Registration Dossier

Administrative data

Description of key information

Based on available data for groups of constituents present in Distilled Tall Oil, a conservative 2-year NOAEL of >200 mg/kg/d is set for the oral route.

An OECD 422 Combined Repeated Dose Toxicity Study with Reproductive/ Developmental Toxicity Screening Study is available for Distilled Tall Oil via the oral route in the diet for Sprague-Dawley rats (Inveresk Research, 2002e). The test was carried out according to the requirements of the guideline and in compliance with GLP. Effects on organ weights and clinical chemistry were observed at 5000 and 20,000 ppm in the diet, and decreased food consumption was also seen at the highest dose level, 20,000 ppm. A NOEL of 1000 ppm in the diet was established for general systemic toxicity in the study. Based on mean body weight and food consumption data over the duration of the study, this is equivalent to approximately 83 mg/kg/d for males and 100 mg/kg/d for females.

In a recently conducted OECD 408 repeated dose toxicity study in the rat (Charles River, 2020), administration of Tall Oil by dietary administration for at least 90 days was well tolerated in rats at levels up to 15,000 ppm and did not result in any adverse test item related (morphologic) alterations. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 15,000 ppm. This dietary level was equivalent to an overall mean daily intake of 1104 mg/kg/d in males and 1221 mg/kg/d in females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21.03.02 to 15.07.02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
no certificate of analysis or details of test substance supplied, however given the nature of the distillation products this deviation was not thought to have affected the integrity of the study.
GLP compliance:
yes
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) Ltd
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: Males: 180-190 g. Females: 113-161 g.
- Fasting period before study: No
- Housing: Initially two per polypropylene cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ±2
- Humidity (%): 50 ±15
- Air changes (per hr): minimum 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 08.04.02 To: 27.05.02
Route of administration:
oral: feed
Vehicle:
acetone
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: An appropriate quantity of the test substance was dissolved in a suitable volume of acetone.


DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Rat and Mouse Breeder diet No. 3 (expanded) SQC
- Storage temperature of food: No data

This solution was added to a suitable quantity of untreated diet, then mixed for about one hour with fan assisted venting to remove the ethanol to form a dose premix. A control premix was prepared using the same proportion of acetone and untreated diet. The diets for the intermediate and high dose groups were prepared by dilution of the dose premix with untreated diet to give the desired concentrations. The low dose diet was prepared by dilution of the high dose diet with untreated diet. The diet premixes were then placed on a Winkworth mixer for approximately 20 minutes. The control diet was prepared by dilution of the control premix with untreated diet such that the diet contained the same proportion of premix as the high dose diet.


VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: No data
- Purity: No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the formulated diets was undertaken with regard to concentration and homogeneity. Diet prepared for Week 1 and Week 4 of treatment was sampled. Triplicate samples of each formulation, including control were taken immediately after preparation.
Duration of treatment / exposure:
The males were treated for at least four weeks overall, starting from two weeks prior to mating until termination. The females were treated for two weeks prior to mating, then through mating, until termination after Day 4 of lactation.
Frequency of treatment:
Continuous in diet.
Remarks:
Doses / Concentrations:
1000 ppm
Basis: nominal in diet
Remarks:
Doses / Concentrations:
5000 ppm
Basis: nominal in diet
Remarks:
Doses / Concentrations:
20000 ppm
Basis: nominal in diet
No. of animals per sex per dose:
Ten
Control animals:
other: acetone in diet
Details on study design:
- Dose selection rationale: The dose levels were selected and agreed following evaluation of existing data and a one week dose range-finding study in rats.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: No satellite groups.
Positive control:
No positve control
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily


BODY WEIGHT: Yes
- Time schedule for examinations: Males: once during the week prior to the commencement of dosing and once weekly thereafter. Females: once during the week prior to commencement of treatment, and weekly thereafter until the start of the mating period, then on Day 0 of gestation (the day of detection of a positive mating sign) followed by Days 7, 14 and 20 of gestation, and then Days 1 and 4 of lactation (where Day 0 is the day of parturition).


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Group mean achieved dosages of test substance calculated: Yes


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: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 5 of dosing for males and on Day 6 of lactation for females.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: Five males and five females
- Parameters checked in table [No.1] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: No
- How many animals: Five males and five females
- Parameters checked in table [No.1] were examined.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2), all animals (males: after 4 weeks treatment; females: after Day 4 of lactation)
HISTOPATHOLOGY: Yes (see table 2), on control and high dose animals.
Other examinations:
No other examinations.
Statistics:
Body weight and food consumption (prior to mating for females), haematology and clinical chemistry data were statistically analysed for homogeneity of variance using the 'F-max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous then Kruskal-Wallis ANOVA was used. Organ weights were also analysed likewise, and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. Histology incidence data were analysed using Fisher's Exact Probability Test. The following pairwise comparisons were performed against the Control group (Group 1): Control group vs Low dose; control group vs intermediate dose; Control group vs high dose. All statistical tests were two-sided and performed at the 5% significance level.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no clinical signs of toxicity or deaths.


BODY WEIGHT AND WEIGHT GAIN: At 20000 ppm there was a transient decrease in weight gain in both sexes. In males decreased weight gain was most notable for over the first week, although absolute weights were significantly lower over the first 3 weeks of treatment. In females there was a notable decrease throughout the pre-mating phase. The resulting deficit in body weight was never regained in either sex. In pregnant females reduced weight gain was evident over Day 7-20 of gestation, compared to the Control animals. There were no effects on body weight at 5000 and 1000 ppm.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): At 20000 ppm food consumption in males was reduced for the first 2 weeks of treatment (attaining significance during Week 1) and in Week 4 (not recorded Week 3 as paired for mating). In females, food consumption was significantly decreased during the premating period. Consumption was also reduced during the first half of the gestation period, compared to the Control
animals. There were no effects on food consumption at 5000 and 1000 ppm.


HAEMATOLOGY (See Table 4): At 20000 ppm there was a non-significant decrease in white blood cells in females.


CLINICAL CHEMISTRY (See Table 4): Alkaline phosphatase levels were significantly increased in females at 5000 and 20000 ppm, and in males at 20000 ppm. In males there was a non-significant increase in levels at 5000 ppm and in females at 1000 ppm there was an equivocal increase, but given the small group size it was considered that the difference was too small to reflect an effect of treatment. Total bilirubin was increased in both sexes at 20000 ppm. In addition, at 20000 ppm, cholesterol levels were increased in males; albumin (and consequently total protein) were reduced in females.


ORGAN WEIGHTS (see Table 3): In males, at 20000 ppm there was a decrease in body weights, with liver weights being essentially similar to Controls. At 5000 and 1000 ppm liver weight was slightly greater than Controls. Following covariance analysis there was a dose related increase in liver weights, with the increases at 5000 and 20000 ppm attaining statistical significance. In females, slight non-significant increases in liver weights following covariance analysis at 5000 and 20000 ppm were too small to attribute to treatment. In males at 20000 ppm spleen weight was notably increased following variance and covariance analysis. Adrenal gland and thymus weights were slightly but significantly decreased. Following covariance analysis adrenal gland weight was still significantly decreased, but for the thymus there was no significant difference from Controls. In females, ovary, adrenal gland and kidney weights were significantly reduced at 5000 and 20000 ppm, with pituitary gland weight reduced at 20000 ppm. Following covariance analysis, kidney and pituitary gland weights were essentially similar to Controls, but a decrease in ovary weight at 20000 ppm, and adrenal gland weight at 5000 and 20000 ppm was still evident, but not significant.


GROSS PATHOLOGY: No findings attributable to treatment.


