Tall oil, sodium salt

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No reproductive toxicity data are available for the registration substance; therefore this endpoint is addressed by a weight of evidence approach for relevant groups of constituents present in the substance.

In a one-generation reproductive toxicity study (Pharmacopathics Research Laboratories, 1977), conducted using a protocol that appears to be similar to the current OECD Test Guideline 415, but pre-dating GLP, a NOAEL was not determined for tall oil fatty acids. It is the reviewer's opinion that the NOAEL can be established as the highest dose tested for F1 animals based on no adverse effects observed. Therefore, the NOAEL is ≥5000 mg/kg bw/day tall oil fatty acids for general and reproductive/developmental toxicity.

In a dietary two-generation reproductive toxicity study on phytosterol esters, conducted using a protocol comparable with OECD 416 and in compliance with GLP (Waalkens-Berendsen et al., 1999), no adverse effects were reported for general systemic toxicity, or reproductive and developmental toxicity in any generation. The highest concentration tested was 8.1% (w/w) phytosterol ester in diet. This gave a NOAEL of 2500-9100 mg/kg bw/day phytosterol ester or 1540-5620 mg/kg bw/day phytosterol.

In a supporting Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test (Inveresk, 2002) conducted to OECD 422 and in compliance with GLP, for distilled tall oil (DTO), the reported no observed effect level (NOEL) for reproductive toxicity was 5000 ppm for both male and female rats. The NOEL was based on 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, there was doubt over the reproducibility of this finding and therefore the NOAEL was 20000 ppm.

An extended one-generation reproductive toxicity study is planned, according to OECD Test Guideline 443, for tall oil rosin (basic test design (Cohorts 1A, and 1B without extension)). When the results of this study are available, their relevance for the assessment of TOS will be considered and the dossier will be updated accordingly.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 March 2002 to 15 July 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature, in the dark, under nitrogen
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The test material was dissolved in acetone.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 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.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species:

rat

Strain:

other: Crl: CD®(SD) IGS BR strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: 180-190 g for males and 113-161 g for females
- Fasting period before study:
- Housing: The animals were housed 2 per cage initially, in polypropylene cages. Male and female cages were racked separately. A few days prior to pairing for mating, males were transferred to individual grid-bottomed cages. Mated females were transferred to individual solid bottomed cages.
- Diet (e.g. ad libitum): Rat and Mouse Breeder diet, ad libitum.
- Water (e.g. ad libitum): domestic mains water ad libitum
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY: No contamination of toxicological significance was reported.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 2 °C
- Humidity (%): 50 +/- 15%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

Route of administration:

oral: feed

Vehicle:

acetone

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 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.

DIET PREPARATION
- Rate of preparation of diet (frequency): Batches of diet were prepared weekly.
- Mixing appropriate amounts with (Type of food): Rat and Mouse Breeder diet
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): acetone

Details on mating procedure:

- M/F ratio per cage: one male to one female
- Length of cohabitation: maximum of 7 consecutive nights.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how): Mated females were transferred to individual solid bottomed cages.
- Any other deviations from standard protocol: no

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. 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.

Duration of treatment / exposure:

The males were treated for at least 4 weeks overall, starting from 2 weeks prior to mating until termination. The females were treated for 2 weeks prior to mating, then through mating until termination after Day 4 of lactation.

Frequency of treatment:

Continuous

Details on study schedule:

- parental animals not mated until 2 weeks after group allocation.
- Age at mating of the mated animals in the study: 10 weeks

Dose / conc.:

0 ppm

Dose / conc.:

1 000 ppm

Dose / conc.:

5 000 ppm

Dose / conc.:

20 000 ppm

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not specified
- Rationale for animal assignment (if not random): random

Positive control:

not used

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked included: All the animals were examined for reaction to treatment. The nature, onset, duration and intensity of any signs were recorded. In addition, all the animals were checked for viability early in the morning and again as late as possible on each day.

DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: Male weights were recorded once during the week prior to the commencement of dosing and once weekly thereafter until termination. Female weights were recorded once during the week prior to the commencement of dosing, and weekly thereafter until the start of the mating period, and 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 = 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/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- For all animals, the weight of food consumed by each cage was recorded once weekly commencing during the week prior to the commencement of dosing, until pairing for mating. All animals were fed ad libitum during mating, but following completion of mating weekly consumption was recommenced for males, until termination. For mated females, the amount of food consumed was recorded over Days 0-7, 7-14 and 14-20 of gestation, and Days 0-4 of lactation.

Oestrous cyclicity (parental animals):

Not specified

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations: testis weight and epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

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

GROSS EXAMINATION OF DEAD PUPS: Yes

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals at the end of 4 weeks of treatment.
- Maternal animals: All surviving animals at day 4 of lactation

GROSS NECROPSY
- See table 1 fo gross necropsy

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1] were prepared for microscopic examination and weighed, respectively. Histological examination was conducted on Control and High dose animals only, the same animals that were used for Laboratory Investigations.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations as follows: macroscopic, external

GROSS NECROPSY
- Gross necropsy was not performed.

HISTOPATHOLOGY / ORGAN WEIGTHS: Not performed

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 as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate.
Histology incidence data were analysed using Fisher’s Exact Probability Test.

