Tall oil

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-10-11 to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Name of substance: Tall Oil
Batch (Lot) Number: AN-400-125
CAS number: 8002-26-4
Expiry date: 01 January 2023 (expiry date)
Physical Description: Amber transparent liquid
Storage Conditions: In freezer (≤ -15°C) protected from light container flushed with nitrogen
Specific gravity / density: 0.942 g/cm3

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Kraton Chemical BV (The Netherlands); Batch No. AN-400-125
- Expiration date of the lot/batch: 2023-01-01
- Purity test date: 2019-01-17

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In freezer (≤ -15°C) protected from light container, flushed with nitrogen
- Stability under test conditions: Stable, maximum temperature: 40°C, maximum duration: 60 minutes
- Solubility and stability of the test substance in the solvent/vehicle: Stable in vehicle (1% (w/v) Aqueous carboxymethyl cellulose with 0.1% (v/v) Tween 80) for at least 24 hours at room temperature protected from light. Confirmed over the concentration range 1 to 200 mg/mL (emulsions) (Test Facility Reference No. 20180610).

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Amber transparent liquid

OTHER SPECIFICS:
- other information: Sample will crystallize upon prolonged storage at ambient or sub-ambient conditions and will become hazy. Sample should become transparent after 4-8 hrs at ambient temperature. Otherwise, heat sample gently (max 40°C) until sample is transparent again. Shake sample bottle gently before use to assure sample homogeneity.

Test animals

Species:

rabbit

Strain:

New Zealand White

Remarks:

time-mated females

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 17-19 weeks old
- Weight at study initiation: between 2942 and 4002 g at the initiation of dosing
- Fasting period before study: Not specified
- Housing: individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles.
- Diet (e.g. ad libitum): Pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Kliba NAFAG Granovit AG, Kaiseraugst, Swizerland) ad libitum throughout the study, except during designated procedures. Additionally, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) was provided during the study period.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles/containers.
- Acclimation period: at least 2 days before the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 20°C
- Humidity (%): 54 to 80%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2019-10-11 To: 2019-11-08

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% (w/v) Aqueous carboxymethyl cellulose with 0.1% (v/v) Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Test material was pre-weighed in amber vials, flushed with nitrogen gas and stored in the freezer (≤-15°C). The pre-weighed test material was removed from the freezer the night before the intended day of use allowing complete thawing of the test material. The dosing formulations were prepared daily as an emulsion and were dosed within 24 hours after completion of the preparation of the test material.

Test material dosing formulations were kept at room temperature and protected from light until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the test material.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 1% (w/v) Aqueous carboxymethyl cellulose with 0.1% (v/v) Tween 80
- Concentration in vehicle: 20, 60, 120 mg/mL for the 100, 300, and 600 mg/Kg/day dose groups, respectively
- Amount of vehicle (if gavage): 5 mL/Kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample Collection and Analysis
Dose formulation samples were collected for analysis as indicated below:

Occasion Concentration Homogeneity
Week 1 all groups Groups 2, 3, and 4a

a: The homogeneity results obtained from the top, middle and bottom for the Group 2, 3 and 4 preparations were averaged and utilized as the concentration results.

Concentration Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for emulsions of target concentration.

Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.

Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Reference No. 20180610) demonstrated that the test material is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Reference No. 20180610.

Details on mating procedure:

Time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France) on Oct 11, 2019. The females arrived on Day 1-4 post-coitum (Day 0 post-coitum is defined as the day of successful mating).

Duration of treatment / exposure:

once daily via oral gavage

Frequency of treatment:

once daily oral gavage for 7 days a week from Day 6 to Day 28 post-coitum, inclusive

Duration of test:

from Day 6 to Day 28 post-coitum, inclusive

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1 (Control)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

22 females per dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral route of exposure was selected because this is the intended route of human exposure during manufacture, handling or use of the test material. The dose levels were selected based on the results of the dose range finding study (Test Facility Reference No. 20180608), and in an attempt to produce graded responses to the test material.

- Rationale for animal assignment: Animals were randomly assigned to groups.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on Day 6 post-coitum and lasting up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 1, 2, 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
- Time schedule for examinations: individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum. In order to monitor the health status, Animal No. 69 was also weighed on Day 11 post-coitum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes (Animals surviving until scheduled euthanasia were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol
® 20%) on Day 29 post-coitum)
- Sacrifice on Day 29 post-coitum
- Organs examined: All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). The liver and uterus were weighed at necropsy for all animals (except for females that were euthanized in poor condition). The adrenal gland, brain, kidneys, ovaries, thymus, spleen, thyroid gland and pituitary gland were weighed 7 days after fixation for all scheduled euthanasia animals. Organs of animals that were sacrificed in extremis were weighed after at least 7 days of fixation. Paired organs were weighed together. Organ weight as a percent of body weight (using the body weight on Day 29 post-coitum) was calculated.

OTHER:
- Clinical Pathology
Blood of all animals (except for animals found dead) was collected on the day of necropsy. Samples were collected from the ear artery.

Haematology: Blood samples at a target volume of 0.5 mL were collected into tubes containing K3-EDTA as anticoagulant. Samples were analyzed for the parameters specified in Table 2 presented in the section ‘Any other information on materials and methods incl. tables’.

Clinical Chemistry: Blood samples at a target volume of 0.5 mL (plasma) were collected into tubes containing Li-Heparin as anticoagulant. Serum samples at a target volume of 0.25 mL were collected in tubes without anticoagulant. Blood samples were processed for plasma or serum (bile acids), which was analyzed for the parameters specified in Table 3 presented in the section ‘Any other information on materials and methods incl. tables’.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes (Each ovary and uterine horn of all animals was dissected and examined as quickly as possible)
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Placental weights; number and distribution of live and dead fetuses; number and distribution of embryo-fetal deaths

Fetal examinations:

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

- External examinations: Yes: [all per litter]
Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight was determined (not for fetuses of animals sacrificed before planned necropsy). For late resorptions and recognizable fetuses of females euthanized in extremis, a gross external examination wasperformed (if possible)

- Soft tissue examinations: Yes: Soft tissue cephalic examination was executed for approximately half of the fetuses of the same litter for all groups. All fetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development.