HISTOPATHOLOGY: No findings attributable to treatment. All histology findings were typical of spontaneously arising background findings in rats of this strain and age.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 ppm
Sex:
male/female
Basis for effect level:
other: Decreased weight gain and food consumption in both sexes at 20000 ppm. Changes in liver function in both sexes at 20000 ppm. At 5000 ppm there was increased liver weight and alkaline phosphatase in both sexes.
Critical effects observed:
not specified

Table 3: Absolute organ weights (following covariance analysis)

 

Males

Females

DAILY DOSE
(ppm)

0

1000

5000

20000

0

1000

5000

20000

NUMBER OF ANIMALS

 10

 10

10 

10 

 10

 10

10 

 10

BODY WEIGHT MEAN (g)a

 458

458 

458 

458 

 310

310 

310 

310 

BRAIN

 

 

 

 

 

 

 

 

Absolute Weighta

g

 2.10

 2.12

2.12 

2.08 

 1.87

1.87 

1.90 

1.96 

ADRENALS

 

 

 

 

 

 

 

 

Absolute Weighta

g

 0.0757

0.0803 

0.0674 

0.0634* 

 0.0911

0.0843 

0.0791 

0.0774 

EPIDIDYMIDES

 

 

 

 

Absolute Weighta

g

 1.2041

1.2032 

1.2048 

1.2047 

n.a.b

n.a.b

n.a.b

n.a.b

HEART

 

 

 

 

 

 

 

 

Absolute Weighta

g

1.72 

1.82 

1.73 

1.72 

 1.12

1.16 

1.25 

1.18 

KIDNEYS

 

 

 

 

 

 

 

 

Absolute Weighta

g

3.77 

3.85 

4.01 

4.07 

 2.59

2.53 

2.47 

2.53 

LIVER

 

 

 

 

 

 

 

 

Absolute Weighta

g

17.88 

18.88 

19.46* 

20.12** 

 16.69

16.16 

17.77 

17.87 

SPLEEN

 

 

 

 

 

 

 

 

Absolute Weighta

g

0.85 

0.83 

0.85 

1.04*** 

 0.59

0.63 

0.68 

0.72 

TESTES

 

 

 

 

Absolute Weighta

g

 3.55

3.51 

3.69 

3.74 

n.a.b

n.a.b

n.a.b

n.a.b

THYROID and PARATHYROID

 

 

 

 

 

 

 

 

Absolute Weighta

g

0.0219 

0.0233 

0.0225 

0.0230 

 0.0166

0.0165 

0.0152 

0.0171 

THYMUS

 

 

 

 

 

 

 

 

Absolute Weighta

g

0.491 

0.415 

0.435 

0.385 

 0.198

0.233 

0.201 

0.242 

OVARIES

 

 

 

 

Absolute Weighta

g

n.a.b

n.a.b

n.a.b

n.a.b

 0.114

0.106 

0.104 

0.097 

UTERUS

 

 

 

 

Absolute Weighta

 

n.a.b

n.a.b

n.a.b

n.a.b

0.53 

0.53 

0.51 

0.50 

a Group means at the end of terminal necropsy are shown.

b n.a. = not applicable

* p0.05, ** p>0.05, ** p0.01, *** p0.001

Table 4: Selected haematology, and clinical chemistry

Doses (ppm)

0

1000

5000

20000

0

1000

5000

20000

male

female

Number of animals/group

10

10

10

10

10

10

10

10

Haematology (week 5 for males; week 7 for females)

 

 

 

 

 

 

 

 

- WBC (GIGA/L)

 14.50

14.33 

15.82 

12.97 

12.99 

12.79 

12.15 

8.99 

Blood chemistry (week 5 for males; week 7 for females)

 

 

 

 

 

 

 

 

- alkaline phosphatise (iu/l)

 683

699 

842 

1891** 

 509

638 

980 *

1649 *** 

- total bilirubin (μmol/l)

 0.6

1.0 

0.9 

1.7 ***

 0.9

0.9 

1.4 

1.9** 

- albumin (g/l)

 41

43 

41 

41 

 40

39 

39 

35*** 

- cholesterol (mmol/l)

 2.0

1.9 

2.0 

2.4 *

 2.4

2.2 

1.9 *

2.7 

- total protein (g/l)

 68

70 

66 

66 

 64

65 

62 

59* 

*P0.05, **P0.01, ***P0.001

 

Conclusions:
In a good quality Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted to OECD test guideline 422, and GLP, the parental NOAEL ,for Tall Oil administered continuously in the diet to rats (Sprague-Dawley), was 1000ppm.
Executive summary:

Four groups of 10 male and 10 female Sprague-Dawley rats received Tall Oil in diets at concentrations of 0, 1000, 5000 and 20000 ppm. The males were dosed for at least four weeks, starting from two weeks prior to mating. The females were dosed from two weeks prior to mating until at least Day 6 of lactation. The animals were monitored for clinical signs, body weight, food consumption,

mating and litter performance. Blood samples were taken from five males (during week five) and five females (lactation Day 6) per group for laboratory investigations. All parental animals were subjected to necropsy, which included weighing of major organs. Histopathology was conducted on tissues from five males from Control and High dose, and seven females from the Control and eight females from the High dose.

At 20000 ppm in-life observations included decreased weight gain and food consumption in both sexes. Increased male liver weight, and increases in bilirubin and alkaline phosphatase were noted in both sexes. In addition, small decreases were noted in adrenal gland weight in both sexes, and in albumin, white blood cell count and ovary weight in females; spleen weight and cholesterol were slightly increased in males.

At 5000 ppm liver weight in males and alkaline phosphatase in both sexes were increased. Female adrenal gland weight was reduced.

In conclusion, under the conditions of this study, toxicity was exhibited at levels of 5000 and 20000 ppm, but there were no clear effects of toxicity at 1000 ppm. Therefore the parental NOAEC was considered to be 1000 ppm.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25/03/19 - 24/09/19
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Appendix to Director General Notification, No. 12-Nousan-8147
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Batch (Lot) Number: AN-400-125
Purity: 100%
Amber transparent liquid
Species:
rat
Strain:
Wistar
Details on species / strain selection:
This strain is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals were acclimatised for 13 days before the start of dosing. A clinical observation was undertaken during this period.

Animals were housed in a temperature range of 18-24 ºC, a relative target humidity of 40-70%, and a 12 hour light/dark cycle.

Rats were housed in groups up to 5 animals of the same sex and same dosing group together in polycarbonate cages.
Route of administration:
oral: feed
Details on route of administration:
The test and control items were administered by inclusion in the diet to the appropriate animals ad libitum from Day 1 for a minimum of 90 days, up to and including the day before scheduled necropsy. The first day diets were available to the animals was designated as Day 1.

The same diets remain in the food hopper for a maximum of one week. On the day of weighing the remaining food in the food hopper, remaining diet was replaced with new diet. The amount of test item incorporated into the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test article intake was estimated based on the body weight and food consumption values.
Vehicle:
acetone
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verification of doses was performed using a Agilent Technologies 7890B Gaschromatograph machine. The injection temperature was 260°C.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Test and control items were administered by inclusion in the diet to the appropriate animals ad libitum from day 1 for a minimum of 90 days- up to, and including the day before scheduled necropsy.
Dose / conc.:
0 ppm
Dose / conc.:
1 500 ppm
Dose / conc.:
5 000 ppm
Dose / conc.:
15 000 ppm
No. of animals per sex per dose:
10/sex/group
Control animals:
yes, concurrent vehicle
Positive control:
None
Observations and examinations performed and frequency:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical observations were performed once daily, once prior to first administration and beginning during the first administration of the test item and lasting up to the day prior to necropsy.

Animals were weighed individually weekly, starting on Day 1. A fasted weight was recorded on the day of necropsy

Food consumption was quantitatively measured weekly starting on Day 1 and continuing weekly throughout the Dosing Period.

Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

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 Main Study animals, and at the end of the Dosing Period in Week 13 in all Group 1 and 4 Main study animals

Functional tests were performed on the first 5 animals per sex per group during Week 12-13. These tests were performed, after completion of clinical observations (including arena observation, if applicable).
Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
• Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Static righting reflex (STATIC R) (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).
• Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). 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 more fine movements like grooming, weaving or movements of the head.

In the absence of a vaginal lavage, the study pathologist determined the estrous stage of all females by examining the reproductive organs.
These data were only used for evaluation of extreme thyroid hormone effects in females and are not included in the results section.
Sacrifice and pathology:
Blood was collected between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) for analysis. A blood smear was prepared from each hematology sample.

Haematology
Blood smears were labeled, stained, and stored. Blood smear from Animal No. 69 (5000 ppm) was evaluated to confirm analyser results.