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)
Viability Index = Number of pups live on Day 4 of lactation/Number live on Day 0

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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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 720 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):

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.
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.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

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):

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.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no histology findings attributed to treatment.
All histology findings were typical of spontaneously arising background findings in rats of this strain and age.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

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.

Dose descriptor:

NOEL

Effect level:

5 000 ppm

Based on:

test mat.

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.

Critical effects observed:

not specified

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Litter survival, as indicated by the birth index and viability index, was similar in all groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no obvious external abnormalities noted in the pups at any of the dose levels applied.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOEL

Generation:

F1

Effect level:

5 000 ppm

Based on:

test mat.

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.

Critical effects observed:

not specified

Reproductive effects observed:

not specified

See attachment for table of results.

Conclusions:

In the Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test for Tall Oil, the reported NOEL for reproductive toxicity was 5000 ppm for both male and female rats. The NOEL was based on 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. The study was conducted according to appropriate OECD Test Guideline and in compliance with GLP.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

Additional obs of oestrous cycling, sexual maturation & histo examinations. No total cauda epididymal sperm no., % progressively mobile sperm, % progressively motile sperm, % morphologically normal sperm & % of sperm with each identified abnormality

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany.
- Females, nulliparous and non-pregnant: yes
- Age at study initiation: (P) 4-5 x weeks (on receipt); (F1) x weeks
- Weight at study initiation: (P) Males: approx. 210 g; Females: approx. 125 g; (F1) Males: approx. 125 g; Females: approx. 112.5 g
- Fasting period before study: No data
- Housing: suspended stainless-steel group cages each with a wire mesh floor and front.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 40-70
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: No data

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): At least every four weeks.
- Mixing appropriate amounts with (Type of food): Commercial rodent diet.
- Storage temperature of food: 2-10°C

Details on mating procedure:

At the end of the 10 week premating period:

- M/F ratio per cage: 1:1
- Length of cohabitation: three weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- The mated F0 females were housed individually in suspended wire mesh cages with wire mesh floor and front.
- mated males were returned to their group cages (four animals per cage).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Several batches of diet were analysed to study homogeneity, stability and content of phytosterol ester.

Phytosterol ester was extracted from the diet with petroleum ether. The concentration of phytosterol ester in thc extracts was determined using HPLC with diode array detection (DAD) at 210 nm. Quantification of phytosterol ester was obtained by comparing the areas of the phytosterol peaks in the samples with those of calibration solutions containing known amounts of phytosterol ester.

Duration of treatment / exposure:

Approximately 32 weeks of treatment

Frequency of treatment:

Continuous in diet

Details on study schedule:

- F1 parental animals not mated until 12 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: at least 15 weeks.

The same procedure was used for the F1 to F2 generation.

Dose / conc.:

1.6 other: % (w/w) phytosterol ester

Remarks:

Equivalent to a concentration of 1.0% (w/w) phytosterol

Dose / conc.:

3.2 other: % (w/w) phytosterol ester

Remarks:

Equivalent to a concentration of 2.0% (w/w) phytosterol

Dose / conc.:

8.1 other: % (w/w) phytosterol ester

Remarks:

Equivalent to a concentration of 5.0% (w/w) phytosterol.
Equivalent to 2500-9100 mg/kg bw/day phytosterol ester.
Equivalent to 1540-5620 mg/kg bw/day phytosterol.

No. of animals per sex per dose:

28

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose used was the same as previously conducted 90-day dietary study.
- Rationale for animal assignment (if not random): Random

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for all animals in the premating period; males weekly thereafter; females on gestation days 0, 7, 14 and 21 and lactation days 1, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

Approximately one week after weaning of their pups, vaginal smears were made from the F0 females for 14 consecutive days. In the F1 animals vaginal opening was scored on post natal day 39.

Sperm parameters (parental animals):

Parameters examined in F0 and F1 male parental generations: testis weight, epididymis weight, prostate weight, seminal vesicles weight, preputial separation was scored in F1 animals on post natal day 31.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: none

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: none

Postmortem examinations (parental animals):

SACRIFICE
At or shortly after weaning 10 male and 10 female F0 and F1 pups per group were subjected to a thorough necropsy. The following tissues were preserved for microscopic examination: ovaries, uterus, vagina, testes, epididymides, seminal vesicles (with coagulating glands and their fluids), prostate, pituitary, adrenals, liver and organs or tissues showing macroscopic abnormalities.

At termination all surviving parent animals of the F0 and F1 generations were killed and the following organs and tissues preserved for microscopic examination: adrenals.
brain, epididymides, liver, ovaries, pituitary, prostate, seminal vesicles with coagulating glands, testes, uterus with cervix (after counting implantation sites), vagina and all gross lesions.

Postmortem examinations (offspring):

On post natal day 21, the F1 pups were weaned and shortly after 28 males and 28 females were selected at random from as many litters as possible in each group to produce the next generation - these animals were approximately 5 weeks old at the beginning of the F1 premating period. Mating of siblings was avoided. From the remaining F1 pups, 10 male and 10 female pups were necropsied thoroughly. The remaining pups were sacrificed after an external examination only. The same procedure was used for the F2 generation.