The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a
mid-coronal slice.

- Skeletal examinations: Yes: [all per litter in Groups 1 and 4]
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and processed for double staining with Alcian Blue 8GX and Alizarin Red S.

Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. Since no possible treatment related effects in the high dose group were seen, skeletal examination was not extended to the fetuses from the low and mid dose group.

A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

- Head examinations: Yes: [all per litter]
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.

Statistics:

For information on statistics, please refer to the section 'Any other information on materials and methods incl. tables’

Indices:

Maternal Indices
Body Weight Gains: Calculated against the body weight on Day 6 post-coitum.

Corrected Body Weight Gains: Body weight determined on Day 29 post-coitum minus the body weight on Day 6post-coitum and the weight of gravid uterus.

Relative Food Consumption: Calculated against the body weight for scheduled intervals.

Organ Weight Relative to Body Weight: Calculated against the body weight on Day 29 post-coitum.

Reproduction and Developmental Variables

1) Preimplantation loss (%): ((number of corpora lutea - number of implantation sites) / (number of corpora lutea)) x 100

2) Post-implantation loss (%): ((number of implantation sites - number of live fetuses) / (number of implantation sites)) x 100

3) Viable fetuses affected/litter (%): ((number of viable fetuses affected/litter) / (number of viable fetuses/litter)) x 100

Historical control data:

Charles River Den Bosch historical data (Study date range: 2014 – 2018) on the background incidence of fetal malformations and developmental variations in this species from the same strain and source has been presented in Appendix 3 of the study report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Reduced faeces production was observed for 9/21 females in the control group, for 10/22 females treated at 100 mg/Kg/day, for 15/21 females at 300 mg/Kg/day and for 14/20 females at 600 mg/Kg/day. Piloerection was observed in 2/20 females at 600 mg/Kg/day while red fluid on the manure tray was observed for 2/22 females at 300 mg/Kg/day. Female Nos. 58 and 83 (300 and 600 mg/Kg/day, respectively) were observed with watery discharge from the nose on a single day. For Female No. 83 this was observed one day prior to the observed slow breathing, but for the other female, no breathing difficulties were observed. From the study daybook, it can be retrieved that for Female No. 58 the presence of watery discharge from the nose was due to the dosing procedure (Watery discharge from the nose was directly observed after extubating), most likely this was also the case for the other animal. Therefore, this was regarded to be unrelated to the test material itself.

Incidental observations included quick breathing, alopecia, missing nails, wounds/scabs and overgrown/broken teeth. These findings occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed and since the effects were also observed in concurrent control animals, these were not considered to be of any toxicological relevance.

Of the animals that were killed in extremis, Female No. 3 in the control group was observed with red fluid on the manure tray, absent food consumption and 5% body weight loss between Days 0 and 6 post-coitum. Necropsy revealed intussusception of the colon, together with many dark red foci on the colon. Female No. 61 in the 300 mg/Kg/day dose group exhibited body weight loss of 6% compared to Day 6 post-coitum and nearly absent food consumption (9 grams in total over ten days). Clinical signs included piloerection on the last two days prior to necropsy and the animal was found lean the day prior to her preterm sacrifice. In addition, severely reduced faeces production was observed for seven days. Relevant gross lesions were an accentuated lobular pattern of the liver and a gelatinous, small thymus. Female No. 69 in the 600 mg/Kg/day exhibited severe body weight loss (up to -14% on Day 11 post-coitum compared to Day 0 post-coitum), absent food consumption and clinical signs including reduced faeces production, diarrhoea, piloerection, hunched posture and lean appearance. Gross lesions included a small spleen. Female No. 70 in the 600 mg/Kg/day dose group was observed to have breathing difficulties(quick breathing, labored respiration and rales), piloerection, severely reduced faeces production and pale appearance one day prior or on the day of early sacrifice, severe body weight loss between Days 12 and 15 post coitum (-7%) and nearly absent food consumption from Day 12 post-coitum onwards. Additionally, increased concentrations of alanine aminotransferase, aspartate aminotransferase, urea, cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, bile acids, potassium and inorganic phosphate were observed. Chloride and calcium concentration were also lower compared to both controls and animals treated at 600 mg/Kg/day.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No treatment-related mortality was observed during the study. However, four animals died during the study period. In the control group, one animal (Female No. 3) was sacrificed in extremis on Day 7 post-coitum. In the 300 mg/Kg/day dose group, one animal (Female No. 61) was sacrificed in extremis on Day 21 post-coitum. Since the effects observed were seen only in a single animal at the mid-dose with no accompanied dose-response, this was considered to be unrelated to treatment. Two females were sacrificed in extremis in the 600 mg/Kg/day dose group. One animal (Female No. 69) was sacrificed in extremis on Day 11 post-coitum and exhibited severe body weight loss. However, since the body weight loss was already present prior to the initiation of dosing, this early sacrifice
was not related to exposure to the test material. The second animal in the high dose group (Female No. 70) was sacrificed in extremis on Day 15 post-coitum. Necropsy confirmed a gavage related trauma as the animal was observed with perforation of the right caudal lobe of the left lung, chest cavity containing watery fluid, black-brown discoloration of the left lung, small left lung, dark-red discoloration of the right lung, adhesion of the pericardium to the heart and thymus, tan discoloration of the thymus, gelatinous thymus and reddish foci on the liver.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects were observed at 100 and 300 mg/Kg/day. At 600 mg/Kg/day, body weight gain was slightly lower compared to controls during the complete treatment period, albeit without reaching statistical significance. No relevant changes were observed in body weight or corrected body weight gain.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Normal values for food consumption and relative food consumption were noted for females treated at 100 mg/Kg/day. Mean food consumption before or after allowance for body weight was lower at 300 and 600 mg/Kg/day compared to concurrent controls, reaching statistical significance on Days 15-18 post-coitum and Days 6-24 post-coitum, respectively. All values remained within available historical control data.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 100 and 300 mg/Kg/day, a statistically significantly lower red blood cell distribution width (RDW) was observed(0.96x of control for both dose levels). In absence of a dose response-relationship this was not considered to be treatment-related. One animal (Female No. 27) in the 100 mg/Kg/day dose group was observed to have an increased reticulocyte count compared to control means. However, as this only concerns one female at the low dose, this was not considered to be treatment-related. Other parameters were within comparable range of concurrent control animals.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 600 mg/Kg/day, triglycerides were statistically significantly increased compared to controls (1.52x of control). Additionally, at 100 and 600 mg/Kg/day, cholesterol levels were statistically significantly higher compared to concurrent controls (1.48x of control for both dose levels). Chloride levels at 100 and 300 mg/Kg/day were statistically significantly increased compared to concurrent controls (1.02x of control). In the absence of a dose response and/or considering the small magnitude of change, this was not considered to be toxicologically relevant.