Haematology parameters:
White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophils (absolute), Haemoglobin, Lymphocytes (absolute), Haematocrit, Monocytes (absolute), Mean corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Reticulocyte (absolute), and Platelets

Coagulation
Blood samples at a target volume of 0.45 mL were collected into tubes containing citrate as anticoagulant. Samples were processed for plasma, and plasma was analyzed for prothrombin time and activated partial thromboplastin time

Clinical chemistry
Blood samples at a target volume of 0.5 mL were collected into tubes containing Li-heparinas anticoagulant. Samples were processed for plasma, and were analysed.
Blood samples at a target volume of 1.0 mL were collected in tubes without anticoagulant. Blood samples were processed for serum (bile acids and thyroid hormone measurement) and analysed

Clinical Chemistry parameters:
Alanine aminotransferase (ALAT) Glucose, Aspartate aminotransferase (ASAT), Cholesterol, Alkaline Phosphatase (ALP), HDL and LDL Cholesterol, Total protein, Triglyceridea (Triglyceride concentration determined from remaining thyroid hormone serum samples), Albumin, Sodium, Bile acids, Potassium, Total Bilirubin, Chloride, Urea, Calcium, Creatinine, and Inorganic Phosphate (Inorg. Phos)

Thyroid hormone
Serum samples at a target volume of 1.0 mL were collected in tubes without anticoagulant (same sample as for bile acid measurement). Blood samples were processed for serum, and were analysed.

Thyroid hormone parameters:
Triiodothyronine (T3), Thyroid-Stimulating Hormone (TSH), Thyroxine (T4)

Terminal procedures
Main study and recovery animals surviving until scheduled euthanasia were weighed, and euthanized using isoflurane, followed by exsanguination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy

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. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

The following organ weights were weighed at necropsy for all scheduled euthanasia animals (paired organs were weighed together)
Brain, Epididymisa, Gland, adrenala, Gland, pituitary, Gland, prostate, Gland, seminal vesicle, Gland, thyroida, Heart, Kidney, Liver, Ovary, Spleen, Testis, Thymus, Uterus/Cervix
Organ to body weight ratio was calculated.

The following tissues were embedded in paraffin (Klinipath, Duiven, The Netherlands), sectioned, mounted on glass slides, and stained with hematoxylin and eosin (Klinipath, Duiven, The Netherlands)

Artery, aorta, Body cavity, nasopharynx, Bone, femur, Bone marrow, Bone, sternum, Brain, Cervix, Epididymisa, Esophagus, Eye, adrenal gland, clitoral gland, harderian gland, lacrimal gland, mammary gland, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidney, Large intestine, cecum, Large intestine, colon, Large intestine, rectum, Larynx, Liver, Lung, Lymph node, mandibular, Lymph node, mesenteric, skeletal muscle, optica nerve, sciatic nerve, Ovary, Pancreas, Skin, Small intestine, duodenum, Small intestine, ileum, Small intestine, jejunum, Spinal cord, Spleen, Stomach, Testisa, Thymus, Tongue, Trachea, Urinary bladder, Uterus, Vagina

All tissues were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported. Tissues that were supposed to be microscopically evaluated per protocol but were not available on the slide (and therefore not evaluated) are listed in Individual Animal Data of the pathology report as not present. These missing tissues did/not affect the outcome or interpretation of the pathology portion of the study because sufficient data was available. A peer review on the histopathology data was performed by a second pathologist.
Statistics:
Body Weight Gains: Calculated against the body weight on Day 1.

Relative Food Consumption: Calculated between at least each scheduled interval.

Test Item Intake: Calculated as concentration of test item in diet against relative food consumption.

Organ Weight Relative to Body Weight: Calculated against the Terminal body weight

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), ratio, percentages, numbers, and/or incidences were reported as appropriate by dataset.

All statistical tests was conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations. The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Non-parametric
Datasets with at least 3 groups were compared using a Steel-test many-to-one rank test).

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

Clinical signs, such as alopecia, scabs and incidental piloerection, noted during the Dosing Period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No effects on body weight and body weight gain were noted in males up to 5000 ppm and in females up to 15000 ppm.
A slightly lower body weight gain was observed in males at 15000 ppm throughout the administration period (-5% compared to controls).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight was similar to the control level over the study period in males and females up to 15000 ppm.

Mean test article intake over the study period tabulated in "Any other information on results incl. tables" section.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no ophthalmology findings at Pretreatment and in Week 13. The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No effects on haematology parameters were noted in males at 1500 ppm and in females up to 15000 ppm.
Lower reticulocyte count was observed in males at 5000 ppm and 15000 ppm (0.83x and 0.84x of controls). In addition, red blood cell distribution width (RDW) was also minimally decreased in males at 15000 ppm (0.92x of controls). These changes werer just marginally outside historical control data, but in absence of any organ weight change or histopathological correlation these changes were not considered to be of toxicological significance.

While few other changes were statistically significant in males at 5000 and/or 15000 ppm, the alterations in haematological parameters were unrelated to administration of the test item due to the minimal magnitude of the change, remained within the historical control range, and/or absence of a clear dose response relationship.

Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No effects on clinical chemistry parameters were noted in males and females at 1500 and 5000 ppm.

At 15000 ppm, an increase in alkaline phosphatase (ALP) activity in males and females (1.53x and 2.06x of controls, respectively) and total bilirubin concentration in males (1.53x of controls) was observed. Furthermore, slight increases in total cholesterol and HDL and LDL cholesterol concentrations were observed in males at 15000 ppm (1.26x, 1.31x 1.60x of controls). In females at 15000 ppm, total cholesterol and HDL cholesterol were also slightly increased (1.16x and 1.28x of controls, respectively). Bile acids was also decreased in in males and females (0.54x and 0.41x of controls, respectively).

Total T3 was increased in males at 15000 ppm (1.50x of controls), which was caused by 4 males showing levels above the concurrent control range (maximally 2.41x of mean control value).

Other values in treated males and females achieving a levle of statistical significance when compared to controls where considered to have arisen as a result of slightly high or low control values, occured in the absence of a dose-related distribution and/or were, given the magnitude of change, considered to be of no toxicological significance.
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 examined animals. Grip strength in animals up to 15000 ppm was similar to control, with the exception of the fore grip strength in males at 15000 ppm, which was slightly lower compared to control. However, as this fore grip strength remained well within the historical control range (633-1649 gram), this isolated finding was therefore considered not to be either test-material related or toxicologically relevant.

Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased absolute liver weight in females at 15000 (22%), and relative liver weight (to body weight) in males and females at 15000 (14 and 23% respectively)

There were no other test item-related organ weight changes. All other organ weight differences observed, were considered incidental and/or related to difference of sexual maturity and unrelated to the administration of Tall Oil.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations.

All 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 item-related microscopic findings after treatment with Tall Oil were noted at 15000 ppm in the liver of females and lung of both sexes.

Multifocal macrophage aggregation in the alveoli of the lung (minimal or slight) was recorded at increased incidence in males and females of the 15000 ppm group. The recorded incidences in the 1500 and/or 5000 ppm groups were comparable with the recorded incidences in the control group, or did not show a dose response and were therefore considered to be unrelated to treatment with the test item.

Hepatocellular hypertrophy of the liver was recorded at minimal degree in females of the 15000 ppm group.
Evaluation of the female reproductive organs for estrus stage revealed no abnormalities.
There were no other test item-related histologic changes. Remaining histologic changes (including the requested adrenal gland of males and kidneys of females) were considered to be within the range of background pathology encountered in rats of this age and strain.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects
Remarks on result:
other: approximate achieved doses of 1104 mg/kg/day in males and 1221 mg/kg/day in females
Key result
Critical effects observed:
no

Test Article Intake

Group

Nominal dietary inclusion level (ppm)

Mean over means intake (mg test item/kg body weight) (mean range indicated within brackets)

Males

Females

2

1500

107 (79-153)

119 (94-152)

3

5000

367 (270-522)

420 (341-526)

4

15000

1104 (837-1506)

1221 (993-1520)

Summary of Body Weights (g) - Males

Treatment

 

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

Day 1

Week 1

Mean

177

176

173

179

St. Dev.

