Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

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. The NOEL for reproductive parameters was 5000 ppm based on a decrease in the number of implant sites (and hence number of pups born). Based on mean body weight and food consumption data over the duration of the study, this is equivalent to approximately 493 mg/kg/d for females. However, the differences compared to the control group were slight.

A Combined Repeat Dose Toxicity Study in Rats with Reproduction/Developmental Screening Test was conducted to GLP according to OECD Test Guideline 422 (Inveresk Research, 2002e). Groups of 10 male and 10 female Sprague-Dawley rats were fed a diet containing tall oil (CAS No 8002-26-4) at concentrations of 0, 1000, 5000 and 20,000 ppm. Males were treated for at least 4 weeks, starting from 2 weeks prior to mating; females were treated from 2 weeks prior to mating until at least Day 6 of lactation. The only indication of reproductive toxicity was a decrease in implant sites at 20,000 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. Litter survival, as indicated by the birth index and viability index, was similar in all groups. There were no histology findings attributed to treatment. The NOEL for reproductive parameters was 5000 ppm. Based on mean body weight and food consumption data over the duration of the study, this is equivalent to approximately 423 mg/kg/d for females.

The OECD 422 study (reliability score 1) was the only study available for DTO for this endpoint. However, data are also available for the constituent parts of DTO and related substances (unpublished laboratory studies and information published in the public domain). The available data are summarised in table 5.13 below.

Table 5.13. Summary of reproductive toxicity data for constituents of DTO

Constituent type

Proportion in DTO (%)

NOAEL mg/kg/d (rat)

Remarks

DTO

100

423

OECD 422 study

Fatty acids

66

≥5000

OECD 416

Resin acids and neutrals

30

275

OECD 421

Sterols

-

>1500

Similar to OECD 416. Calculated as phytosterol.

These data are sufficient to demonstrate that none of the constituents of DTO require classification for reproductive toxicity. Any effects that were observed on reproductive parameters in the available studies were minor and associated with dose levels where systemic effects were observed.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
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:
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.
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 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
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 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
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 10 weeks
Remarks:
Doses / Concentrations:
1000 ppm
Basis: nominal in diet
Remarks:
Doses / Concentrations:
5000 ppm
Basis: nominal in diet
Remarks:
Doses / Concentrations:
20000
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 positive control
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
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
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.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): The achieved intake in the first week of treatment for males and females at 20000 ppm was lower than the second week. For males and females at the low and intermediate dose levels, intake was higher than in the following weeks (as expected). At other times, the achieved intake was essentially proportional to the diet concentrations.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): Not examined


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): Not examined


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): 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.

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 ppm (nominal)
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.
Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING): Litter survival, as indicated by the birth index and viability index, was similar in all groups.


CLINICAL SIGNS (OFFSPRING): No data


BODY WEIGHT (OFFSPRING): At 20000 p.p.m. mean litter weights were slightly reduced compared to the Controls, reflecting the decrease in litter size.


SEXUAL MATURATION (OFFSPRING): Not examined


ORGAN WEIGHTS (OFFSPRING): Not examined


GROSS PATHOLOGY (OFFSPRING): No abnormal findings.


HISTOPATHOLOGY (OFFSPRING): Not conducted
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
ca. 5 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: 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.
Reproductive effects observed:
not specified
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.

Effect on fertility: via oral route
Dose descriptor:
NOAEL
483 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The only available study for this endpoint is the OECD 422 study with DTO (Inveresk Research, 2002e). Groups of 10 male and 10 female Sprague-Dawley rats were fed a diet containing tall oil (CAS No 8002-26-4) at concentrations of 0, 1000, 5000 and 20,000 ppm. Males were treated for at least 4 weeks, starting from 2 weeks prior to mating; females were treated from 2 weeks prior to mating until at least Day 6 of lactation. The only indication of reproductive toxicity was a decrease in implant sites at 20,000 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. Litter survival, as indicated by the birth index and viability index, was similar in all groups. There were no histology findings attributed to treatment. The NOEL for reproductive parameters was 5000 ppm. Based on mean body weight and food consumption data over the duration of the study, this is equivalent to approximately 423 mg/kg/d for females.