Statistics:

Body weight, food consumption and food efficiency data were subjected to one way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. Fisher's exact probability test was used to evaluate the numbers of mated and pregnant females, females with liveborn pups, females surviving delivery, females with (all) stillborn pups. live born and stillborn pups, pups lost at various stages, pups surviving 21 days, and male pups on postnatal days 1 and 21. Duration of gestation, litter size, and number of implantation sites per litter were evaluated by Kruskal Wallis non-parametric analysis of variance followed by Mann-Whitney U test. For evaluation of the incidence of pathological changes and clinical signs, Fisher's exact probability test was used. The time of achievement of preputial separation and vaginal opening was analysed with ANOVA followed by Dunnett's multiple comparison test. The chi -square test was used to evaluate the length of oestrus.

Reproductive indices:

For the assessment of fertility and reproductive performance the mating, fertility, fecundity, gestation and live births indices were calculated, and the sex ratio of pups was determined.

Offspring viability indices:

Viability and lactation indices were calculated.

Clinical signs:

no effects observed

Description (incidence and severity):

No further details available.

Mortality:

no mortality observed

Description (incidence):

No further details available.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males: decreases were observed in weeks 2-7 and on occasions in weeks 5-15 and 18-19 in the high dose group.
Females: no statistically significant differences.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were inconsistent differences (increases and decreases) between control and treated groups.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related observations (no further details available).

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 8.1 other: % (w/w) phytosterol ester; equivalent to 2500-9100 mg/kg bw/day phytosterol ester, or 1540-5620 mg/kg bw/day phytosterol

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No further details available.

Mortality:

no mortality observed

Description (incidence):

No further details available.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males: decreases were observed in weeks 2-7 and on occasions in weeks 5-15 and 18-19 in the high dose group, and decreases in weeks 6-9 and 11-14 of the low dose group. These changes were statistically significant.
Females: no statistically significant differences, except for day 7 of lactation in the high dose F1 generation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were inconsistent differences (increases and decreases) between control and treated groups.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In males there was a small but statistically significant increase in the relative brain weight in low and mid dose groups only.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No further details available.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No further details available.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 8.1 other: % (w/w) phytosterol ester; equivalent to 2500-9100 mg/kg bw/day phytosterol ester or 1540-5620 phytosterol

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No further details available.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Pup mortality was increased on post natal day 4. Although the differences reached statistical significance, the values were within the historical control limits.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No further details available.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was an inconsistent pattern of food consumption. The findings were not adverse.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 8.1 other: % (w/w) phytosterol ester; equivalent to 2500-9100 mg/kg bw/day phytosterol ester and 1540-5620 mg/kg bw/day phytosterol

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Pup mortality was increased in the mid and high dose groups compared with the controls; however, the values fell within the historical control limits.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

>= 8.1 other: % (w/w) phytosterol ester; equivalent to 2500-9100 mg/kg bw/day phytosterol ester and 1540-5620 mg/kg bw/day phytosterol

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Table 1 Intake of phytosterol ester in parent animals

			Phytosterol esters (% in diet)
Generation	Sex	Period	1.6	3.2	8.1
			Phytosterol ester (g/kg bw/day)
F0	Males	Premating weeks 1 -10	0.9 -0.5	1.8 -1.6	4.4 -2.7
	Females	Premating weeks 1 -10	1.1 -0.6	2.1 -1.3	5.6 -3.4
		Gestation week 1	0.8	1.5	3.8
		Gestation week 2	0.8	1.5	3.9
		Gestation week 3	0.5	1.0	2.5
		Lactation week 1	1.1	2.1	5.7
		Lactation week 2	1.8	3.5	9.1
		Lactation week 3*	2.3	4.5	12.6
F1	Males	Premating weeks 1 -10	1.3 -0.6	2.6 -1.1	6.2 -2.8
	Females	Premating weeks 1 -10	1.3 -0.6	2.6 -1.2	6.5 -3.3
		Gestation week 1	0.7	1.4	3.6
		Gestation week 2	0.7	1.4	3.6
		Gestation week 3	0.5	0.9	2.3
		Lactation week 1	1.0	2.1	4.8
		Lactation week 2	1.7	3.4	8.5
		Lactation week 3*	2.1	4.4	10.9

* As pups started to eat on approximately post natal day 14, test substance intake in lactation week 3 is unrealistic.

Values are group means; during premating, n=7 cages/group/sex; during gestation and lactation, n=19 -27 female rats/group.

Table 2 Reproductive performance of female rats

		Phytosterol esters (% of diet)
Parameter	Generation	0	1.6	3.2	8.1
Mating index (%)^1	F0	100	100	100	100
	F1	100	100	100	96
Fertility index (%)^2	F0	100	96	86	93
	F1	96	93	96	89
Fecundity index (%)^3	F0	100	96	86	93
	F1	96	93	96	93
Gestation index (%)^4	F0	100	100	96	96
	F1	100	100	96	100
Precoital time (days)^5	F0	3.3 ± 0.65	3.7 ± 0.73	2.6 ± 0.23	2.4 ± 0.54
	F1	2.4 ± 0.28	3.3 ± 0.51	2.6 ± 0.20	3.6 ± 0.63
Gestation time (days)^5	F0	21.2 ± 0.11	21.4 ± 0.12	21.3 ±  0.14	21.4 ± 0.12
	F1	21.2 ± 0.12	21.4 ± 0.11	21.4 ± 0.11	21.2 ± 0.11
Post implantation loss/animal (%)^5,6	F0	15.31 ± 4.24	19.01 ± 3.83	19.33 ± 5.29	21.66 ± 5.18
	F1	6.44 ± 1.43	13.05 ± 2.21	11.21 ± 3.78	10.81 ± 1.54

