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Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21st March 2002 to 26th February 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
It was based on the version of the test guideline available at the time (Adopted 22nd March 1996). There are some differences in the information reported compared with stipulations in the current version of OECD 422 (Adopted 29th July 2016). These are: • Functional Observations not performed • Thyroid hormones not measured • Study terminated on PND6, rather than continuing until minimally PND13 • Anogenital distance was not reported There were no deviations from the study plan that impacted upon the integrity of the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Qualifier:
equivalent or similar 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.
Principles of method if other than guideline:
N/A
GLP compliance:
yes
Limit test:
no
Justification for study design:
The dose levels were selected and agreed with the Sponsor, following evaluation of existing toxicological data. This included data from a one-week dose range finding study in rats carried out under a separate contract and project number at Inveresk (Inveresk Project No. 493160).

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
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
At room temperature, in the dark, under nitrogen>

STABILITY UNDER TEST CONDITIONS
Not evaluated during study.

FORM AS APPLIED IN THE TEST
Batches of diet were prepared weekly.
An appropriate quantity of the test item was dissolved in a suitable volume of acetone. This solution was added to a suitable quantity of untreated diet, then mixed for ca 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 ca 20 min. 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.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat is a standard rodent species for the reproduction toxicity testing in animals required by regulatory authorities. The normal processes of reproduction in the rat are well documented in the test laboratory.
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 exposure:
The test material was administered orally because this is a possible route of exposure in humans. Administration via the diet is an established experimental routine in rats.

The dose levels were selected and agreed with the Sponsor, following evaluation of existing toxicological data. This included data from a one-week dose range finding study in rats carried out under a separate contract and project number at Inveresk (Inveresk Project No. 493160).
Details on mating procedure:
Pairing was on a one male to one female basis, within the same treatment group. Each female was transferred to the cage of an appropriate co-group male near the end of the working day, and remained there until mating was detected. Vaginal lavages were taken early each morning commencing on the day of pairing, until mating was detected, and the day of observation of a copulatory plug in situ and/or sperm in the lavage was designated Day 0 of gestation. Each female remained with its first designated male for a maximum of 7 consecutive nights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the formulated diets was undertaken regarding 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. The samples were analysed by the Inveresk Product Chemistry Laboratory using a method previously validated in the Inveresk laboratory under a separate protocol and contract (Inveresk Project No. 491836).
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
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 10 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Remarks:
Group 1 (control)
Dose / conc.:
1 000 ppm (nominal)
Remarks:
Group 2
Dose / conc.:
5 000 ppm (nominal)
Remarks:
Group 3
Dose / conc.:
20 000 ppm (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
Ten
Control animals:
yes, concurrent vehicle
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 positive control

Examinations

Parental animals: Observations and examinations:
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: During Week 5 of dosing for males and on Day 6 of lactation for females.
- Animals fasted: No
- How many animals: Five males and five females
- Parameters checked in table No.1 were examined.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical abnormalities.


GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following four weeks of treatment.
- Maternal animals: All surviving animals after four days of lactation.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 2 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- All of the F1 offspring.
- These animals were subjected to postmortem examinations: externally visible abnormalities only.


GROSS NECROPSY: None


HISTOPATHOLOGY / ORGAN WEIGTHS: None
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.
Reproductive indices:
Fertility Index (female) = Number Pregnant/Number Paired
Fertility Index (male) = Number Siring a Litter/Number paired
Gestation Index = Number bearing live pups/Number pregnant
Birth Index = Total number of pups born (live and dead)/number of implantation scars
Live Birth Index = Number of pups live on Day 0 of lactation/Total number born (live and dead)
Offspring viability indices:
Viability Index = Number of pups live on Day 4 of lactation/number live on Day 0

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
All clinical observations were considered to be consistent with those normally seen in rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
N/A
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 3-Table 5 for data.

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 obvious effects of treatment at 5000 or 1000 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
FOOD CONSUMPTION

Please see Table 6 to Table 8 for data.

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 pre-mating period. Consumption was also reduced during the first half of the gestation period, compared to the Control animals.

There were no obvious effects of treatment at 5000 or 1000 ppm.