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

Description (incidence and severity):

Liver:body weight ratios were increased with statistical significance for females treated at 300 and 600 mg/Kg/day (1.1x of control for both dose levels). Additionally, ovary weights (absolute only) were increased with statistical significance in females treated at 100 mg/Kg/day(1.1x of control). However, in the absence of a dose-response relationship, this was not considered to be treatment-related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to be treatment-related. Two non-pregnant females (Female No. 58 at 300 mg/Kg/day and Female No. 81 at 600 mg/Kg/day) were observed with ovaries that were reduced in size. At the incidence observed and in the absence of a treatment related effect on ovary weights, this was considered unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

All surviving females were pregnant and had litters with viable fetuses.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The numbers of pre- and post-implantation loss in the control and test groups were comparable and in the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Females sacrificed in extremis were all pregnant at the time of necropsy. All remaining females were pregnant and had litters with viable fetuses.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Female Nos. 27 (100 mg/Kg/day), 58 (300 mg/Kg/day), and 71, 81 and 88 (600 mg/Kg/day) were nongravid. As dosing was only initiated after the implantation occurred, these were not considered to be treatment-related.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 600 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse treatment-related systemic toxicity effects obseved at the highest dose tested.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no effects on fetal body weights (both sexes) observed subsequent to treatment up to 300 mg/Kg/day. At 600 mg/Kg/day, fetal body weights (both males and females) were lower compared to controls, however without reaching statistical significance and values remaining within the available historical control data.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The numbers of fetuses (litters) available for fetal morphological examination were 206 (21), 186 (21), 187 (20) and 176 (17) in the control, 100, 300 and 600 mg/Kg/day groups, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 600 mg/Kg/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no test material-related effects on litter size in any dose group.

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External variations were not observed and there were no test material-related effects on external morphology following treatment up to 600 mg/Kg/day. At 300 mg/Kg/day, a facial cleft occurred in one fetus (Fetus No.A056-05). In addition, the late resorption in litter A045 was observed with exencephaly and gastroschisis. At 100 mg/Kg/day, two malformations occurred that both affected the same fetus (No. A030-01). This fetus missed the head and had two flexed carpals. Due to the single occurrence and occurrence at low and mid dose levels, all these malformations were considered chance findings.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on skeletal morphology were observed at 600 mg/Kg/day. Skeletal malformations occurred in two control and two high-dose fetuses. The high-dose fetuses were observed with sternal anomaly (A087-01) or vertebral centra anomaly (A082-08), in addition to the visceral malformations that were observed in these fetuses. The single occurrence and fact that these skeletal malformations are among historical control data does not indicate a treatment effect and were therefore, considered chance findings.

Two control fetuses were observed with costal cartilage anomaly (A006-07) or bent limb bones (A014-04) and were as such considered to be spontaneous in origin. Of the variations, unossified tarsals occurred more frequently at 600 mg/Kg/day than at the control level. Incidences were 1.2% and 2.3% per litter in Groups 1 and 4, respectively. Although this increase was not statistically significant, the high dose value was above the historical control maximum value of 2.1% per litter. All other skeletal variations that occurred were considered to be unrelated to treatment as they occurred infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on visceral morphology were observed following treatment up to 600 mg/Kg/day. Visceral malformations occurred in 6 (4), 2 (2), 4 (3) and 3 (3) fetuses (litters) in the control, 100, 300 and 600 mg/Kg/day groups, respectively.

At 600 mg/Kg/day, one fetus (No. A087-01) was observed with the following malformations: malpositioned kidney, retroesophageal aortic arch and left carotid originating from the pulmonary trunk. In addition to this fetus, two other fetuses at this dose level were observed with malformations. One fetus (No.A082-08) had a narrow aortic arch and atrial septum defect, and another fetus (No. A086-07) exhibited internal hydrocephaly. Two of the above malformations were also observed in fetuses at 300 mg/Kg/day, namely malpositioned kidney (Fetus Nos. A047-05 and A047-06) and internal hydrocephaly (Fetus No. A045-10). The other affected fetus in this group had transposition of the great vessels and was also noticed with a facial cleft (Fetus No. A056-05).

At 100 mg/Kg/day, the fetus without head and flexed carpals (A030-01) appeared to have abnormal liver lobation and diaphragmatic hernia. The latter anomaly also occurred in another fetus (No. A042-03) of this group, whereas abnormal liver lobation was observed in four control fetuses (Nos. A004-01, A004-02, A004-07 and A007-13). Two other control fetuses had either tetralogy of Fallot or malpositioned testis (Nos. A012-01 and A013-01, respectively). Although two fetuses were observed with internal hydrocephaly at the mid and high dose groups (A045-10 at 300 mg/Kg/day and A086-07 at 600 mg/Kg/day), both dams originating from different litters and were bred by different males, this malformation was considered to be unrelated to treatment. This malformation is at a low incidence present in the historical control data. Moreover, in the last six prenatal developmental studies in rabbits, this malformation was observed in four studies in which five fetuses were observed with internal hydrocephaly. Based on this recent trend, this malformation was considered to be within the historical context.

