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Repeated dose toxicity: oral

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
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

Test material

Constituent 1
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Tall Oil
- Substance type: Complex mixture
- Physical state: dark brown liquid
- Analytical purity: No data
- Composition of test material, percentage of components: Not supplied
- Purity test date: No data
- Lot/batch No.: 7252-30
- Expiration date of the lot/batch: 15.05.05
- Stability under test conditions: No data
- Storage condition of test material: Room temperature, dark, under nitrogen

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.