1 (no. of females mated/no. of females placed with males) X 100

2 (no. of females pregnant/no. of females placed with males) X 100

3 (no. of females pregnant/no. of females mated) X 100

4 (no. of females with live pups/no. of females pregnant) X 100

5 Values are means ± SEM; statistical test: Kruskal-Wallis + Mann-Whitney U test

6 Total post implantation loss = ((no. of implantation sites/no. of pups born alive)/no. of implantation sites) X 100

Table 3 Summary of offspring data

		Phytosterol esters (% of diet)
Parameter	Generation	0	1.6	3.2	8.1
Pups delivered (total)^1	F0	10.39 ± 0.37	10.26 ± 0.44	10.33 ± 0.48	9.68 ± 0.49
	F1	10.56 ± 0.28	9.73 ± 0.44	9.96 ± 0.40	10.60 ± 0.27
Live birth index (%)^2,3	F0	92	92	92	93
	F1	100	98	99	98
Pup mortality day 1 (%)^2	F0	8.2	8.3	8.5	7.0
	F1	0	1.6	1.5	1.5
Viability index day 4 (%)^2,4	F0	93.3	87.4*	84.6**	84.0**
	F1	98.9	97.2	95.8*	89.7***
Viability index day 21 (%)^2,5	F0	98	100	100	99
	F1	99	99	99	100
Dams with no deaths, day 21^6	F0	20	17	13	14
	F1	21	21	20	18
Dams with 1 or more deaths, day 21^6	F0	7	10	11	11
	F1	6	5	7	7
Whole litter losses^2	F0	3	3	2	5
	F1	0	0	1	0
Sex ratio day 1 (%)^7	F0	51	53	52	49
	F1	53	50	48	60

1 Values are means ± SEM per litter, statistical test: Kruskal-Wallis + Mann-Whitney U test

2 Statistical test: Fisher's exact test (*P<0.05; **P<0.01; ***P<0.001)

3 (no. of pups born alive/no. of pups born) X 100

4 (no. of pups at day 4/no. of live pups at day 1) X 100

5 (no. of pups at day 21/no. of live pups at day 4) X 100

6 Litter data analysed by Kruskal-Wallis

7 % of male pups of total live pups at day 1

Conclusions:

In a dietary two-generation reproductive toxicity study conducted using a protocol comparable with OECD 416 and in compliance with GLP (reliability 1) no adverse effects were reported for general systemic toxicity, or reproductive and developmental toxicity in any generation. The highest concentration tested was 8.1% (w/w) phytosterol ester in diet. This gave a NOAEL of 2500-9100 mg/kg bw/day phytosterol ester or 1540-5620 mg/kg bw/day phytosterol.

Reference 3

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

approximately 5 months

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Lack of detail in methods used; post-mortem examinations were not performed on parental animals; results reported for F1 only

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Principles of method if other than guideline:

The study is reported to be a two-generation reproductive toxicity study, but the F1 animals were not mated to produce an F2 generation, so the study is in actual fact a one-generation reproductive toxicity study.

GLP compliance:

no

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 Laboratories, USA
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 80 days
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: individually in wire mesh cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Diets were prepared weekly by mixing the correct amount of the respective compounds with the appropriate amount of basic diet in a mixer.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Charles River, 19 RF, rat, mouse, hamster meal.
- Storage temperature of food: not specified

Details on mating procedure:

- M/F ratio per cage: 1:2
- Length of cohabitation: 20 days
- Proof of pregnancy: not specified
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged: individually in plastic cages
- Any other deviations from standard protocol: none

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Not specified

Duration of treatment / exposure:

F0: until weaning of F1
F1: until sexual maturation

Frequency of treatment:

Continuous

Details on study schedule:

Twenty male and 20 female rats from each group (with the members of each sex coming from a different dam) were selected shortly after weaning and were carried to sexual maturity. The F0 parents and all remaining offspring were subsequently destroyed .

Dose / conc.:

5 other: % tall oil fatty acid

Remarks:

Low dose (5%) Tall Oil Fatty Acid (FA3) Group 5.0 kg. of the Tall Oil Diet plus 5.0 kg. of Charles River 19RF meal.

Dose / conc.:

10 other: % tall oil fatty acid

Remarks:

High Dose (10%) Tall Oil Fatty Acid (FA3) Group 1.5 kg. of Tall Oil Fatty Acid plus 13.5 kg. of Charles River 19RF meal.

Dose / conc.:

5 other: % oleic acid

Remarks:

Low Dose (5%) Oleic Acid Group 5.0 kg. of the 10% Oleic Acid Diet plus 5.0 kg. of Charles River 19RF meal.

Dose / conc.:

10 other: % oleic acid

Remarks:

High Dose (10%) Oleic Acid Group 1.5 kg. Oleic Acid plus 13.5 kg. of Charles River 19RF meal.