11.7

10.0

7.9

5.7

N

10

10

10

10

 

Day 8

Week 2

Mean

227

222

218

217

St. Dev.

15.4

14.4

11.9

6.9

N

10

10

10

10

 

Day 15

Week 3

Mean

265

261

260

257

St. Dev.

18.6

20.0

15.9

8.0

N

10

10

10

10

 

Day 22

Week 4

Mean

291

287

286

281

St. Dev.

21.7

26.3

21.0

9.3

N

10

10

10

10

 

Day 29

Week 5

Mean

315

311

315

308

St. Dev.

23.4

31.5

23.8

10.0

N

10

10

10

10

 

Day 36

Week 6

Mean

337

334

335

331

St. Dev.

26.7

33.3

25.7

19.5

N

10

10

10

10

 

Day 43

Week 7

Mean

354

350

352

346

St. Dev.

29.2

35.4

28.7

10.9

N

10

10

10

10

 

Day 50

Week 8

Mean

370

363

367

359

St. Dev.

30.9

38.1

30.9

11.6

N

10

10

10

10

 

Day 57

Week 9

Mean

378

371

376

365

St. Dev.

31.1

37.9

34.2

12.2

N

10

10

10

10

 

Day 64

Week 10

Mean

387

385

387

374

St. Dev.

34.7

40.0

35.2

14.5

N

10

10

10

10

 

Day 71

Week 11

Mean

397

391

393

383

St. Dev.

34.3

40.9

36.4

15.7

N

10

10

10

10

 

Day 78

Week 12

Mean

404

400

401

389

St. Dev.

33.6

41.4

34.4

15.0

N

10

10

10

10

 

Day 85

Week 13

Mean

415

407

411

399

St. Dev.

34.7

38.9

35.2

14.1

N

10

10

10

10

 

Day 91

Week 13

Mean

416

408

406

397

St. Dev.

35.1

40.0

35.7

16.5

N

10

10

10

10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Summary of Body Weights (g) - Females

Treatment

 

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

Day 1

Week 1

Mean

131

132

137

132

St. Dev.

4.9

8.2

7.1

7.4

N

10

10

10

10

 

Day 8

Week 2

Mean

150

153

158

149

St. Dev.

8.4

8.8

7.1

10.5

N

10

10

10

10

 

Day 15

Week 3

Mean

169

173

178

166

St. Dev.

8.2

8.6

7.8

10.4

N

10

10

10

10

 

Day 22

Week 4

Mean

180

183

191*

177

St. Dev.

9.3

8.9

7.1

10.1

N

10

10

10

10

 

Day 29

Week 5

Mean

193

194

202

190

St. Dev.

9.7

10.1

7.4

9.0

N

10

10

10

10

 

Day 36

Week 6

Mean

200

205

211

200

St. Dev.

12.3

10.2

10.7

8.5

N

10

10

10

10

 

Day 43

Week 7

Mean

212

215

220

206

St. Dev.

10.8

11.6

10.3

11.8

N

10

10

10

10

 

Day 50

Week 8

Mean

218

223

224

212

St. Dev.

12.5

11.8

10.4

13.0

N

10

10

10

10

 

Day 57

Week 9

Mean

219

222

226

213

St. Dev.

11.9

14.1

9.2

9.5

N

10

10

10

10

 

Day 64

Week 10

Mean

219

226

231

216

St. Dev.

12.7

12.6

12.1

9.7

N

10

10

10

10

 

Day 71

Week 11

Mean

223

232

234

220

St. Dev.

11.6

13.1

11.6

11.4

N

10

10

10

10

 

Day 78

Week 12

Mean

229

234

238

223

St. Dev.

12.3

13.4

10.9

10.7

N

10

10

10

10

 

Day 85

Week 13

Mean

232

234

242

227

St. Dev.

12.5

14.8

11.6

12.9

N

10

10

10

10

 

Day 91

Week 13

Mean

227

235

240

225

St. Dev.

11.3

14.2

11.4

10.8

N

10

10

10

10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Summary of Body Weight Gain (%) - Males

Treatment

 

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

Day 1

Week 1

Mean

0

0

0

0

St. Dev.

0.0

0.0

0.0

0.0

N

10

10

10

10

 

Day 8

Week 2

Mean

28

26

26

21**

St. Dev.

1.9

2.7

1.7

3.0

N

10

10

10

10

 

Day 15

Week 3

Mean

50

48

50

43**

St. Dev.

3.7

4.9

3.6

4.6

N

10

10

10

10

 

Day 22

Week 4

Mean

64

62

65

57*

St. Dev.

3.9

8.9

5.8

5.8

N

10

10

10

10

 

Day 29

Week 5

Mean

78

76

82

72

St. Dev.

5.6

12.6

7.7

7.1

N

10

10

10

10

 

Day 36

Week 6

Mean

90

89

93

85

St. Dev.

6.6

13.1

8.5

10.2

N

10

10

10

10

 

Day 43

Week 7

Mean

100

98

103

93

St. Dev.

7.0

14.4

10.5

7.9

N

10

10

10

10

 

Day 50

Week 8

Mean

109

106

111

101

St. Dev.

8.4

15.8

11.8

8.5

N

10

10

10

10

 

Day 57

Week 9

Mean

113

110

117

104

St. Dev.

9.0

16.1

13.1

9.5

N

10

10

10

10

 

Day 64

Week 10

Mean

118

118

123

109

St. Dev.

10.3

17.3

13.8

10.4

N

10

10

10

10

 

Day 71

Week 11

Mean

124

122

126

114

St. Dev.

10.6

17.6

14.4

10.7

N

10

10

10

10

 

Day 78

Week 12

Mean

128

127

131

117

St. Dev.

10.5

17.5

14.2

10.4

N

10

10

10

10

 

Day 85

Week 13

Mean

134

131

137

123

St. Dev.

11.5

16.4

14.3

10.3

N

10

10

10

10

 

Day 91

Week 13

Mean

135

131

134

121

St. Dev.

10.8

16.5

14.6

10.9

N

10

10

10

10

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Summary of Body Weight Gain (%) - Females

Treatment

 

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

Day 1

Week 1

Mean

0

0

0

0

St. Dev.

0.0

0.0

0.0

0.0

N

10

10

10

10

 

Day 8

Week 2

Mean

14

17

16

12

St. Dev.

3.5

3.4

3.3

3.3

N

10

10

10

10

 

Day 15

Week 3

Mean

29

32

30

26

St. Dev.

3.1

3.3

4.4

3.2

N

10

10

10

10

 

Day 22

Week 4

Mean

37

39

39

34

St. Dev.

3.4

4.9

5.1

3.1

N

10

10

10

10

 

Day 29

Week 5

Mean

47

47

47

43

St. Dev.

3.7

5.2

4.6

5.5

N

10

10

10

10

 

Day 36

Week 6

Mean

52

56

54

51

St. Dev.

5.2

6.9

7.4

4.8

N

10

10

10

10

 

Day 43

Week 7

Mean

62

64

61

56

St. Dev.

4.4

7.4

7.5

8.0

N

10

10

10

10

 

Day 50

Week 8

Mean

66

70

63

60

St. Dev.

5.9

8.8

6.6

8.6

N

10

10

10

10

 

Day 57

Week 9

Mean

67

69

65

61

St. Dev.

4.9

9.8

6.2

6.5

N

10

10

10

10

 

Day 64

Week 10

Mean

66

72

69

63

St. Dev.

6.6

7.7

8.1

6.1

N

10

10

10

10

 

Day 71

Week 11

Mean

70

77

71

67

St. Dev.

5.3

8.6

8.5

6.5

N

10

10

10

10

 

Day 78

Week 12

Mean

74

78

74

69

St. Dev.

5.8

7.8

7.3

8.1

N

10

10

10

10

 

Day 85

Week 13

Mean

77

78

77

72

St. Dev.

6.9

7.6

7.5

9.0

N

10

10

10

10

 

Day 91

Week 13

Mean

73

78

76

70

St. Dev.