COMPOUND INTAKE

Please see Table 9 for data.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 10 & Table 11 for data.

At 20000 ppm, there was a non-significant decrease In white blood cells in females. Any other intergroup differences were not considered to reflect an effect of treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 12 & Table 13 for data.

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 sjze, 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.
Endocrine findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All histology findings were typical of spontaneously arising background findings in rats of this strain and age.
Other effects:
not examined
Description (incidence and severity):
N/A

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
N/A
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
N/A
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 16 to Table 18 for data.

Mating performance was not affected by treatment. There were no obvious effects on the duration of gestation at any of the dose levels applied.

At 20000 ppm mean number of implant sites per pregnancy was marginally decreased and hence mean total number of pups born was lower than that of all other dose groups. However, due to the very slight differences compared to the Control group, there is some doubt as to the reproducibility of this finding.

Details on results (P0)

N/A

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
ca. 1 000 ppm (nominal)
Based on:
test mat.
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.

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Description (incidence and severity):
N/A
Mortality / viability:
not specified
Description (incidence and severity):
N/A
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 19 for data.

At 20000 ppm, mean litter weights were slightly reduced compared to the Controls, reflecting the decrease In litter size.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
N/A
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Sexual maturation:
not examined
Description (incidence and severity):
N/A
Anogenital distance (AGD):
not examined
Description (incidence and severity):
N/A
Nipple retention in male pups:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
N/A
Gross pathological findings:
no effects observed
Description (incidence and severity):
N/A
Histopathological findings:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined
Description (incidence and severity):
N/A

Details on results (F1)

N/A

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 20 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 3                Body Weights & Body Weight Gain (g). Male



































































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



BODY WEIGHT



DAY 0



MEAN


SD


N



320


17


10



313


9


10



316


11


10



313


10


10



DAY 7



MEAN


SD


N



374


26


10



369


16


10



373


16


10



351**


13


10



DAY 14



MEAN


SD


N



414


34


10



408


17


10



415


20


10



386*


18


10



DAY 21



MEAN


SD


N



447


37


10



437


19


10



444


26


10



413**


22


10



DAY 28



MEAN


SD


N



468


45


10



460


23


10



471


28


10



434


29


10



BODY WEIGHT GAIN



DAY 0 to DAY 28



MEAN


SD


N



149


30


10



147


16


10



154


20


10



121*


24


10



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 4                Body Weights & Body Weight Gain (g). Female – Pre-mating



















































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



BODY WEIGHT



DAY 0



MEAN


SD


N



194


9


10



199


15


10



197


8


10



188


9


10



DAY 7



MEAN


SD


N



217


13


10



221


18


10



219


10


10



200**


9


10



DAY 14



MEAN


SD


N



237


16


10



240


22


10



237


10


10



214**


15


10



BODY WEIGHT GAIN



DAY 0 to DAY 14



MEAN


SD


N



43


9


10



41


12


10



40


4


10



25**


12


10



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 5                Mean Body Weights & Mean Body Weight Gain (g). Female – Gestation and Lactation














































































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



BODY WEIGHT



DAY 0


Of gestation



MEAN



245



247



240



218



DAY 7


Of gestation



MEAN



286



285



275



256



DAY 14


Of gestation



MEAN



329



320



317



288



DAY 20


Of gestation



MEAN


 



410



407



388



345



BODY WEIGHT GAIN



DAY 0 to DAY 20



MEAN


(% of Control)



165


-



160


97



148


90



127


77



BODY WEIGHT



DAY 1


Of lactation



MEAN



298



286



273



261



DAY 4


Of Lactation



MEAN



319



313



300



281



 


 


Table 6                Food Consumption (g/animal/day). Male





































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



DAYS 0-7



MEAN


SD


N (CAGE)



31.5


2.2


5



30.9


2.0


5



32.6


1.6


5



27.2**


2.2


5



DAYS 7-14



MEAN


SD


N (CAGE)



35.5


3.9


5



34.1


1.9


5



34.9


1.3


5



32.1


2.4


5



DAYS 21-28



MEAN


SD


N (CAGE)