No. of animals per sex per dose:

15 male and 30 female rats per group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Not specified
- Rationale for animal assignment (if not random): Not specified

Positive control:

Not used

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Not specified

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Not specified

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not specified

Oestrous cyclicity (parental animals):

Not specified

Sperm parameters (parental animals):

Not specified

Litter observations:

STANDARDISATION OF LITTERS
- Performed post-weaning
- If yes, maximum of 20 pups/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain,

GROSS EXAMINATION OF DEAD PUPS: Yes

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

CLINICAL CHEMISTRY: (a) FBS; (b) BUN; (c) SCOT; and (e) SC,PT

HEMATOLOGY: (a) Hematocrit; (b) Hemoglobin; (c) White Blood Cell Count; and (d) Red Blood Cell Morphology

URINALYSIS: (a) Color; (b) Appearance; (c) Reaction; (d) Specific Gravity; (e) Protein; (f) Sugar; (g) WBC/hpf; and (h) RBC/hpf.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed post weaning.
- Maternal animals: All surviving animals were sacrificed post-weaning.

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examinations

HISTOPATHOLOGY / ORGAN WEIGHTS: Histopathology was not performed.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at weaning.
- These animals were subjected to postmortem examinations as follows: macroscopic and microscopic

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examinations of (k) Spleen; (1) Adrenals; (m) Pancreas; (n) Stomach; (o) Intestines; (p) Lymph Nodes; (q) Bladder; (r) Gonads; (s) Skin; (t) Bone and marrow; (u) Nerve and Muscle; and (v) Any unusual lesions . .

HISTOPATHOLOGY / ORGAN WEIGHTS:
- the following organs were weighed from male and ten female rats from each group in the FI generation: Thyroids; (b) Heart; (c) Liver; (d) Adrenals; (e) Kidneys; and (f) Gonads. These organs were weighed after fixation in 10% buffered formalin.
- histology was performed on the collected tissues.

Statistics:

Statistical analysis was performed on clinical chemistry, hematology and organ weights. All calculations were confirmed by a second individual to assure their accuracy.

Reproductive indices:

gestation index or lactation index.

Offspring viability indices:

Not included

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Remarks on result:

other: No results were reported for parental animals

Critical effects observed:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs specified.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No mortality mentioned in the study report.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

F1 Control Group: The male pups averaged 39.9 grams in weight while the female pups averaged 38.3 grams.
F1 5% Tall Oil (FA3) Group: The average weight for the male pups was 43.0 grams while for the female pups it was 43.1 grams.
FI 10% Tall Oil (FA3) Group: The male pups had an average weight of 44.8 grams while the female pups averaged 43.3 grams.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

All rats examined had normal blood glucose results and blood urea nitrogen

Urinalysis findings:

no effects observed

Description (incidence and severity):

Several male rats in all groups exhibited various degrees of protein in their urine. This was not considered to be dose related.

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The livers of the 10% male oleic acid weighed less than the male control group. The adrenals of the male FA3 and and 5% oleic acid groups weighed statistically more than the male control group. The liver weight of the 10% female oleic acid group as well as the liver and kidney of the 10% and FA3 groups weight statistically more than the controls. All statistical significance as stated above, however, was lost when compared to our historical control data.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were several animals with pathological findings such as respiratory disease and renal disease which are endemically found in this strain of rat. There was no compound related pathology in any of the rats examined.

Description (incidence and severity):

Reproductive performance:
F1 5% Tall Oil Group: There were four litters with stillborn pups out of 20 cast with a total number of eight stillborn pups (average of 0.4 pup per litter) .
F1 10% Tall Oil (FA3) Group: There was one litter with one stillborn pup out of 19 cast with a total number of three stillborn pups (average of 0.2 pup per litter) .
F1 5% Oleic Acid Control Group: There were four litters with stillborn pups out of 19 cast with a total number of 13 stillborn pups (average of 0.7 pup per litter) .
F110% Oleic Acid Control Group: There with stillborn pups out of 20 cast with four stillborn pups (average of 0.2 pup were three litters a total number of per litter).
Based on our in-house experience, these data are neither statistically nor biologically significant between any 0B the Tall Oil (FA—3) groups and any of the negative or Oleic Acid control groups.
No statistical significance or biological significance between any of the tall oil groups and the negative group in gestation index or lactation index.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 5 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects were reported.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

In a one-generation reproductive toxicity study, not conducted according to Guideline or GLP, no NOAEL was determined. It is the reviewer's opinion that the NOAEL can be established as the highest dose tested for F1 animals based on no adverse effects observed. Therefore, the NOAEL is ≥5000 mg/kg bw/day for general and reproductive/developmental toxicity.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Tall Oil Soap is a complex UVCB substance containing a range of constituent types. The principal constituents are sodium salts of saturated and unsaturated C14-C20 fatty acids (5-45% w/w, typical ca. 26%), rosin acid sodium salts (10-40% w/w, typical ca. 23%) and sterols (1-10%, typical ca. 3.5%). Other neutral constituents such as rosin alcohol and aldehyde isomers are also present at 0 – 3% w/w (typically ca. 1.3%).

In order to reduce in vivo testing in vertebrates, the REACH Regulation requires registrants to consider alternative methods to fulfil Annex requirements. For endpoints where measured data for substance are not available, a weight of evidence approach has therefore been followed based on available data for groups of constituents.