5.7

7.8

9.5

7.2

N

10

10

10

10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Summary of Ophthalmoscopic Observations

 

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

Males

Pre-treatment

 

      No findings

8/10

10/10

10/10

10/10

      Persistent Pupillary Membrane

2/10

0/10

0/10

0/10

End of Treatment

 

 

 

 

       No findings

10/10

 

 

10/10

Females

Pre-treatment

 

 

 

 

       No findings

10/10

10/10

9/10

9/10

       Focal Corneal Opacity

0/10

0/10

1/10

1/10

End of Treatment

 

 

 

 

       No findings

10/10

 

 

10/10

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level


Haematology summary – Males

End of treatment

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

WBC

10E9/L

Mean

7.2

6.2

6.3

5.8

St. Dev

1.4

1.6

1.7

1.6

N

10

10

10

9

Neutrophils

10E8/L

Mean

1.0

0.9

0.8

0.8

St. Dev

0.4

0.3

0.3

0.4

N

10

10

10

9

Lymphocytes

10E9/L

Mean

5.9

5.1

5.3

4.8

St. Dev

1.2

1.5

1.5

1.3

N

10

10

10

9

Monocytes

10E9/L

Mean

0.1

0.1

0.1

0.1

St. Dev

0.1

0.0

0.1

0.0

N

10

10

10

9

Eosinophils

10E9/L

Mean

0.1

0.1

0.1

0.1

St. Dev

0.0

0.0

0.0

0.1

N

10

10

10

9

Basophils

10E9/L

Mean

0.0

0.0

0.0

0.0

St. Dev

0.0

0.0

0.0

0.0

N

10

10

10

9

Red Blood Cells

10E12/L

Mean

9.08

8.94

8.90

8.85

St. Dev

0.42

0.24

0.42

0.33

N

10

10

10

9

Reticulocytes

10E9/L

Mean

165.2

164.4

137.0

139.8

St. Dev

30.5

24.2

45.0

20.1

N

10

10

10

9

RDW

%

Mean

11.9

11.6

11.4

11.0**

St. Dev

0.6

0.4

0.8

0.3

N

10

10

10

9

Haemoglobin

Mmol/L

Mean

10.3

10.1

9.9*

9.7**

St. Dev

0.4

0.2

0.3

0.2

N

10

10

10

9

Haematocrit

L/L

Mean

0.477

0.470

0.464

0.460

St. Dev

0.017

0.010

0.018

0.012

N

10

10

10

9

MCV

fL

Mean

52.5

52.6

52.1

52.0

St. Dev

1.5

0.9

0.8

1.1

N

10

10

10

9

MCH

Fmol

Mean

1.13

1.13

1.12

1.10

St. Dev

0.05

0.03

0.03

0.03

N

10

10

10

9

MCHC

Mmol/L

Mean

21.54

21.47

21.39

21.05*

St. Dev

0.52

0.42

0.37

0.30

N

10

10

10

9

Platelets

10E9/L

Mean

710

680

680

723

St. Dev

70

75

75

81

N

10

10

10

9

PT

s

Mean

17.8

17.1

17.6

17.8

St. Dev

0.9

0.6

1.0

1.0

N

10

10

10

9

APTT

s

Mean

17.7

17.9

18.1

16.7

St. Dev

3.4

2.8

2.7

3.5

N

10

10

10

9

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Haematology summary – Females

End of treatment

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

WBC

10E9/L

Mean

6.7

4.3**

3.9**

5.4

St. Dev

1.6

1.4

1.3

1.1

N

10

10

10

10

Neutrophils

10E8/L

Mean

0.7

0.6

0.5

0.6

St. Dev

0.2

0.2

0.2

0.3

N

105.8

10

10

10

Lymphocytes

10E9/L

Mean

1.5

3.6 +

3.3++

4.6

St. Dev

0.1

1.2

1.1

1.1

N

10

10

10

10

Monocytes

10E9/L

Mean

0.1

0.1 +

0.1

0.1

St. Dev

0.0

0.0

0.0

0.0

N

10

10

10

10

Eosinophils

10E9/L

Mean

0.1

0.1

0.1

0.1

St. Dev

0.0

0.0

0.0

0.0

N

10

10

10

10

Basophils

10E9/L

Mean

0.0

0.0

0.0

0.0

St. Dev

0.0

0.0

0.0

0.0

N

10

10

10

10

Red Blood Cells

10E12/L

Mean

8.28

7.91*

8.11

8.14

St. Dev

0.26

0.21

0.36

0.36

N

10

10

10

10

Reticulocytes

10E9/L

Mean

187.8

195.0

165.2

158.3

St. Dev

29.4

25.8

15.7

23.3

N

10

10

10

10

RDW

%

Mean

10.3

10.4

10.3

10.2

St. Dev

0.3

0.2

0.4

0.5

N

10

10

10

10

Haemoglobin

Mmol/L

Mean

9.5

9.0 **

9.2*

9.2

St. Dev

0.3

0.3

0.3

0.3

N

10

10

10

10

Haematocrit

L/L

Mean

0.118

0.433*

0.434

0.435

St. Dev

0.014

0.012

0.011

0.015

N

10

10

10

10

MCV

fL

Mean

54.1

54.7

53.6

53.6

St. Dev

0.9

1.2

1.9

1.3

N

10

10

10

10

MCH

Fmol

Mean

1.15

1.14

1.14

1.14

St. Dev

0.03

0.03

0.06

0.03

N

10

10

10

10

MCHC

Mmol/L

Mean

21.26

20.84

21.24

21.21

St. Dev

0.38

0.39

0.40

0.35

N

10

10

10

10

Platelets

10E9/L

Mean

835

7.3*

707*

795

St. Dev

139

69

114

105

N

10

10

10

10

PT

s

Mean

17.7

17.4

17.2

17.3

St. Dev

0.3

0.8

0.7

0.6

N

10

10

10

10

APTT

s

Mean

18.5

16.9

17.0

17.0

St. Dev

1.2

2.3

2.1

2.0

N

10

10

10

10

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Clinical Biochemistry Summary – Males

End of treatment

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

ALAT

U/L

Mean

46.2

47.0

48.9

54.0

St. Dev

8.4

10.8

8.8

15.0

N

10

10

10

10

ASAT

U/L

Mean

84.1

79.9

81.2

82.6

St. Dev

13.8

10.6

936

7.7

N

10

10

10

10

ALP

U/L

Mean

130

125

155

199**

St. Dev

45

24

43

42

N

10

10

10

10

Total Protein

g/L

Mean

66.6

65.6

64.4

65.6

St. Dev

2.0

1.8

2.1

2.0

N

10

10

10

10

Albumin

g/L

Mean

33.5

33.5

32.7

33.3

St. Dev

0.9

1.1

1.1

0.6

N

10

10

10

10

Total Bilirubin

μmol/L

Mean

2.9

2.9

3.3

3.5*

St. Dev

0.4

0.4

0.5

0.6

N

10

10

10

10

Urea

mmol/L

Mean

7.4

7.9

7.2

8.0

St. Dev

1.4

1.7

1.9

0.9

N

10

10

10

10

Creatinine

μmol/L

Mean

39.7

42.7**

41.9

44.4**

St. Dev

1.9

1.7

2.5

1.8

N

10

10

10

10

Glucose

mmol/L

Mean

11.89

11.02

10.07*

10.46

St. Dev

2.00

1.66

1.29

1.26

N

10

10

10

10

Cholesterol

mmol/L

Mean

2.29

2.31

2.29

2.88**

St. Dev

0.28

0.23

0.21

0.43

N

10

10

10

10

HDL cholesterol

mmol/L

Mean

0.94

0.97

0.89

1.23**

St. Dev

0.12

0.14

0.10

0.15

N

10

10

10

10

LDL Cholesterol

mmol/L

Mean

0.30

0.30

0.30

0.48**

St. Dev

0.04

0.04

0.05

0.11

N

10

10

10

10

Triglycerides

mmol/L

Mean

1.16

0.86

1.00

0.78*

St. Dev

0.21

0.15

0.43

0.23

N

10

10

10

10

Bile Acids

μmole/L

Mean

20.3

25.3

15.5

10.9*

St. Dev

6.1

10.8

5.6

4.5

N

10

10

10

10

Sodium

mmol/L

Mean

140.6

141.7*

141.8*

140.5

St. Dev

0.9

0.6

1.1

1.0

N

10

10

10

10

Potassium

mmol/L

Mean

3.84

3.85

3.97

4.01

St. Dev

0.12

0.0

0.19

0.22

N

10

10

10

10

Chloride

mmol/L

Mean

102

103*

103**

103*

St. Dev

0

1

1

1

N

10

10

10

10

Calcium

mmol/L

Mean

2.66

2.58*

2.61

2.64

St. Dev

0.07

0.04

0.04

0.08

N

10

10

10

10

Inorg. Phos

mmol/L

Mean

1.56

1.47

1.53

1.57

St. Dev

0.20

0.14

0.12

0.21

N

10

10

10

10

TSH

ulU/mL

Mean

0.129

0.309*

0.201

0.16.