32.0


2.8


5



31.5


1.5


5



32.4


2.5


5



29.9


1.9


5



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 7                Food Consumption (g/animal/day). Female – pre-mating





























 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



DAYS 0-7



MEAN


SD


N (CAGE)



21.1


1.0


5



21.7


1.2


5



20.5


2.0


5



17.2***


1.3


5



DAYS 7-14



MEAN


SD


N (CAGE)



22.9


1.3


5



22.6


1.1


5



21.6


1.5


5



20.3**


1.5


5



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 8                Mean Food Consumption (g/animal/day). Female – Gestation and Lactation













































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



DAYS 0-7


Of gestation



MEAN



26.9



26.5



26.2



23.9



DAYS 7-14


Of gestation



MEAN



29.9



30.0



27.9



26.7



DAYS 14-20


Of gestation



MEAN



31.8



32.8



32.6



30.1



DAYS 0-4


Of lactation



MEAN



33.9



31.9



31.6



30.1



 


Table 9                Group Mean Achieved Dosage of Test Item (mg/kg/day)








































































 



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



MALES



WEEK 1



91



473



1639



WEEK 2



88



443



1742



WEEK 4



70



354



1412



FEMALES



WEEK 1



103



493



1773



WEEK 2



98



474



1961



DAYS 0-7


(OF GESTATION)



100



509



2017



DAYS 7-14


(OF GESTATION)



99



471



1963



DAYS 14-20


(OF GESTATION



90



462



1902



DAYS 1-4


(OF LACTATION)



107



551



2221



 


Table 10             Haematology, Week 5. Males






































































































































 



 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



Hb



MEAN


SD


N



16.1


0.6


5



16.1


0.4


5



15.5


0.1


5



15.7


0.5


5



RBC



MEAN


SD


N



8.28


0.32


5



8.34


0.23


5



7.95


0.27


5



8.10


0.25


5



Hct



MEAN


SD


N



0.453


0.019


5



0.452


0.014


5



0.442


0.004


5



0.440


0.015


5



MCH



MEAN


SD


N



19.5


0.4


5



19.3


0.5


5



19.5


0.7


5



19.4


0.3


5



MCV



MEAN


SD


N



54.7


1.5


5



54.2


1.4


5



55.5


1.7


5



54.3


0.9


5



MCHC



MEAN


SD


N



35.6


0.3


5



35.7


0.4


5



35.1*


0.2


5



35.7


0.4


5



WBC



MEAN


SD


N



14.50


2.21


5



14.33


2.85


5



15.82


4.05


5



12.97


3.93


5



Neut



MEAN


SD


N



1.54


0.37


5



2.30


1.46


5



1.57


0.49


5



1.49


0.74


5



Lymp



MEAN


SD


N



12.39


1.96


5



11.41


3.59


5



13.71


4.23


5



10.91


3.02


5



Mono



MEAN


SD


N



0.25


0.07


5



0.28


0.10


5



0.20


0.02


5



0.22


0.11


5



Eos



MEAN


SD


N



0.19


0.05


5



0.17


0.07


5



0.16


0.07


5



0.15


0.10


5



Baso



MEAN


SD


N



0.04


0.02


5



0.04


0.02


5



0.06


0.03


5



0.04


0.02


5



LUC



MEAN


SD


N



0.09


0.03


5



0.12


0.03


5



0.12


0.05


5



0.16


0.10


5



Plat



MEAN


SD


N



1023


102


5



975


122


5



967


119


5



994


195


5



PT



MEAN


SD


N



13


1


5



13


1


5



13


1


5



12*


1


5



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


 


Table 11             Haematology, Week 7. Females






































































































































 



 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



Hb



MEAN


SD


N



14.0


1.0


5



13.9


0.6


5



13.2


0.4


5



13.0


1.7


5



RBC



MEAN


SD


N



7.30


0.46


5



7.13


0.31


5



6.88


0.29


5



6.87


0.85


5



Hct



MEAN


SD


N



0.394


0.029


5



0.390


0.021


5



0.370


0.014


5



0.365


0.048


5



MCH



MEAN


SD


N



19.1


0.6


5



19.5


0.2


5



19.2


0.4


5



19.0


0.4


5



MCV



MEAN


SD


N



54.0


1.4


5



54.7


1.3


5



53.8


1.3


5



53.2


1.6


5



MCHC



MEAN


SD


N



35.4


0.6


5



35.7


0.7


5



35.7


0.4


5



35.7


0.3


5



WBC



MEAN


SD


N



12.99


3.46


5



12.79


3.85


5



12.15


2.84


5


 