The registrants of TOS consider that the available information for constituents of the substance are sufficient for the purposes of hazard assessment and risk characterisation and that further animal testing is not warranted.

Reliable reproductive toxicity data and other supporting information are available, or studies are in progress, for substances (including UVCBs) that are representative of these four main constituent groups, correlating to Blocks, 1, 2, 9 and 11 in the substance definition (Section 1.2) and environmental assessment.

In addition, TOS contains 25-45% (typical ca. 32%) w/w/ water and cellulose fibre, lignin and oligomeric acids at 3 – 20% w/w (typical ca. 10%). These are not considered further for the assessment of human health hazards.

Other minor constituent groups, for which relevant measured data have not been identified, are typically present at concentrations that are below the threshold to be taken into consideration in the rules for classification and labelling of mixtures for reproductive toxicity according to Regulation (EC) No 1272/2008 (Table 3.7.3.2). These constituent groups are therefore not assessed separately since the conditions of use specified for the whole substance or Blocks 1, 2 and 11 would be sufficient to control any risk relating to hazards that have not been identified for the remaining blocks.

Sodium salts of fatty acids and rosin acids are considered to be toxicologically equivalent to the acid forms for the purpose of this discussion, since following in vivo dosing via the oral route, the salts and acids would be fully dissociated.

Block 1: Fatty acids

Tall oil fatty acids (TOFA, CAS 61790-12-3) is exempt from registration under REACH in accordance with Annex V of the Regulation. It is a non-hazardous product containing fatty acids in the range C14 to C24. As such, the fatty acid constituents of CTO and TOS can also be considered non-hazardous. There is a one-generation reproductive toxicity study on TOFA.

In a one-generation reproductive toxicity study (described as two-generation, but only one-generation by current standards) conducted using a protocol that appears to be similar to the current OECD Test Guideline 415, but pre-dating GLP, 15 male and 30 female rats per group were administered 5% tall oil fatty acid, 10% tall oil fatty acid, 5% oleic acid control and 10% oleic acid control daily in diet (Pharmacopathics Research Laboratories, 1977). All animals were mated at 100 days of age. The offspring were weaned onto the corresponding diet. Twenty male and 20 female rats from each group were selected shortly after weaning and were carried to sexual maturation. the F0 parents and all remaining offspring were subsequently sacrificed. No examinations were reported on parental animals.

No dose-related changes were observed in litters with stillborn pups and average stillborn pups per litter for F1 group. Average weaning weight of treated F1 pups was similar to that of control group. No changes were noted in clinical chemistry parameters examined for F1. One female of the 10% oleic acid control group had a haematocrit value of 58%; its bone marrow, however, was histologically normal. Several rats in all groups had high white blood cell counts. Their bone marrow was also histologically normal. Several male rats in all groups exhibited various degrees of protein in their urine. This was not considered to be dose-related. There were several animals with pathological findings such as respiratory disease and renal disease which are endemically found in this strain of rat. No NOAEL was determined in this study. It is the reviewer's opinion that the NOAEL for effects on reproductive parameters can be established as 10% tall oil fatty acid (equivalent to ≥5000 mg/kg bw/day, the highest dose tested; only data for F1 animals was reported).

Block 2: Rosin acids and Block 9: Rosin alcohol and aldehyde isomers

An extended one-generation reproductive toxicity study is in progress for Tall Oil Rosin, according to OECD 443. This substance represents the neutral form of the rosin acid sodium salts present at 10-40% by weight in the registration substance.

The results of this study will be added to the weight of evidence discussion when these become available.

In a screening study (reported in the disseminated REACH dossier for rosin), four groups of 10 male and 10 female Sprague-Dawley rats received rosin via the 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).

Block 11: Sterols

The sterol constituents of TOS are also present in many other plant species, including foodstuffs. In particular β-sitosterol is added at significant levels to foods due to its cholesterol-lowering properties. Plant sterols have been studied in repeated dose and reproductive toxicity tests.

 

In a dietary two-generation reproductive toxicity study conducted using a protocol comparable with OECD 416 and in compliance with GLP (Waalkens-Berendsen et al., 1999) no adverse effects were reported for general systemic toxicity, or reproductive and developmental toxicity in any generation. The highest concentration tested was 8.1% (w/w) phytosterol ester in diet. This gave a NOAEL of 2500-9100 mg/kg bw/day phytosterol ester or 1540-5620 mg/kg bw/day phytosterol for fertility and reproductive toxicity.

These good quality studies used sterol/fatty acid esters as test material, since this is the chemical form in which they are extracted from plants. TOS itself contains mainly sodium salts of the fatty acids and “free” sterol. However, this does not negate use of the data to support REACH registrations of CTO-derived materials because in vivo, the esters are hydrolysed by pancreatic carboxy ester lipase, therefore the toxicological properties of the free sterols are of most relevance (EC, 2000).

The composition of the tested material was as shown in Tables 5.9.3 and 5.9.4 below.