The single occurrence and/or group distribution of these malformations does not indicate a test material-relationship and all except two (great vessel transposition and malposition of the left carotid) were observed previously in historical control fetuses and were therefore considered incidental findings. All variations observed were considered unrelated to treatment with the test material as they occurred in the absence of a dose-related trend, infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

There were no effects on placenta weights (both sexes) observed following treatment up to 300 mg/Kg/day. At 600 mg/Kg/day, placenta weights (both males and females) were lower compared to controls, however without reaching statistical significance.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse treatment-related developmental toxicity effects obseved at the highest dose tested.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 6. Summary of Body Weights (gram): F0 Generation
		Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg
Post Coitum
Day 0	Mean	3504	3508	3563	3527
	St. Dev.	257.6	302.4	319.1	315.8
	N	22	21	21	19
Day 6	Mean	3483	3530	3522	3418
	St. Dev.	242.6	271.6	248.2	243.3
	N	22	21	21	19
Day 9	Mean	3528	3621	3583	3453
	St. Dev.	248.5	275.2	271.1	256.1
	N	21	21	21	19
Day 12	Mean	3597	3686	3651	3497
	St. Dev.	228.0	275.0	261.5	235.3
	N	21	21	21	18
Day 15	Mean	3696	3781	3726	3566
	St. Dev.	223.5	268.4	264.7	258.0
	N	21	21	21	18
Day 18	Mean	3725	3823	3748	3646
	St. Dev.	221.3	283.9	251.0	221.6
	N	21	21	21	17
Day 21	Mean	3767	3853	3772	3649
	St. Dev.	233.7	272.4	252.8	233.7
	N	21	21	21	17
Day 24	Mean	3817	3878	3824	3695
	St. Dev.	231.7	252.2	253.6	256.5
	N	21	21	20	17
Day 27	Mean	3867	3946	3867	3734
	St. Dev.	205.6	245.5	231.2	287.9
	N	21	21	20	17
Day 29	Mean	3910	3991	3925	3792
	St. Dev.	204.4	240.9	237.8	309.0
	N	21	21	20	17

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 7. Summary of Body Weight Gain (%): F0 Generation
		Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg
Post Coitum
Day 6	Mean	0	0	0	0
	St. Dev.	0.0	0.0	0.0	0.0
	N	22	21	21	19
Day 9	Mean	2	3	2	1
	St. Dev.	1.0	1.5	2.1	2.5
	N	21	21	21	19
Day 12	Mean	4	4	4	2
	St. Dev.	1.4	1.9	2.4	2.6
	N	21	21	21	18
Day 15	Mean	7	7	6	4
	St. Dev.	2.1	2.4	3.6	4.2
	N	21	21	21	18
Day 18	Mean	8	8	7	6
	St. Dev.	2.5	2.8	3.9	4.7
	N	21	21	21	17
Day 21	Mean	9	9	7	6
	St. Dev.	2.5	2.7	4.9	5.3
	N	21	21	21	17
Day 24	Mean	10	10	9	8
	St. Dev.	3.8	3.1	4.2	6.1
	N	21	21	20	17
Day 27	Mean	12	12	10	9
	St. Dev.	4.2	3.5	5.1	8.4
	N	21	21	20	17
Day 29	Mean	13	13	12	11
	St. Dev.	5.4	4.2	5.4	9.2
	N	21	21	20	17

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 8. Summary of Food Consumption (g/animal/day): F0 Generation
		Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg
Post Coitum
Day 6-9	Mean	149	158	135	99**
	St. Dev.	40.9	39.2	40.3	44.5
	N	22	21	21	19
Day 9-12	Mean	148	149	136	103**
	St. Dev.	23.3	26.1	34.8	40.7
	N	21	21	21	19
Day 12-15	Mean	121	124	95	77**
	St. Dev.	30.1	32.7	48.3	36.9
	N	21	21	21	18
Day 15-18	Mean	146	143	109**	98**
	St. Dev.	22.3	31.9	44.2	52.0
	N	21	21	21	18
Day 18-21	Mean	150	151	124	113*
	St. Dev.	29.0	31.7	43.9	47.5
	N	21	21	21	17
Day 21-24	Mean	132	115	108	99**
	St. Dev.	27.8	29.3	38.4	33.3
	N	21	21	21	17
Day 24-27	Mean	111	114	98	95
	St. Dev.	35.8	36.3	29.7	43.6
	N	21	21	20	17
Day 27-29	Mean	109	110	109	115
	St. Dev.	49.7	32.7	28.5	42.5
	N	21	21	20	17
Mean of Means		133	133	114	100

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 9. Summary of Relative Food Consumption (g/Kg Body Weight/day): F0 Generation
		Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg
Post Coitum
Day 6-9	Mean	44	43	37	29**
	St. Dev.	6.0	10.1	10.4	12.8
	N	21	21	21	19
Day 9-12	Mean	41	40	37	31**
	St. Dev.	5.3	6.0	8.8	9.2
	N	21	21	21	18
Day 12-15	Mean	33	33	25*	21**
	St. Dev.	7.6	7.6	12.0	10.5
	N	21	21	21	18
Day 15-18	Mean	39	37	29**	28**
	St. Dev.	5.8	7.4	11.5	12.8
	N	21	21	21	17
Day 18-21	Mean	40	39	33	31*
	St. Dev.	6.6	7.7	11.4	12.7
	N	21	21	21	17
Day 21-24	Mean	35	30	29	27**
	St. Dev.	7.3	6.7	7.0	8.7
	N	21	21	20	17
Day 24-27	Mean	29	29	25	25
	St. Dev.	9.4	9.2	7.7	11.1
	N	21	21	20	17
Day 27-29	Mean	28	28	28	30
	St. Dev.	12.6	8.3	7.0	10.7
	N	21	21	20	17
Mean of Means		36	35	31	28