8.89


2.28


5



Neut



MEAN


SD


N



3.84


1.68


5



3.30


1.19


5



3.62


1.09


5



2.44


0.98


5



Lymp



MEAN


SD


N



8.55


2.89


5



8.78


2.85


5



7.99


2.05


5



6.04


1.83


5



Mono



MEAN


SD


N



0.28


0.13


5



0.34


0.22


5



0.26


0.04


5



0.15*


0.04


5



Eos



MEAN


SD


N



0.18


0.07


5



0.15


0.07


5



0.14


0.03


5



0.17


0.17


5



Baso



MEAN


SD


N



0.03


0.03


5



0.04


0.03


5



0.03


0.01


5



0.02


0.01


5



LUC



MEAN


SD


N



0.11


0.06


5



0.17


0.10


5



0.12


0.07


5



0.08


0.02


5



Plat



MEAN


SD


N



1073


133


5



1061


145


5



1110


170


5



906


478


5



PT



MEAN


SD


N



14


1


5



14


1


4



14


1


4



13


2


3



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 12             Clinical Chemistry, Week 5. Male














































































































































 



 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



Urea



MEAN


SD


N



7.3


0.5


5



7.4


1.1


5



6.5


0.5


5



6.4


0.6


5



Glu



MEAN


SD


N



7.92


0.42


5



7.95


0.76


5



8.12


0.88


5



7.90


0.29


5



AST



MEAN


SD


N



86


8


5



92


15


5



92


6


5



83


5


5



ALT



MEAN


SD


N



59


2


5



71


8


5



70


11


5



65


9


5



AP



MEAN


SD


N



683


39


5



699


137


5



842


183


5



1891**


905


5



Na



MEAN


SD


N



144


1


5



144


2


5



143


2


5



142


3


5



k



MEAN


SD


N



4.9


0.2


5



5.0


0.1


5



4.9


0.3


5



5.0


0.2


5



Cl



MEAN


SD


N



101


1


5



102


2


5



101


2


5



101


4


5



TP



MEAN


SD


N



68


2


5



70


2


5



66


2


5



66


2


5



Alb



MEAN


SD


N



41


1


5



43


1


5



41


1


5



41


2


5



AG-R



MEAN


SD


N



1.6


0.2


5



1.6


0.1


5



1.6


0.3


5



1.7


0.1


5



Chol



MEAN


SD


N



2.0


0.4


5



1.9


0.3


5



2.0


0.3


5



2.4*


0.1


5



Crea



MEAN


SD


N



51


2


5



53


2


5



52


3


5



56


4


5



Ca



MEAN


SD


N



2.91


0.06


5



2.95


0.07


5



2.94


0.07


5



2.94


0.07


5



Phos



MEAN


SD


N



2.01


1.13


5



2.58


0.09


5



2.60


0.14


5



2.08


1.17


5



T. Bi



MEAN


SD


N



0.6


0.2


5



1.0


0.6


5



0.9


0.2


5



1.7***


0.4


5



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 13             Clinical Chemistry, Week 7. Female














































































































































 