St. Dev

0.134

0.192

0.198

0.094

N

10

10

10

10

Total T3

ng/dL

Mean

47.4

47.6

53.4

71.1*

St. Dev

4.4

4.4

10.2

25.3

N

10

10

10

10

Total T4

μg/dL

Mean

4.73

4.51

4.30

4.70

St. Dev

0.59

0.77

0.73

1.12

N

10

10

10

10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Clinical Biochemistry Summary – Females

End of treatment

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

ALAT

U/L

Mean

48.7

36.5

43.2

52.2

St. Dev

14.0

7.9

13.6

15.4

N

10

10

10

10

ASAT

U/L

Mean

81.5

77.5

87.4

81.0

St. Dev

5.8

4.4

7.1

8.1

N

10

10

10

10

ALP

U/L

Mean

53

49

59

109**

St. Dev

14

12

15

54

N

10

10

10

10

Total Protein

g/L

Mean

69.4

69.1

68.1

68.1

St. Dev

1.9

2.2

2.2

2.2

N

10

10

10

10

Albumin

g/L

Mean

36.8

36.4

36.3

35.8

St. Dev

1.3

1.8

1.3

1.4

N

10

10

10

10

Total Bilirubin

μmol/L

Mean

3.3

3.5

3.6

3.7

St. Dev

0.4

0.4

0.7

0.3

N

10

10

10

10

Urea

mmol/L

Mean

7.3

7.4

7.2

7.6

St. Dev

1.4

1.1

0.9

1.0

N

10

10

10

10

Creatinine

μmol/L

Mean

38.8

45.4**

43.2*

43.3*

St. Dev

3.0

4.1

3.8

2.7

N

10

10

10

10

Glucose

mmol/L

Mean

9.27

8.87

8.20

8.63

St. Dev

1.79

1.53

0.90

1.26

N

10

10

10

10

Cholesterol

mmol/L

Mean

2.17

2.20

2.22

2.51

St. Dev

0.30

0.35

0.37

0.22

N

10

10

10

10

HDL cholesterol

mmol/L

Mean

0.94

0.95

0.98

1.20**

St. Dev

0.17

0.19

0.21

0.14

N

10

10

10

10

LDL Cholesterol

mmol/L

Mean

0.28

0.28

0.26

0.32

St. Dev

0.04

0.06

0.05

0.02

N

10

10

10

10

Triglycerides

mmol/L

Mean

0.66

0.59

0.74

0.79

St. Dev

0.27

0.16

0.25

0.21

N

10

10

10

10

Bile Acids

μmole/L

Mean

19.2

13.2

15.2

7.9**

St. Dev

13.1

5.4

4.1

3.6

N

10

10

10

10

Sodium

mmol/L

Mean

140.2

140.7

140.6

140.4

St. Dev

0.7

0.6

1.0

0.4

N

10

10

10

10

Potassium

mmol/L

Mean

3.77

3.45*

3.53*

3.57

St. Dev

0.14

0.22

0.17

0.16

N

10

10

10

10

Chloride

mmol/L

Mean

103

105**

104*

104

St. Dev

1

1

1

1

N

10

10

10

10

Calcium

mmol/L

Mean

2.72

2.60**

2.59**

2.71

St. Dev

0.04

0.06

0.04

0.03

N

10

10

10

10

Inorg. Phos

mmol/L

Mean

1.56

1.14**

1.11**

1.51

St. Dev

0.13

0.19

0.14

0.19

N

10

10

10

10

TSH

ulU/mL

Mean

0.036

0.119*

0.103

0.109*

St. Dev

0.015

0.089

0.064

0.073

N

10

10

10

10

Total T3

ng/dL

Mean

49.2

51.6

49.1

43.7

St. Dev

7.9

7.8

4.8

2.3

N

10

10

10

10

Total T4

μg/dL

Mean

1.95

2.94*

2.55

2.37

St. Dev

0.63

1.01

0.77

0.049

N

10

10

10

10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Summary of Macroscopic Findings

 

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

Males

End of Treatment

 

Animals examined

10

10

10

10

Animals without findings

6

9

8

6

Animals affected

4

1

2

4

 

Stomach: Thickened

1

0

0

0

Seminal vesicles: Reduced size

2

1

1

0

Preputial glands: Focus/Foci

0

0

0

1

Thyroid gland: Discolouration

0

0

0

1

Thymus: Focus/Foci

1

0

1

1

Mandibular lymph Node: Enlarged

0

0

0

1

Females

End of Treatment

 

 

 

 

Animals examined

10

10

10

10

Animals without findings

8

6

6

7

Animals affected

2

4

4

3

 

Kidneys: Pelvic dilation

0

0

1

0

Uterus: Contains fluid

1

2

4

2

Clitoral glands: Focus/Foci

0

1

0

1

Thymus: Focus/Foci

0

1

0

0

Mandibular lymph Node: Discolouration

0

0

0

1

Eyes: Exophthalmus

0

1

0

0

Body cavities: Nodule(s)

1

0

0

0

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level    

Summary of Organ Weights (g) - Males

 

 

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

End of Treatment

Body Weight (g)

Mean

396

387

384

382

St. Dev.

34

38

33

18

N

10

10

10

10

 

Brain (g)

Mean

2.04

2.07

2.06

2.07

St. Dev.

0.07

0.08

0.07

0.07

N

10

10

10

10

 

Pituitary (g)

Mean

0.009

0.009

0.009

0.009

St. Dev.

0.001

0.001

0.002

0.001

N

10

10

10

10

 

Heart (g)

Mean

1.020

1.018

0.968

0.976

St. Dev.

0.086

0.110

0.075

0.036

N

10

10

10

10

 

Liver (g)

Mean

9.32

9.19

8.91

10.18

St. Dev.

1.24

1.42

1.09

0.86

N

10

10

10

10

 

Thyroids (g)

Mean

0.018

0.019

0.021

0.020

St. Dev.

0.002

0.005

0.005

0.003

N

10

10

10

10

 

Thymus (g)

Mean

0.301

0.258

0.264

0.300

St. Dev.

0.070

0.083

0.063

0.065

N

10

10

10

10

 

Kidneys (g)

Mean

2.38

2.39

2.39

2.40

St. Dev.

0.24

0.28

0.19

0.19

N

10

10

10

10

 

Adrenals (g)

Mean

0.056

0.053

0.055

0.051

St. Dev.

0.006

0.007

0.004

0.006

N

10

10

10

10

 

Spleen (g)

Mean

0.569

0.577

0.580

0.553

St. Dev.

0.059

0.085

0.063

0.053

N

10

10

10

10

 

Testes (g)

Mean

3.68

3.66

3.62

3.64

St. Dev.

0.30

0.30

0.25

0.17

N

10

10

10

10

 

Prostate (g)

Mean

0.908

0.805

0.843

0.790

St. Dev.

0.149

0.188

0.101

0.135

N

10

10

10

10

 

Epididymides (g)

Mean

1.195

1.187

1.147

1.178

St. Dev.

0.085

0.084

0.091

0.053

N

10

10

10

10

 

Seminal Vesicle (g)

Mean

1.219

1.251

1.223

1.300

St. Dev.

0.181

0.220

0.212

0.616

N

10

10

10

10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Summary of Organ / Body weight Ratios (%) - Males

 

 

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

End of Treatment

Body Weight (g)

Mean

396

387

384

382

St. Dev.