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 10. Summary of Haematology Parameters: F0 Generation
		Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg
End of Treatment
WBC 10E9/L	Mean	5.9	6.1	6.9	6.7
	St. Dev.	1.4	1.5	1.7	2.0
	N	21	19	19	15
Heterophils 10E9/L	Mean	1.2	1.2	1.3	1.5
	St. Dev.	0.2	0.3	0.4	0.6
	N	21	19	19	15
Lymphocytes 10E9/L	Mean	4.4	4.5	5.2	4.8
	St. Dev.	1.2	1.3	1.6	1.5
	N	21	19	19	15
Monocytes 10E9/L	Mean	0.1	0.1	0.1	0.2
	St. Dev.	0.1	0.0	0.1	0.1
	N	21	19	19	15
Eosinophils 10E9/L	Mean	0.0	0.0	0.0	0.1
	St. Dev.	0.0	0.0	0.0	0.0
	N	21	19	19	15
Basophils 10E9/L	Mean	0.1	0.2	0.1	0.2
	St. Dev.	0.0	0.1	0.0	0.1
	N	21	19	19	15
Large Unstained Cells (LUC) 10E9/L	Mean	0.0	0.0	0.0	0.0
	St. Dev.	0.0	0.0	0.0	0.0
	N	21	19	19	15
Red Blood Cells 10E12/L	Mean	6.02	5.97	6.11	6.12
	St. Dev.	0.51	0.33	0.51	0.41
	N	21	19	19	15
Reticulocytes 10E9/L	Mean	65.7	81.4	55.3	64.0
	St. Dev.	39.0	65.0	27.6	31.8
	N	21	19	19	15
RDW %	Mean	13.8	13.3*	13.3*	13.7
	St. Dev.	0.7	0.7	0.5	0.5
	N	21	19	19	15
Haemoglobin mmol/L	Mean	7.8	7.8	7.9	7.8
	St. Dev.	0.6	0.4	0.5	0.5
	N	21	19	19	15
Haematocrit L/L	Mean	0.385	0.382	0.390	0.380
	St. Dev.	0.032	0.021	0.027	0.022
	N	21	19	19	15
MCV fL	Mean	63.9	63.9	64.0	62.2
	St. Dev.	2.4	1.6	2.7	2.5
	N	21	19	19	15
MCH fmol	Mean	1.29	1.30	1.29	1.27
	St. Dev.	0.05	0.04	0.05	0.06
	N	21	19	19	15
MCHC mmol/L	Mean	20.22	20.37	20.24	20.42
	St. Dev.	0.43	0.42	0.38	0.50
	N	21	19	19	15
Platelets 10E9/L	Mean	408	423	377	411
	St. Dev.	109	86	128	114
	N	21	19	19	15

+/++ Steel-test significant at 5% (+) or 1% (++) level

* / ** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 11. Summary of Clinical Chemistry Parameters: F0 Generation
		Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg
End of Treatment
Alanine aminotransferase U/L	Mean	30.9	32.9	24.2	27.9
	St. Dev.	12.4	13.7	6.4	10.8
	N	21	22	20	17
Aspartate aminotransferase U/L	Mean	44.1	43.5	33.7	53.4
	St. Dev.	29.3	33.9	11.5	54.0
	N	21	22	20	17
Alkaline Phosphatase U/L	Mean	43	40	43	48
	St. Dev.	20	21	24	34
	N	21	22	20	17
Total Protein g/L	Mean	42.3	43.1	41.0	42.2
	St. Dev.	3.4	4.9	2.6	3.6
	N	21	22	20	17
Albumin g/L	Mean	29.6	29.8	28.2	28.6
	St. Dev.	2.6	3.7	2.0	2.6
	N	21	22	20	17
Total bilirubin µmol/L	Mean	2.5	2.5	2.5	2.5
	St. Dev.	0.4	0.4	0.4	0.3
	N	21	22	20	17
Urea mmol/L	Mean	7.3	7.1	7.1	7.2
	St. Dev.	1.2	0.8	1.5	1.5
	N	21	22	20	17
Creatinine µmol/L	Mean	96.5	96.1	100.6	100.6
	St. Dev.	9.1	10.8	10.8	11.3
	N	21	22	20	17
Glucose mmol/L	Mean	7.08	7.28	7.34	7.23
	St. Dev.	0.60	0.56	0.54	0.62
	N	21	22	20	17
Cholesterol mmol/L	Mean	0.25	0.37*	0.30	0.37*
	St. Dev.	0.11	0.20	0.09	0.10
	N	21	22	20	17
HDL cholesterol mmol/L	Mean	0.09	0.14	0.11	0.14
	St. Dev.	0.04	0.13	0.03	0.05
	N	21	22	20	17
LDL cholesterol mmol/L	Mean	0.10	0.13	0.10	0.14
	St. Dev.	0.05	0.09	0.04	0.05
	N	21	22	20	17
Triglycerides mmol/L	Mean	0.64	0.68	0.72	0.97**
	St. Dev.	0.18	0.19	0.21	0.52
	N	21	22	20	17
Bile Acids µmol/L	Mean	7.2	6.4	5.7	5.9
	St. Dev.	3.2	5.2	3.8	2.9
	N	21	22	20	17
Sodium mmol/L	Mean	140.9	141.5	142.2	141.3
	St. Dev.	2.1	1.7	2.1	2.3
	N	21	22	20	17
Potassium mmol/L	Mean	5.10	5.19	5.22	5.27
	St. Dev.	0.55	0.57	0.41	0.64
	N	21	22	20	17
Chloride mmol/L	Mean	106	108*	108*	107
	St. Dev.	3	2	2	3
	N	21	22	20	17
Calcium mmol/L	Mean	3.14	3.18	3.17	3.16
	St. Dev.	0.8	0.14	0.10	0.22
	N	21	22	20	17
Inorganic Phosphate mmol/L	Mean	1.49	1.47	1.49	1.46
	St. Dev.	0.20	0.15	0.14	0.22
	N	21	22	20	17

* / ** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table12. Summary of Macroscopic Findings: F0 Generation
	Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg
Intercurrent Death
Animals Examined	1		1	2
Animals Affected	1		1	2
Heart
Grown together with:	0		0	1
Lungs
Reduced in size	0		0	1
Perforation(s)	0		0	1
Discolouration	0		0	1
Colon
Intussusception	1		0	0
Focus/foci	1		0	0
Liver
Accentuated lobular pattern	0		1	0
Focus/foci	0		0	1
Spleen
Reduced in size	0		0	1
Thymus
Reduced in size	0		1	0
Discolouration	0		0	1
Gelatinous	0		1	1
Skin
Scab formation	0		1	0
Body cavities
Contains fluid	0		0	1
End of Treatment
Animals Examined	21	22	21	20
Animals without findings	11	14	8	13
Animals Affected	10	8	13	7
Lungs
Focus/foci	4	4	3	3
Discolouration	1	0	0	1
Liver
Accentuated lobular pattern	2	1	4	1
Nodule(s)	0	0	0	1
Gall Bladder
Agenesis	0	0	1	0
Ovaries
Reduced in size	0	0	1	1
Thyroid Gland
Reduced in size	1	1	0	0
Adrenal Glands
Focus/foci	0	0	0	1
Reduced in size	1	0	0	0
Spleen
Constricted	1	1	0	0
Ectopic splenic issue	2	3	1	1
Reduced in size	0	1	0	0
Thymus
Discolouration	0	0	1	0
Skin
Alopecia	1	0	0	1
Scab formation	0	0	1	0
Bone
Broken	0	0	2	0

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Table 13. Summary of Organ Weights (gram): Pregnant Females
		Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg
End of Treatment
Body Weight (gram)	Mean	3910	3991	3925	3792
	St. Dev.	204	235	238	309
	N	21	22	20	17
Brain (gram)	Mean	11.9	12.2	11.9	12.0
	St. Dev.	0.7	0.8	0.6	0.6
	N	21	22	20	17
Pituitary (gram)	Mean	0.049	0.052	0.048	0.053
	St. Dev.	0.009	0.012	0.016	0.012
	N	21	22	20	17
Liver (gram)	Mean	81.3	88.7	89.4	88.6
	St. Dev.	8.2	13.9	10.8	12.0
	N	21	22	20	17
Thyroids (gram)	Mean	0.399	0.373	0.405	0.368
	St. Dev.	0.095	0.081	0.103	0.099
	N	21	22	20	17
Thymus (gram)	Mean	3.04	2.99	2.99	2.59
	St. Dev.	0.64	0.65	0.90	0.63
	N	21	22	20	16
Kidneys (gram)	Mean	19.00	19.71	18.79	18.99
	St. Dev.	2.16	2.48	1.73	1.74
	N	21	22	20	17
Adrenals (gram)	Mean	0.273	0.261	0.277	0.280
	St. Dev.	0.063	0.063	0.046	0.079
	N	21	22	20	17
Spleen (gram)	Mean	1.99	1.99	2.00	2.00
	St. Dev.	0.49	0.66	0.58	0.64
	N	21	22	20	17
Ovaries (gram)	Mean	0.773	0.877*	0.818	0.797
	St. Dev.	0.115	0.164	0.086	0.165
	N	21	22	20	17

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 14. Summary of Organ/ Body Weight Rations (%): Pregnant Females
		Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg
End of Treatment
Body Weight (gram)	Mean	3910	3991	3925	3792
	St. Dev.	204	235	238	309
	N	21	22	20	17
Brain (%)	Mean	0.3	0.3	0.3	0.3
	St. Dev.	0.0	0.0	0.0	0.0
	N	21	22	20	17
Pituitary (%)	Mean	0.001	0.001	0.001	0.001
	St. Dev.	0.000	0.000	0.000	0.000
	N	21	22	20	17
Liver (%)	Mean	2.1	2.2	2.3*	2.3**
	St. Dev.	0.2	0.3	0.2	0.2
	N	21	22	20	17
Thyroids (%)	Mean	0.010	0.009	0.010	0.010
	St. Dev.	0.002	0.002	0.003	0.003
	N	21	22	20	17
Thymus (%)	Mean	0.08	0.08	0.08	0.07
	St. Dev.	0.02	0.02	0.02	0.02
	N	21	22	20	16
Kidneys (%)	Mean	0.49	0.49	0.48	0.50
	St. Dev.	0.04	0.04	0.04	0.04
	N	21	22	20	17
Adrenals (%)	Mean	0.007	0.007	0.007	0.007
	St. Dev.	0.002	0.002	0.001	0.002
	N	21	22	20	17
Spleen (%)	Mean	0.05	0.05	0.05	0.05
	St. Dev.	0.01	0.02	0.01	0.01
	N	21	22	20	17
Ovaries (%)	Mean	0.020	0.022	0.021	0.021
	St. Dev.	0.003	0.004	0.002	0.004
	N	21	22	20	17

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 15. Summary of Maternal Survival and Pregnancy Status
	Group 1 Control		Group 2 100 mg/Kg		Group 3 300 mg/Kg		Group 4 600 mg/Kg
	No.	%	No.	%	No.	%	No.	%
Females on Study	22		22		22		22
Females that Aborted or Delivered	0	0.0	0	0.0	0	0.0	0	0.0
Females that Died	0	0.0	0	0.0	0	0.0	0	0.0
Females that Aborted	0	0.0	0	0.0	0	0.0	0	0.0
Non gravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	0	0.0	0	0.0	0	0.0
Females that were Euthanized	1	4.5	0	0.0	1	4.5	2	9.1
Non gravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	1	100.0	0	0.0	1	100.0	2	100.0
Females examined at Scheduled Necropsy	21	95.5	22	100.0	21	95.5	20	90.9
Non gravid	0	0.0	1	4.5	1	4.8	3	15.0
Gravid	21	100.0	21	95.5	20	95.2	17	85.0
with Resorptions only	0	0.0	0	0.0	0	0.0	0	0.0
with Viable fetuses	21	100.0	21	100.0	20	100.0	17	100.0
Total Females Gravid	22	100.0	21	95.5	21	95.5	19	86.4