 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



Urea



MEAN


SD


N



10.7


1.2


5



11.1


2.7


5



11.8


1.2


5



10.5


0.7


5



Glu



MEAN


SD


N



6.68


0.90


5



6.65


0.70


5



6.00


0.35


5



6.44


0.77


5



AST



MEAN


SD


N



96


17


5



88


17


5



98


8


5



84


14


5



ALT



MEAN


SD


N



105


23


5



114


31


5



128


28


5



113


21


5



AP



MEAN


SD


N



509


203


5



638


152


5



980*


378


5



1649***


929


5



Na



MEAN


SD


N



145


3


5



141


3


5



144


1


5



142


3


5



k



MEAN


SD


N



5.3


0.7


5



5.6


0.3


5



5.9


0.4


5



5.9


0.5


5



Cl



MEAN


SD


N



106


4


5



103


4


5



105


2


5



104


1


5



TP



MEAN


SD


N



64


1


5



65


4


5



62


2


5



59*


4


5



Alb



MEAN


SD


N



40


1


5



39


2


5



39


2


5



35***


1


5



AG-R



MEAN


SD


N



1.6


0.1


5



1.5


0.1


5



1.7


0.2


5



1.4*


0.1


5



Chol



MEAN


SD


N



2.4


0.5


5



2.2


0.5


5



1.9*


0.1


5



2.7


0.3


5



Crea



MEAN


SD


N



59


4


5



58


2


5



62


4


5



63


3


5



Ca



MEAN


SD


N



2.84


0.12


5



2.90


0.06


5



2.79


0.08


5



2.77


0.09


5



Phos



MEAN


SD


N



1.89


0.27


5



1.93


0.31


5



1.58


0.21


5



1.86


0.24


5



T. Bi



MEAN


SD


N



0.9


0.2


5



0.9


0.6


5



1.4


0.6


5



1.9**


0.5


5



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 14             Absolute Organ Weights (Following Covariance Analysis). Males





































































































































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



BODY WEIGHT



MEAN


SE


N



458


11


10



458


11


10



458


11


10



458


11


10



ADRENAL GLANDS



MEAN


SE


N



0.0757


0.0036


10



0.0803


0.0036


10



0.0674


0.0036


10



0.0634*


0.0038


10



BRAIN



MEAN


SE


N



2.10


0.03


10



2.12


0.03


10



2.12


0.03


10



2.08


0.03


10



EPIDIDYMIDES



MEAN


SE


N



1.2041


0.0292


10



1.2032


0.0287


10



1.2048


0.0291


10



1.2047


0.0311


10



HEART



MEAN


SE


N



1.72


0.06


10



1.82


0.06


10



1.73


0.06


10



1.72


0.06


10



KIDNEYS



MEAN


SE


N



3.77


0.10


10



3.85


0.10


10



4.01


0.10


10



4.07


0.11


10



LIVER



MEAN


SE


N



17.88


0.53


10



18.88


0.52


10



19.46*


0.53


10



20.12**


0.56


10



LUNG



MEAN


SE


N



1.82


0.06


9



1.86


0.05


10



1.76


0.05


10



1.75


0.06


10



PITUITARY GLAND



MEAN


SE


N



0.0013


0.0001


10



0.012


0.001


10



0.013


0.001


10



0.011


0.001


10



PROSTATE



MEAN


SE


N



0.771


0.046


10



0.777


0.045


10



0.704


0.046


10



0.761


0.049


10



SPLEEN



MEAN


SE


N



0.85


0.03


10



0.83


0.03


10



0.85


0.03


10



1.04***


0.03


10



SALIVARY GLANDS



MEAN


SE


N



0.7770


0.0240


10



0.7572


0.0236


10



0.7501


0.0239


10



0.7978


0.0255


10



TESTES



MEAN


SE


N



3.55


0.08


10



3.51


0.07


10



3.69


0.08


10



3.74


0.08


10



THYMUS



MEAN


SE


N



0.491


0.032


10



0.415


0.031


10



0.435


0.032


10



0.385


0.034


10



THYROID GLANDS



MEAN


SE


N



0.0219


0.0010


10



0.0233


0.0009


10



0.0225


0.0010


9



0.0230


0.0010


10



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 15             Absolute Organ Weights (Following Covariance Analysis). Females





























































































