Data are also available for the constituent parts of DTO and related substances (unpublished laboratory studies and information published in the public domain). The available data are summarised in the table below.

Tall oil Fatty Acid - In a reproductive/developmental toxicity study Sprague-Dawley rats (30 female and 15 male/dose) were administered TOFA (CAS 61790-12-3) via the diet at 0, 5 and 10 % (~ 0, 2500 and 5000 mg/kg-bw/day) starting at 20 days pre-mating (parental generation) and continuing through weaning of the F1 generation. After weaning, 20 F1 males and 20 F1 females per group were maintained on the parental diet. At 100 days of age, these rats were mated and were allowed to deliver pups (F2). The F2 generation was followed until weaning. The results showed there to be no treatment-related effects on reproductive performance or development. NOAEL (systemic toxicity) ≥5000 mg/kg bw/day, NOAEL (developmental/reproductive toxicity) ≥5000 mg/kg bw/day

Sterols -In a published study (Waalkens-Beredsenet al.1999), phytosterol esters 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.There were no effects on reproduction, on the development of the offspring, or on sexual maturation.A NOEL of 8.1% (PE) was identified in the 90 day study, which is equivalent to a dose of 6.6 g/kg/bw/day PE or 4.1 g phytosterol/kg bw/day.

Rsin acids and other neutrals - In a Robust summary report (PCA 2004) which outlined a reliable screening reproductive/developmental study by Clubb S and Sutherland JR (2002; rel 1) four groups of 10 male and 10 female Sprague-Dawley rats received rosinviathe diet at concentrations of 0, 1000, 3000 and 10000 ppm; the approximate doses were 0, 105, 275, or 825 mg/kg/day. No treatment related effects were observed in the pups at any dose level. The NOAEL for adults was considered to be 1000 ppm (105 mg/kg/day), due to slight weight changes at 3000 ppm and the NOAEL for reproductive/developmental effects was 3000 ppm (275 mg/kg/day).

Constituent type

Proportion in DTO (%)

NOAEL mg/kg/d (rat)

Remarks

DTO

100

423

OECD 422 study

Fatty acids

66

≥5000

OECD 416

Resin acids and neutrals

30

275

OECD 421

Sterols

-

>1500

Similar to OECD 416. Calculated as phytosterol.

 

These data are sufficient to demonstrate that none of the constituents of DTO require classification for reproductive toxicity. Any effects that were observed on reproductive parameters in the available studies were minor and associated with dose levels where systemic effects were observed.

Effects on developmental toxicity

Description of key information

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. No effects on developmental parameters were observed up to the highest dose tested, therefore the NOAEL for developmental toxicity is at least 20,000 ppm in the diet.

The OECD 422 study (reliability score 1) was the only study available for DTO for this endpoint. However, data are also available for the constituent parts of DTO and related substances (unpublished laboratory studies and information published in the public domain). The available data are summarised in the table below.

 

Table 5.14: Summary of developmental toxicity data for constituents of DTO

Constituent type

Proportion in DTO (%)

NOAEL mg/kg/d (rat)

Remarks

DTO

100

423

OECD 422 study

Fatty acids

66

≥5000

OECD 416

Resin acids and neutrals

30

275

OECD 421

Sterols

-

>2400

OECD 414

Fatty acids

No pre-natal developmental studies have been identified for Tall Oil Fatty Acids. However, no abnormalities were detected in either the F1 or the F2 pups in a 2-generation reproductive toxicity study (see Section 4.1). There is no concern regarding developmental toxicity for the fatty acid constituents of CTO and CTOS.

 

Sterols

The available developmental toxicity study was carried out using stanol esters (i.e. the hydrogenated form of the sterol). Both stanols and sterols are reported to have the same cholesterol-lowering properties. Given the very close structural similarity between the stanols and sterols, the mode of action for any developmental toxicity could reasonably be expected to be the same. In the absence of developmental effects both in this study and in a 2-generation reproductive toxicity study with sterol esters, it should be acceptable to read across the result on stanol esters to the corresponding sterol esters.