Table 16. Summary of Fetal Data at Necropsy
Group		Sex		Viable Fetuses	Dead Fetuses	Resorptions		Post Implantation Loss	Implantation Sites	Corpora Lutea	Pre Implantation Loss	Fetal Weight in grams	No. of Gravid Females
		M	F			Early	Late
1 Control	Total	107	99	206	0	10	5	15	221	226	5	NA	21
	Mean	5.1	4.7	9.8	0.0	0.5	0.2	0.7	10.5	10.8	0.2	40.0
	S.D.	1.76	1.71	2.62	0.00	1.17	0.62	1.31	2.34	2.34	0.77	5.60
2 100 mg/Kg	Total	104	82	186	0	8	7	15	201	212	11	NA	21
	Mean	5.0	3.9	8.9	0.0	0.4	0.3	0.7	9.6	10.1	0.5	42.7
	S.D.	2.09	1.73	2.35	0.00	0.80	0.73	1.01	2.18	1.41	1.57	4.14
3 300 mg/Kg	Total	98	89	187	0	7	10	17	204	219	15	NA	20
	Mean	4.9	4.5	9.4	0.0	0.4	0.5	0.9	10.2	11.0	0.8	41.0
	S.D.	2.17	1.79	2.11	0.00	0.75	0.76	0.99	1.88	1.43	1.71	5.25
4 600 mg/Kg	Total	86	90	176	0	5	5	10	186	191	5	NA	17
	Mean	5.1	5.3	10.4	0.0	0.3	0.3	0.6	10.9	11.2	0.3	36.8
	S.D.	1.34	1.61	1.80	0.00	0.59	0.47	0.62	1.78	1.52	0.59	6.66

None significantly different from control group

NA = Not applicable

Mean Number of Viable Fetuses, Mean Number of Implantation Sites, Mean Number of Corpora Lutea, Fetal Weights compared using Dunnett’s Test.

Table 17. Summary of Fetal Data at Necropsy (% per Litter)
		Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg
Corpora Lutea	Mean	10.8	10.1	11.0	11.2
	St. Dev.	2.34	1.41	1.43	1.52
	N	21	21	20	17
Implantation Sites	Mean	10.5	9.6	10.2	10.9
	St. Dev.	2.34	2.18	1.88	1.78
	N	21	21	20	17
Viable Fetuses (%)	Mean	93.1	92.5	91.6	94.6
	St. Dev.	12.51	10.38	9.94	6.60
	N	21	21	20	17
Dead Fetuses (%)	Mean	0.0	0.0	0.0	0.0
	St. Dev.	0.00	0.00	0.00	0.00
	N	21	21	20	17
Early Resorptions (%)	Mean	5.0	4.0	3.5	3.0
	St. Dev.	11.57	8.78	7.98	6.64
	N	21	21	20	17
Late Resorptions (%)	Mean	1.9	3.5	4.9	2.4
	St. Dev.	5.13	7.19	7.35	3.85
	N	21	21	20	17
Total Resorptions (%)	Mean	6.9	7.5	8.4	5.4
	St. Dev.	12.51	10.38	9.94	6.60
	N	21	21	20	17
Pre-Implantation Loss (%)	Mean	2.1	5.5	6.4	2.9
	St. Dev.	7.03	17.18	15.11	5.79
	N	21	21	20	17
Post-Implantation Loss (%)	Mean	6.9	7.5	8.4	5.4
	St. Dev.	12.51	10.38	9.94	6.60
	N	21	21	20	17
Males (%)	Mean	52.0	56.6	51.0	49.1
	St. Dev.	11.2	19.65	20.95	10.76
	N	21	21	20	17
Females (%)	Mean	48.0	43.4	49.0	50.9
	St. Dev.	11.2	19.65	20.95	10.76
	N	21	21	20	17
Male Fetal Weights (g)	Mean	40.0	44.0	40.5	37.7
	St. Dev.	5.42	4.62	4.85	7.29
	N	21	21	19	17
Female Fetal Weights (g)	Mean	39.6	40.6	41.3	36.0
	St. Dev.	6.38	3.85	6.29	6.74
	N	21	21	20	17
Combined Fetal Weights (g)	Mean	40.0	42.7	41.0	36.8
	St. Dev.	5.60	4.14	5.25	6.66
	N	21	21	20	17
Male Placenta (g)	Mean	4.65	4.70	4.69	4.29
	St. Dev.	0.874	1.330	0.925	1.315
	N	21	21	19	17
Female Placenta (g)	Mean	4.43	4.20	4.55	3.88
	St. Dev.	1.070	0.809	0.950	1.181
	N	21	20	20	17

Proportional (%) Data Compared Using the Mann-Whitney Test

Fetal Weights, Corpora Lutea, and Implantation Sites compared using Dunnett’s Test