34

38

33

18

N

10

10

10

10

 

Brain (%)

Mean

0.52

0.54

0.54

0.54

St. Dev.

0.04

0.04

0.04

0.03

N

10

10

10

10

 

Pituitary (%)

Mean

0.002

0.002

0.002

0.002

St. Dev.

0.000

0.000

0.000

0.000

N

10

10

10

10

 

Heart (%)

Mean

0.258

0.263

0.253

0.255

St. Dev.

0.012

0.010

0.012

0.008

N

10

10

10

10

 

Liver (%)

Mean

2.34

2.37

2.32

2.66**

St. Dev.

0.17

0.18

0.14

0.18

N

10

10

10

10

 

Thyroids (%)

Mean

0.005

0.005

0.005

0.005

St. Dev.

0.000

0.001

0.001

0.001

N

10

10

10

10

 

Thymus (%)

Mean

0.076

0.067

0.069

0.079

St. Dev.

0.016

0.018

0.016

0.017

N

10

10

10

10

 

Kidneys (%)

Mean

0.60

0.62

0.62

0.63

St. Dev.

0.02

0.03

0.03

0.04

N

10

10

10

10

 

Adrenals (%)

Mean

0.014

0.014

0.014

0.013

St. Dev.

0.002

0.002

0.001

0.002

N

10

10

10

10

 

Spleen (%)

Mean

0.144

0.149

0.151

0.145

St. Dev.

0.018

0.019

0.015

0.012

N

10

10

10

10

 

Testes (%)

Mean

0.93

0.96

0.95

0.96

St. Dev.

0.10

0.13

0.09

0.06

N

10

10

10

10

 

Prostate (%)

Mean

0.230

0.208

0.220

0.207

St. Dev.

0.041

0.043

0.025

0.038

N

10

10

10

10

 

Epididymides

Mean

0.304

0.309

0.300

0.309

St. Dev.

0.038

0.036

0.024

0.022

N

10

10

10

10

 

Seminal Vesicle (%)

Mean

0.309

0.325

0.320

0.341

St. Dev.

0.054

0.058

0.059

0.047

N

10

10

10

10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Summary of Organ Weights (g) - Females

 

 

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

End of Treatment

Body Weight (g)

Mean

217

223

229

214

St. Dev.

11

14

10

9

N

10

10

9

10

 

Brain (g)

Mean

1.89

1.88

1.89

1.89

St. Dev.

0.08

0.05

0.06

0.08

N

10

10

10

10

 

Pituitary (g)

Mean

0.013

0.014

0.012

0.011

St. Dev.

0.002

0.004

0.002

0.003

N

10

10

10

10

 

Heart (g)

Mean

0.704

0.729

0.710

0.654

St. Dev.

0.066

0.087

0.051

0.042

N

10

10

10

10

 

Liver (g)

Mean

5.27

5.66

5.70

6.42**

St. Dev.

0.40

0.58

0.40

0.53

N

9

10

10

10

 

Thyroids (g)

Mean

0.014

0.015

0.015

0.014

St. Dev.

0.003

0.002

0.003

0.022

N

10

10

10

10

 

Thymus (g)

Mean

0.270

0.264

0.275

0.269

St. Dev.

0.035

0.036

0.055

0.055

N

10

10

10

10

 

Kidneys (g)

Mean

1.53

1.52

1.55

1.50

St. Dev.

0.12

0.11

0.11

0.12

N

10

10

10

10

 

Adrenals (g)

Mean

0.067

0.070

0.065

0.058

St. Dev.

0.008

0.007

0.011

0.008

N

10

10

10

10

 

Spleen (g)

Mean

0.377

0.422

0.390

0.393

St. Dev.

0.039

0.051

0.047

0.043

N

10

10

10

10

 

Ovaries (g)

Mean

0.133

0.145

0.148

0.140

St. Dev.

0.020

0.021

0.018

0.021

N

10

10

10

10

 

Uterus (g)

Mean

0.669

0.628

0.765

0.662

St. Dev.

0.400

0.199

0.389

0.445

N

10

10

10

10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Summary of Organ / Body weight Ratios (%) - Females

 

 

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

End of Treatment

Body Weight (g)

Mean

217

223

229

214

St. Dev.

11

14

10

9

N

10

10

9

10

 

Brain (%)

Mean

0.87

0.85

0.83

0.88

St. Dev.

0.04

0.05

0.03

0.03

N

10

10

9

10

 

Pituitary (%)

Mean

0.006

0.006

0.005

0.005

St. Dev.

0.001

0.002

0.001

0.001

N

10

10

9

10

 

Heart (%)

Mean

0.326

0.327

0.314

0.305

St. Dev.

0.037

0.042

0.013

0.015

N

10

10

9

10

 

Liver (%)

Mean

2.44

2.54

2.52

3.00**

St. Dev.

0.17

0.20

0.12

0.28

N

9

10

9

10

 

Thyroids (%)

Mean

0.006

0.007

0.007

0.007

St. Dev.

0.001

0.001

0.001

0.001

N

10

10

9

10

 

Thymus (%)

Mean

0.124

0.118

0.125

0.126

St. Dev.

0.015

0.014

0.020

0.027

N

10

10

9

10

 

Kidneys (%)

Mean

0.70

0.68

0.69

0.70

St. Dev.

0.06

0.05

0.04

0.06

N

10

10

9

10

 

Adrenals (%)

Mean

0.031

0.031

0.028

0.027

St. Dev.

0.003

0.003

0.004

0.004

N

10

10

9

10

 

Spleen (%)

Mean

0.174

0.189

0.169

0.183

St. Dev.

0.016

0.022

0.019

0.107

N

10

10

9

10

 

Ovaries (%)

Mean

0.061

0.065

0.064

0.065

St. Dev.

0.010

0.011

0.006

0.008

N

10

10

9

10

 

Uterus (%)

Mean

0.307

0.283

0.344

0.309

St. Dev.

0.177

0.093

0.167

0.206

N

10

10

9

10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Mean percentage liver weight differences from control groups

Dose level (ppm)

Males

Females

1500

5000

15000

1500

5000

15000

LIVER

Absolute

-1

-4

9

7

8

22**

Relative to body weight

1

-1

14**

4

3

23**

*: P<0.05, **:P<0.01

Summary of Treatment-Related Microscopic Finding

 

Males

Females

Dose Level (ppm)

0

1500

5000

15000

0

1500

5000

15000

Lunga

10

10

10

10

10

10

10

10

Macrophage aggregation, multifocal

 

 

 

 

 

 

 

 

    Minimal

1

-

1

3

2

1

2

6

    Slight

-

-

1

3

-

-

1

-

 

Livera

10

10

10

10

10

10

10

10

Hepatocellular hypertrophy

 

 

 

 

 

 

 

 

    Minimal

-

-

-

-

-

-

-

4

a Number of tissues examined from each group.

Summary of Functional Observations

 

 

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

Males

End of Treatment

 

Hearing

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Pupil L

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Pupil R

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Static R

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Grip Fore

Gram

Mean

1263

1211

1094

956*

St. Dev

61

270

218

120

N

5

5

5

5

 

Grip Hind

Gram

Mean

493

592

515

520

St. Dev

62

115

47

76

N

5

5

5

5

Females

End of Treatment

 

Hearing

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Pupil L

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Pupil R

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Static R

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Grip Fore

Gram

Mean

980

850

747

882

St. Dev

129

102

147

182

N

5

5

5

5

 

Grip Hind

Gram

Mean

363

381

396

422

St. Dev

41

68

93

60

N

5

5

5

5

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Summary of Motor Activity

 

Group 1

Control

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

Males

Total Movements

 

Mean

4535

4340

3552

4174

N

5

5

5

5

St. Dev

929

1039

731

372

Ambulations

 

 

 

 

Mean

950

924

697

846

N

5

5

5

5

St. Dev

385

264

272

112

Females

Total Movements

 

 

 

 

Mean

4536

5543

4666

4490

N

5

5

5

5

St. Dev

866

766

927

1538

Ambulations

 

 

 

 

Mean

1224

1530

1140

1208

N

5

5

5

5

St. Dev

294

315

321

612

* indicates a p-value <0.05, ** indicates a p-value <0.01

MEAN and ST. DEV values are calculated per group, from each animal's Total Movements over all interval

 

Conclusions:
In conclusion, administration of Tall Oil by dietary administration for at least 90 days was generally well tolerated in Han Wistar rats at levels up to 15000 ppm and did not result in any adverse test item-related (morphologic) alterations.