Table 5.9.3: Gross composition of the test material

Constituent	Composition (% w/w)
Total sterol	62.0
Total fatty acid	38.2
Free sterol (as % of the total mixture)	8.4
Free fatty acid	<0.3

Table 5.9.4: Sterol profile of the test material

Sterol Constituent	% w/w	Fatty acid profile	% w/w
Cholesterol	0.4	C16:0	9.6
Brassicasterol	1.1	C18:0	4.1
Campesterol	25.8	C18:1	21.6
Stigmasterol	21.6	C18:2	64.6
β-Sitosterol	48.7
β-Sitostanol	1.8
Unknowns	0.8

Distilled tall oil (CAS 8002-26-4, EC No. 232-304-6)

In addition to the studies described above, screening data are available on the related substance Distilled Tall Oil (DTO). DTO is obtained by physical distillation of CTO and the two substances therefore share many of the same constituents in different proportions. Direct read-across from DTO to CTO and CTOS is not appropriate because DTO does not contain any sterol constituents and has lower neutral content overall. However, an existing OECD 422 combined repeated dose toxicity test and reproductive/developmental screening test adds weight of evidence to the absence of adverse effects on fertility and reproductive parameters following repeated exposure to CTO and TOS.

In the Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening test (Inveresk, 2002), conducted according to OECD 422 and in compliance with GLP, four groups of 10 male and 10 female Sprague-Dawley rats received tall oil via the diet 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 and five females per group for haematology and clinical chemistry investigations. Necropsy was conducted on all animals, which included weighing of major organs. Histopathology was conducted on tissues from five males from control and high dose groups, and seven females from the control and eight females from the high dose groups.

The NOEL was based on 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 with controls, there was doubt over the reproducibility of this finding and therefore the author of the IUCLID study summary concluded that the NOAEL for fertility and reproductive toxicity in this study is ≥20000 ppm (equivalent to approximately 1598 mg/kg bw/day for males and 1923 mg/kg bw/day for females; the highest dose tested).

Conclusions for reproductive toxicity of TOS

Reproductive toxicity data on the constituents of TOS (tall oil fatty acids and sterols) together with supporting data on DTO and rosin, lead to the conclusion that TOS does not require classification for reproductive toxicity.

Effects on developmental toxicity

Description of key information

The following information is taken into account for any hazard / risk assessment:

No developmental toxicity data are available for the registration substance; therefore, this endpoint is addressed by a weight of evidence approach for relevant groups of constituents present in the substance.

In a prenatal developmental toxicity study conducted to OECD 414 and in compliance with GLP (Slesinski et al., 1999) administration of stanol fatty acid esters in the diet of Wistar rats throughout gestation days 0-21 did not cause any general adverse effects on maternal animals or developmental toxicity in maternal animals or foetuses. The NOAEL from this study was at least 6200 mg/kg bw/day (the highest dose tested).

In a prenatal developmental toxicity study (Envigo, 2017) conducted to OECD 414 and in compliance with GLP, the oral administration of Rosin (CAS 8050-09-7) to pregnant rats by continuous dietary exposure during gestation days 3 to 19, at concentrations of 2500, 5000 or 7500 ppm was associated with lower maternal body weight gain during gestation and an initial effect on food consumption at 7500 ppm and lower maternal body weight gain at 5000 ppm. No similar effects were apparent at 2500 ppm (equivalent to a mean achieved dosage of 199.3 mg/kg bw/day) which was considered to represent the NOAEL for the pregnant female. The ‘No Observed Adverse Effect Level’ (NOAEL) for foetal developmental toxicity was therefore considered to be 5000 ppm (equivalent to a mean achieved dosage of 387.2 mg/kg bw/day) based on reduced foetal and placental weights indicated at 7500ppm.

In a supporting Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test conducted to OECD 422 and in compliance with GLP, for distilled tall oil, the reported NOEL for reproductive and developmental toxicity was 5000 ppm for both male and female rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

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

Tall Oil Soap is a complex UVCB substance containing a range of constituent types. The principal constituents are sodium salts of saturated and unsaturated C14-C20 fatty acids (5-45% w/w, typical ca. 26%), rosin acid sodium salts (10-40% w/w, typical ca. 23%) and sterols (1-10%, typical ca. 3.5%). Other neutral constituents such as rosin alcohol and aldehyde isomers are also present at 0– 3% w/w (typically ca. 1.3%).

In order to reduce in vivo testing in vertebrates, the REACH Regulation requires registrants to consider alternative methods to fulfil Annex requirements. For endpoints where measured data for substance are not available, a weight of evidence approach has therefore been followed based on available data for groups of constituents.

The registrants of TOS consider that the available information for constituents of the substance are sufficient for the purposes of hazard assessment and risk characterisation and that further animal testing is not warranted.

Reliable developmental toxicity data and other supporting information are available for substances (including UVCBs) that are representative of these four main constituent groups, correlating to Blocks, 1, 2, 9 and 11 in the substance definition (Section 1.2) and environmental assessment.

In addition, TOS contains 25-45% (typical ca. 32%) w/w/ water and cellulose fibre, lignin and oligomeric acids at 3 – 20% w/w (typical ca. 10%). These are not considered further for the assessment of human health hazards.

Other minor constituent groups, for which relevant measured data have not been identified, are typically present at concentrations that are below the threshold to be taken into consideration in the rules for classification and labelling of mixtures for developmental toxicity according to Regulation (EC) No 1272/2008 (Table 3.7.3.2). These constituent groups are therefore not assessed separately since the conditions of use specified for the whole substance or Blocks 1, 2 and 11 would be sufficient to control any risk relating to hazards that have not been identified for the remaining blocks.