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



BODY WEIGHT



MEAN


SE


N



310


7


10



310


7


10



310


7


10



310


8


8



ADRENAL GLANDS



MEAN


SE


N



0.0911


0.0037


10



0.0843


0.0032


10



0.0791


0.0032


10



0.0774


0.0042


8



BRAIN



MEAN


SE


N



1.87


0.02


10



1.87


0.02


10



1.90


0.02


10



1.96


0.03


8



HEART



MEAN


SE


N



1.12


0.04


10



1.16


0.04


10



1.25


0.04


10



1.18


0.05


8



KIDNEYS



MEAN


SE


N



2.59


0.05


10



2.53


0.05


10



2.47


0.05


10



2.53


0.06


8



LIVER



MEAN


SE


N



16.69


0.48


10



16.16


0.43


10



17.77


0.43


10



17.87


0.55


8



LUNG



MEAN


SE


N



1.32


0.04


9



1.38


0.03


10



1.29


0.03


10



1.37


0.04


8



OVARIES



MEAN


SE


N



0.114


0.005


10



0.106


0.004


10



0.104


0.004


10



0.097


0.006


8



PITUITARY GLAND



MEAN


SE


N



0.014


0.001


10



0.016


0.001


10



0.015


0.001


10



0.013


0.001


8



SPLEEN



MEAN


SE


N



0.59


0.03


10



0.63


0.03


10



0.68


0.03


10



0.72


0.03


8



SALIVARY GLANDS



MEAN


SE


N



0.6038


0.0208


10



0.6027


0.0184


10



0.6121


0.0185


10



0.5755


0.0238


8



THYMUS



MEAN


SE


N



0.198


0.024


10



0.233


0.021


10



0.201


0.021


10



0.242


0.028


8



THYROID GLANDS



MEAN


SE


N



0.0166


0.0010


9



0.0165


0.0009


10



0.0152


0.0009


10



0.0171


0.0011


8



UTERUS



MEAN


SE


N



0.53


0.02


10



0.53


0.02


10



0.51


0.02


10



0.50


0.03


8



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 16             Mating performance and fertility indices





































































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



Number of males paired



10



10



10



10



Number of siring males



10



10



10



8



Number of females paired



10



10



10



10



Pregnant females



10



10



10



8



Number of females producing a liver litter



10



10



10



8



Fertility Index, males (%)



100



100



100



80



Fertility Index, females (%)



100



100



100



80



Gestation Index (%)



100



100



100



100



 


 


Table 17             Implantation sites summary





























 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



IMPLANTATIONS



MEAN


SD


N



13.4


3.6


10



15.4


2.5


10



14.2


3.8


10



12.1


1.5


10


      

 


 


Table 18             Developmental data























































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



Mean number of live pups per litter ± SD, Day 0



12.4 ± 3.5



14.7 ± 2.5



13.1 ± 3.4



11.4 ± 1.7



Mean number of live pups per litter ± SD, Day 1



12.3 ± 3.3



14.0 ± 3.9



13.0 ± 3.4



11.4 ± 1.7



Mean number of live pups per litter ± SD, Day 4



12.3 ± 3.3



13.1 ± 4.7



13.0 ± 3.4



11.4 ± 1.7



Birth index (%)



90



96



93



97



Live birth index (%)



99



100



100



97



Viability index (%)



99



88



99



100



 


 


Table 19             Group Mean Litter and Pup Weight (g) ± SD


 



































































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



LITTER



Day 1



84 ± 17



85 ± 21



82 ± 16



79 ± 9



Day 4



126 ± 23



124 ± 50



121 ± 22



117 ± 12



Mean of Litter Mean Pup Weight



MALES



Day 1



7.2 ± 0.9



6.4 ± 0.9



6.7 ± 1.2



7.2 ± 0.8



Day 4



10.8 ± 2.0



9.7 ± 1.9



10.1 ± 2.2



10.7 ± 1.4



FEMALES



Day 1



6.9 ± 1.2



5.9 ± 0.8



6.2 ± 1.0



6.8 ± 0.6



Day 4



10.5 ± 2.2



9.0 ± 2.6



9.4 ± 1.7



10.2 ± 1.2



 

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. The NOAEL for reproductive toxicity was 5000 ppm.
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.

The only indication of reproductive toxicity was a marginal decrease in implant sites at 20000 ppm with a corresponding decrease in the mean total number of pups born compared to all other dose groups. However, due to the very slight differences compared to the Control group, there is some doubt as to the reproducibility of this finding. 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 NOAEL was considered to be 1000 ppm. For reproductive parameters the NOEL was considered to be 5000 ppm.