 

These data are sufficient to demonstrate that none of the constituents of DTO require classification for developmental toxicity. Any effects that were observed on developmental parameters in the available studies were minor and associated with dose levels where systemic effects were observed.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
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:
equivalent or similar to guideline
Guideline:
other: OECD 422
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
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 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.
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
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.
Maternal 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


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.


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.
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
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.
Indices:
No further information
Historical control data:
No data
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.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 ppm (nominal)
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 20 000 ppm (nominal)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No signs of developmental toxicity observed.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 20 000 ppm (nominal)
Basis for effect level:
other: teratogenicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 20 000 ppm (nominal)
Basis for effect level:
other: fetotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 20 000 ppm (nominal)
Basis for effect level:
other: embryotoxicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
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 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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A Combined Repeat Dose Toxicity Study in Rats with Reproduction/Developmental Screening Test was conducted to GLP according to OECD Test Guideline 422 (Inveresk Research, 2002e), as discussed above for fertility. There were no obvious external abnormalities in any pups at any of the dose levels tested. A slight decrease in mean litter weight was noted at 20,000 ppm, reflecting the decrease in litter size at this dose level. The NOAEL for developmental toxicity was therefore at least 20,000 ppm.

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: In a reproductive/developmental toxicity study Sprague-Dawley rats (30 female and 15 male/dose) were administered TOFA (CAS 61790-12-3) via the diet at 0, 5 and 10 % (~ 0, 2500 and 5000 mg/kg-bw/day) starting at 20 days pre-mating (parental generation) and continuing through weaning of the F1 generation. After weaning, 20 F1 males and 20 F1 females per group were maintained on the parental diet. At 100 days of age, these rats were mated and were allowed to deliver pups (F2). The F2 generation was followed until weaning. The results showed there to be no treatment-related effects on reproductive performance or development.NOAEL (systemic toxicity) ≥5000 mg/kg bw/day.NOAEL (developmental/reproductive toxicity) ≥5000 mg/kg bw/day.

Sterols: In a published study (Waalkens-Beredsen et al. 1999), phytosterol esters 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. There were no effects on reproduction, on the development of the offspring, or on sexual maturation. A NOEL of 8.1% (PE) was identified in the 90 day study, which is equivalent to a dose of 6.6 g/kg/bw/day PE or 4.1 g phytosterol/kg bw/day.

Resin acids and other neutrals: In a Robust summary report (PCA 2004) which outlined a reliable screening reproductive/developmental study by Clubb S and Sutherland JR (2002; rel 1) four groups of 10 male and 10 female Sprague-Dawley rats received rosin (CAS No. 8050-09-7) viathe diet at concentrations of 0, 1000, 3000 and 10000 ppm; the approximate doses were 0, 105, 275, or 825 mg/kg/day. No treatment related effects were observed in the pups at any dose level. The NOAEL for adults was considered to be 1000 ppm (105 mg/kg/day), due to slight weight changes at 3000 pppm and the NOAEL for reproductive/developmental effects was 3000 ppm (275 mg/kg/day).

 

In summary,

Constituent type

Proportion in DTO (%)

NOAEL mg/kg/d (rat)

Remarks

DTO

100

423

OECD 422 study

Fatty acids

66

≥5000

OECD 416

Resin acids and neutrals

30

275

OECD 421

Sterols

-

>2400

OECD 414

 

These data are sufficient to demonstrate that none of the constituents of DTO require classification for developmental toxicity. Any effects that were observed on developmental parameters in the available studies were minor and associated with dose levels where systemic effects were observed.

Justification for classification or non-classification

Based on the available data, Distilled Tall Oil is not classified for reproductive or developmental toxicity according to the criteria set out in EU Regulation 1272/2008.

According to ECHA communication CCH-D-2114412576-50-01/F (attached in Section 13 of the dossier) received on July 09, 2018, OECD 443 and 414 (Rat and Rabbit) studies will commence in the near future. This section will be updated upon receipt of the final reports from the CRO.

Additional information