Sodium salts of fatty acids and rosin acids are considered to be toxicologically equivalent to the acid forms for the purpose of this discussion, since following in vivo dosing via the oral route, the salts and acids would be fully dissociated.

Block 1: Fatty acids

Tall oil fatty acids (TOFA, CAS 61790-12-3) is exempt from registration under REACH in accordance with Annex V of the Regulation. It is a non-hazardous product containing fatty acids in the range C14 to C24. As such, the fatty acid constituents of CTO and TOS can also be considered non-hazardous. There are no developmental toxicity data for TOFA; however, in a one-generation reproductive toxicity study on TOFA, no adverse developmental toxicity was observed.

Block 2: Rosin acids and Block 9: Rosin alcohol and aldehyde isomers

In a prenatal developmental toxicity study (Envigo 2017) conducted to OECD 414 and in compliance with GLP, the oral administration of Rosin (CAS 8050-09-7) to pregnant rats by continuous dietary exposure during gestation days 3 to 19, at concentrations of 2500, 5000 or 7500 ppm was associated with lower maternal body weight gain during gestation and an initial effect on food consumption at 7500 ppm and lower maternal body weight gain at 5000 ppm. No similar effects were apparent at 2500 ppm (equivalent to a mean achieved dosage of 199.3 mg/kg bw/day) which was considered to represent the NOAEL for the pregnant female.

In-utero survival of the developing conceptus was unaffected by maternal exposure at 7500 ppm, although reduced foetal and placental weights indicated an adverse effect on foetal growth. The absence of any structural defects indicated that development was unaffected at this dietary exposure level. At 5000 or 2500 ppm no adverse treatment-related changes were detected in the offspring parameters measured or on embryofoetal development. The ‘No Observed Adverse Effect Level’ (NOAEL) for foetal developmental toxicity was therefore considered to be 5000 ppm (equivalent to a mean achieved dosage of 387.2 mg/kg bw/day), representing the neutral form of the rosin acid sodium salts present at 10-40% by weight in the registration substance.

Block 11: 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 (see below), it should be acceptable to read across the result on stanol esters to the corresponding sterol esters.

 

In a prenatal developmental toxicity study conducted to OECD 414 and in compliance with GLP (Slesinski et al., 1999) administration of stanol fatty acid esters in the diet of Wistar rats throughout gestation days 0-21 did not cause any general adverse effects on maternal animals or developmental toxicity in maternal animals or fetuses. The NOAEL from this study was at least 6200 mg/kg bw/day (the highest dose tested). The test material contained 57.08% total stanols/100g fat (68% sitostanol, 30% campestanol, 2% unsaturated sterol), 41.96% fatty acids and 2% unsaturated sterols and unknowns. Approximately 93.4% of the esterified groups was C-18 fatty acids, with 3.6% C-16, 2.1% C-20 and 0.9% other fatty acid esters.

In a dietary two-generation reproductive toxicity study conducted using a protocol comparable with OECD 416 and in compliance with GLP (Waalkens-Berendsen et al., 1999) no adverse effects were reported for general systemic toxicity, or reproductive and developmental toxicity in any generation. The highest concentration tested was 8.1% (w/w) phytosterol ester in diet. This gave a NOAEL of 2500-9100 mg/kg bw/day phytosterol ester or 1540-5620 mg/kg bw/day phytosterol for fertility and reproductive toxicity.

Distilled tall oil (CAS 8002-26-4, EC No. 232-304-6)

In addition to the data described above, screening data are available for the related substance Distilled Tall Oil (DTO). DTO is obtained by physical distillation of CTO and the two substances therefore share many of the same constituents in different proportions. Direct read-across from DTO to CTO and TOS is not appropriate because DTO does not contain any sterol constituents and has lower neutral content overall. However, an existing OECD 422 combined repeated dose toxicity test and reproductive/developmental screening test adds weight of evidence to the absence of adverse effects developmental parameters following repeated exposure to CTO and TOS.

In the Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening, conducted according to appropriate OECD Test guideline and in compliance with GLP, four groups of 10 male and 10 female Sprague-Dawley rats received the test item via the diet at concentrations of 0, 1000, 5000 and 20000 ppm (Inveresk, 2002). The males were dosed for at least 4 weeks, starting from 2 weeks prior to mating. The females were dosed from 2 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 5 males and 5 females per group for laboratory investigations. Males were sampled during Week 5: females were sampled on Day 6 of lactation. All animals were subjected to necropsy, which included weighing of major organs.  Histopathology was conducted on tissues from 5 males from control and high dose, and 7 females from the Control and 8 females from the high dose. No adverse effects on developmental parameters were observed. Therefore, under the conditions of this study the NOEL for developmental toxicity (including during lactation) was considered to be 20000 ppm (equivalent to approximately 1598 mg/kg bw/day for males and 1972 mg/kg bw/day for females; the highest dose tested).

Conclusions for developmental toxicity of TOS

Developmental toxicity data on the constituents of TOS (rosin acids and neutrals and sterols) together with supporting data on DTO, lead to the conclusion that TOS does not require classification for developmental toxicity.

Justification for classification or non-classification

Based on data for constituents of TOS no classification for reproductive or developmental toxicity is proposed in accordance with Regulation (EC) No 1272/2008.