Therefore, based on these results, the No-Observed-Adverse-Effect Level (NOAEL) was considered to be 15000 ppm (which equates to approximate achieved doses of 1104 mg/kg/day in males and 1221 mg/kg/day in females).
Executive summary:

The objective of this study was to determine the potential toxicity of Tall Oil, when given via diet administration for 90 days to Wistar Han rats. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.

 

The study design was as follows:

 

Table 1: Experimental Design

Group No.

Test Item ID.

Dose level (ppm)

No. of Animals

Male

Female

1

-

0 (Vehicle)

10

10

2

Tall Oil

1500

10

10

3

5000

10

10

4

15000

10

10

Table 2: Test Article Intake

Group

Nominal dietary inclusion level (ppm)

Mean over means intake (mg test item/kg body weight) (mean range indicated within brackets)

Males

Females

2

1500

107 (79-153)

119 (94-152)

3

5000

367 (270-522)

420 (341-526)

4

15000

1104 (837-1506)

1221 (993-1520)

 

Chemical analyses of dietary preparations were conducted three times to assess accuracy and homogeneity. Additional analysis for stability was done in Week 13.

The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, ophthalmology, functional observations, estrous stage determination, clinical pathology parameters (hematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations. Dietary analyses confirmed that formulations of test item in diets were prepared accurately and homogenously in Weeks 1 and 6. The concentrations analyzed in the diets prepared in Week 13 were not in agreement with target concentrations (i.e. mean accuracies between 73 and 79%). Therefore, as concentrations in Week 13 were only below the acceptable range (80%-120%) for up to a maximum of 7% and taken into account that the Weeks 1 and 6 diets which were analyzed were within specification, it was concluded that the diets were accurately prepared throughout the administration period.

At 1500 ppm, no test item-related changes were observed.

At 5000 ppm, the only observed test item-related finding was lower reticulocyte count in males which was, at the severity observed and the absence of histopathological correlation, considered not to be adverse.

At 15000 ppm, multiple test item-related effects were seen, which were considered not to be adverse. Firstly, a lower body weight gain was observed in males throughout the administration period. Affected test item-related hematology changes consisted of slightly lower reticulocyte count and decreased red blood cell distribution width in males. The body weight and hematology effects were only slight and in absence of a histopathological correlation considered not to be adverse. 

At clinical chemistry, an increase in alkaline phosphatase activity was noted in males and females, which in females correlates with the hepatocellular hypertrophy of the liver. Furthermore, increases in total bilirubin concentration in males and total cholesterol and HDL and/or LDL cholesterol concentrations in males and females and decreases in bile acids concentration in males and females were observed. Finally, total T3 was increased in males at 15000 ppm. The bilirubin, cholesterol, bile acids and T3 findings were in absence of a histopathological correlation or any effects in the other thyroid hormones considered not to be adverse. At histopathological examination, minimal hepatocellular hypertrophy of the liver was observed in females, which correlated with increased liver weights and higher alkaline phosphatase activity and total bilirubin concentration. There was no microscopic correlate for the observed increased liver weight in males. These findings were at the observed severity and/or magnitude and in absence of any concomitant degenerative finding considered nonadverse and is a commonly observed adaptive response associated with the metabolism of xenobiotics or their metabolites. In the lung, multifocal macrophage aggregation was observed in males and females. The exacerbation in incidence of this finding at low degree of both sexes was regarded as test item-related and in absence of any other test item-related lung finding, was considered not to be adverse.

No test item-related or toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, food consumption, functional observations, ophthalmoscopy, coagulation parameters and macroscopic examination). In conclusion, administration of Tall Oil by dietary administration for at least 90 days was generally well tolerated in Han Wistar rats at levels up to 15000 ppm and did not result in any adverse test item-related (morphologic) alterations. Therefore, based on these results, the No-Observed-Adverse-Effect Level (NOAEL) was considered to be 15000 ppm (which equates to approximate achieved doses of 1104 mg/kg/day in males and 1221 mg/kg/day in females).

Endpoint conclusion
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
chronic
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

In addition, data are also available for the constituent parts of DTO and related substances (unpublished laboratory studies and information published in the public domain). These are as follows -

Fatty acids: Three repeated dose toxicity studies are reported under the US EPA HPV program for TOFA. The study of longest duration, 90-days, is considered to be the key study for this endpoint. During the 90 day study administration of up to 25% (approximately 12,500 mg/kg bw/day) Tall Oil Fatty Acids (TOFA) in the diet of Charles River rats daily for 90 days did not produce any adverse effects. The NOAEL is greater than or equal to 12,500 mg/kg bw/day.

Resin acids and neutrals: A number of repeated dose toxicity studies (90-day and 2-year) are available in rats and dogs for Tall Oil Rosin and other related types of rosin. Although these tests do not meet current test guidelines in all respects, the overall lack of toxicity in two species with all product types indicates that further testing would not be appropriate. Only the rat data are discussed further here, since this is the preferred species for testing according to REACH. The following oral dietary studies have been performed: Tall oil Rosin (90 day, Calandra, 1960a, Kay 1962a), Gum rosin (90 day & 2 year, calandra 1960c, Kay 1962b), n-wood rosin (90 day & 2 year study, Calandra 1960d, Kay 1962c).Studies were performed at dose levels up to 1.0% in the diet, prepared as a suspension in corn oil prior to mixing with feed.

Examinations performed were: Body weight gain, mortality and abnormal behavioural reactions on a daily basis; Haematological studies and urine analysis; Liver and kidney function tests, gross and microscopic pathologic studies (comprising heart, aorta, trachea, lungs, liver, gall bladder, pancreas, oesophagus, stomach, small & large intestine, spleen, lymph nodes, kidney, urinary bladder, gonads, prostate, uterus, pituitary, adrenal gland, salivary gland, thyroid gland, parathyroid gland, skeletal muscle, bone marrow, peripheral nerve, spinal cord and brain).

In all cases, rats fed diets containing 1% test substance displayed a significant growth depression throughout the major part of the study. No adverse effects were apparent at lower dietary levels. The growth effect observed among animals at the 1% dietary level was attributable to the palatability of the test diet. Animal survival was not however influenced by the ingestion of the test material at any dose level. As a result, the reported NOAEL for the studies was 0.05% or 0.2% in the diet (equivalent to approximately 50 or 200 mg/kg/day), depending on the dose levels used. However, for the purposes of human hazard assessment, the reduced growth observed for the high dose group is not relevant, since this is related to exposure route. The NOAEL can therefore be considered to be >200 mg/kg/day for risk characterisation purposes.

Sterols: In a published study (Hepburn et al., 1999), phytosterol esters (PE) were added to the diet of male and female Wistar derived rats over a period of 90 days, to give concentrations of 0.16, 1.6, 3.2, and 8.1% (w/w) equivalent to phytosterol concentrations of 0.1, 1.0, 2.0, and 5.0 % (w/w) respectively. The results of this study showed there to be no clinical signs or effects on survival attributed to the administration of the test material. A NOEL of 8.1% (PE) was identified in the study, which is equivalent to a dose of 6.6 g/kg/bw/day PE or 4.1 g phytosterol/kg bw/day.

In summary,

Constituent type

Proportion in DTO (%)

NOAEL (mg/kg/day, rat)

Duration

Remarks

DTO

100

>1000

90 days

OECD 408

Fatty acids

66

≥12,500

90 days

Resin acids and neutrals

30

>200

2 year

Weight of evidence from a number of 90-day and 2-year studies

Sterols

-

≥4100

90 days

 

These data are sufficient to demonstrate that none of the constituents of DTO require classification for specific target organ toxicity following repeated oral exposure, and to allow quantitative Derived No Effect Level to be set for risk characterisation.

Justification for classification or non-classification

The available data for DTO and constituents of DTO indicate that classification for specific target organ toxicity is not required according to the criteria set out in Regulation EC No. 1272/2008.