiltrations are persistent in the mandibular salivary gland of males from the 1000 mg/kg bw/day satellite group only. This may also confirm that males are more sensitive than females. In females, such lesions appeared to be reversible due to their absence in the 1000 mg/kg bw/day satellite group. The NOAEL for males and females is 62.6 mg/kg bw/day.

Justification for classification or non-classification

The assessment of whether STOT-RE classification is required was carried out on the basis of available data. In the 90-day repeated-dose toxicity study according to OECD 408, dose-dependent histopathological changes in the salivary gland were observed. The results revealed severe lesions at 1000 mg/kg bw/day, slight lesions at 250 mg/kg bw/day and no effects at 62.5 mg/kg bw/day.

No other available study provides indications that the registered substance has an effect on the salivary glands. 

Consideration of the findings in relation to CLP

Classification for systemic target organ toxicity-repeated exposure (STOT-RE) may be warranted on the “basis of evidence from studies in experimental animals [that a substance] can be presumed to have the potential to be harmful to human health following repeated exposure.” The determination of whether a substance should be classified into category 1 or category 2 is made on the basis of whether significant and/or severe toxic effects occur at low exposures (≤ 10 mg/kg bw/day; Category 1) or significant toxic effects occur at moderate exposures (≤ 100 mg/kg bw/day; Category 2). These guidance values apply to oral studies in rat of 90 days’ duration.

Considering the severity of the observed histopathological changes in the salivary gland at the dose levels tested in the available 90-day study, it becomes clear that the observed findings do not support a STOT-RE classification of the registered substance. Severe histopathological effects to the salivary gland were noted at the limit dose which is 10-times higher than the CLP-based cut-off value of 100 mg/kg bw/day for a Cat. 2 classification. At a dose 2.5 times higher than this guidance value, only slight lesions were reported. At a dose of 62.5 mg/kg bw/day - the dose closest to the cut-off value for Cat. 2 - no effects were observed. Thus, no significant toxicity of the salivary glands is to be expected near the cut-off values for STOT-RE Cat 2.

Therefore, it can be concluded that the substance CARDOLITE NC-513 (EC/List no. 701-477-4) does not need to be classified when considering the criteria outlined in Annex I of 1272/2008/EC.