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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21.03.02 to 15.07.02
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 Type:
study report

Materials and methods

Test guideline
equivalent or similar to guideline
other: OECD 422
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:
Limit test:

Test material

Constituent 1
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

Details on test animals or test system and environmental conditions:
- 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

- 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
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: An appropriate quantity of the test substance was dissolved in a suitable volume of acetone.

- 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.

- 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:
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.
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.
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
Duration of test:
At least four weeks
Doses / concentrationsopen allclose all
Doses / Concentrations:
1000 ppm
Basis: nominal in diet
Doses / Concentrations:
5000 ppm
Basis: nominal in diet
Doses / Concentrations:
20000 ppm
Basis: nominal in diet
No. of animals per sex per dose:
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.


Maternal examinations:
- Time schedule: Daily

- Time schedule: Daily

- 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 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

- 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

- 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.

- 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.

- Male animals: All surviving animals following four weeks of treatment.
- Maternal animals: All surviving animals after four days of lactation.

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

The tissues indicated in Table 2 were prepared for microscopic examination and weighed, respectively.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:
- External examinations: Yes, gross external abnormalities only
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
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.
No further information
Historical control data:
No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There was a notable decrease in body weights throughout the pre-mating phase. The resulting deficit in body weight was never regained. In pregnant females reduced weight gain was evident over Day 7-20 of gestation, compared to the Control animals. 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.
At 20000 ppm there was a non-significant decrease in white blood cells. Alkaline phosphatase levels were significantly increased at 5000 and 20000 ppm, and total bilirubin was increased at 20000 ppm (indicative of effects on liver function). In addition, at 20000 ppm albumin (and consequently total protein) was reduced. Small decreases were noted in adrenal gland and ovary weights at 20000 ppm. At 5000 ppm adrenal gland weight was reduced.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
Effect level:
ca. 1 000 ppm (nominal)
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
Effect level:
>= 20 000 ppm (nominal)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No signs of developmental toxicity observed.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
Effect level:
>= 20 000 ppm (nominal)
Basis for effect level:
other: teratogenicity
Key result
Dose descriptor:
Effect level:
>= 20 000 ppm (nominal)
Basis for effect level:
other: fetotoxicity
Key result
Dose descriptor:
Effect level:
>= 20 000 ppm (nominal)
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Key result
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:

Applicant's summary and conclusion

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 developmental toxicity was >= 20000 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. There were no external abnormalities in the offspring at any dose. 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 developmental toxicity parameters the NOAEL was considered to be >= 20000 ppm.