None significantly different from control group

Table 18. Summary of Fetuses and Litters with Malformations (Absolute No.)
Dose Group:	Fetuses				Litters
	Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg	Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg
Malformations
Number Examined Externally	206	186	187	176	21	21	20	17
Acrania	0	1	0	0	0	1	0	0
Carpal and/or Tarsal Flexure	0	1	0	0	0	1	0	0
Facial Cleft	0	0	1	0	0	0	1	0
Number Examined Viscerally	206	186	187	176	21	21	20	17
Hydrocephaly – internal	0	0	1	1	0	0	1	1
Kidney(s) – malpositioned	0	0	2	1	0	0	1	1
Liver – abnormal lobation	4	1	0	0	2	1	0	0
Diaphragmatic hernia	0	2	0	0	0	2	0	0
Testis – malpositioned	1	0	0	0	1	0	0	0
Teratology of Fallot	1	0	0	0	1	0	0	0
Transposition of the great vessels	0	0	1	0	0	0	1	0
Aortic arch – narrow	0	0	0	1	0	0	0	1
Atria septum defect	0	0	0	1	0	0	0	1
Aortic arch – retroesophageal	0	0	0	1	0	0	0	1
Left carotid originating from the pulmonary trunk	0	0	0	1	0	0	0	1
Number examined skeletally	206	-	-	176	21	-	-	17
Coastal cartilage anomaly	1	-	-	0	1	-	-	0
Sternal anomaly	0	-	-	1	0	-	-	1
Bent limb bone(s)	1	-	-	0	1	-	-	0
Vertebral centra anomaly	0	-	-	1	0	-	-	1
Total number with malformations
External	0	1	1	0	0	1	1	0
Soft tissue	6	2	4	3	4	2	3	3
Skeletal	2	-	-	2	2	-	-	2
Combined	8	2	4	3	6	2	3	3

Table 19. Summary of Fetuses and Litters with Variations (Absolute No.)
Dose Group:	Fetuses				Litters
	Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg	Group 1 Control	Group 2 100 mg/Kg	Group 3 300 mg/Kg	Group 4 600 mg/Kg
Variations
Number Examined Externally	206	186	187	176	21	21	20	17
Number with Findings	0	0	0	0	0	0	0	0
Number Examined Viscerally	206	186	187	176	21	21	20	17
Ovary – Cyst(s)	2	2	1	0	2	2	1	0
Left carotid – originating from brachiocephalic trunk	11	7	6	6	5	4	3	3
Retrocaval ureter	5	9	4	1	4	6	3	1
Aortic arch – supernumerary artery	0	0	3	1	0	0	3	1
Lung – absent accessory lobe	2	7	4	1	2	4	2	1
Right subclavian –retroesophageal	3	0	0	0	2	0	0	0
Liver – Cyst(s)	2	0	0	1	2	0	0	1
Gall bladder – absent or small	3	0	0	1	1	0	0	1
Lung – discoloured	0	0	0	1	0	0	0	1
Gall bladder – bilobed	0	0	1	0	0	0	1	0
Liver – small supernumerary lobe(s)	0	1	0	0	0	1	0	0
Number examined skeletally	206	-	-	176	21	-	-	17
13^thRudimentary Rib(s)	22	-	-	17	15	-	-	10
13^thFull Rib(s)	118	-	-	102	21	-	-	15
Sternebra(e) malaligned	20	-	-	11	9	-	-	6
Metacarpal(s) and/or Metatarsal(s) unossified	7	-	-	14	5	-	-	7
Sternebra(e) #5 and/or #6 unossified	39	-	-	28	10	-	-	14
Pelvic girdle – caudal shift	53	-	-	63	16	-	-	14
Sternebrae – fused	0	-	-	2	0	-	-	2
Reduced ossification of the skull	1	-	-	1	1	-	-	1
Tarsal(s) unossified	2	-	-	4	2	-	-	3
Pubis – unossified	2	-	-	2	2	-	-	2
Sternebra (A) - wide	1	-	-	0	1	-	-	0
Hyoid body and/or arches unossified	1	-	-	1	1	-	-	1
Sternebra(e) branched	1	-	-	0	1	-	-	0
Skull – supernumerary site	1	-	-	1	1	-	-	1
Supernumerary sternebra	1	-	-	0	1	-	-	0
Vertebral centra – reduced ossification	0	-	-	2	0	-	-	2
Caudal vertebral anomaly	2	-	-	3	2	-	-	3
Vertebral centra anomaly	1	-	-	0	1	-	-	0
Rib(s) – short	1	-	-	0	1	-	-	0
Skull bone – unossified line	0	-	-	1	0	-	-	1

Applicant's summary and conclusion

Conclusions:

Based on the lack of adverse treatment-related effects observed at the highest dose tested in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Tall Oil in rabbits was determined to be 600 mg/Kg/day.

Executive summary:

A key Guideline OECD 414 prenatal developmental toxicity study was conducted to evaluate the potential of the test material (Tall Oil) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested. Time-mated female New Zealand White rabbits (22 females / dose) were exposed to the test material (in 1% (w/v) Aqueous carboxymethyl cellulose with 0.1% (v/v) Tween 80 vehicle) at 0, 100, 300, or 600 mg/Kg/day via oral gavage once daily, 7 days a week, from Day 6 to Day 28 post-coitum, inclusive.

Parameters such as mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, organ weights, number of corpora lutea, (gravid) uterine weight and uterine contents were evaluated in this study for the F0-generation. For the F1 generation, the parameters evaluated included the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, placenta weights, sex ratio, and external, visceral and skeletal malformations and developmental variations.

 

No treatment-related mortality was observed during the study period. However, four animals were sacrificed in extremis (one each in the control and 300 mg/Kg/day group, and two in the 600 mg/Kg/day group). Red fluid on the manure tray was observed for two females in the 300 mg/Kg/day dose group. However, as both females were pregnant with viable fetuses only, this observation was considered to be of no toxicological relevance.Piloerection was observed in 2 of 20 females surviving until scheduled necropsy in the 600 mg/Kg/day dose group. Additionally, reduced faeces production was observed with a dose-relationship concerning duration, severity and incidence.

 

Lower mean food consumption was observed in animals at 300 and 600 mg/Kg/day accompanied by a slightly lower body weight gain at 600 mg/Kg/day. As values remained within available historical control data and as the test material is an oily substance, it is most likely that this decrease in food consumption was caused by the high caloric value of the substance, instead of being a result of toxicity. The increase in triglycerides levels was also considered to be an effect of the characteristics of the test material itself, and not a result of treatment-related toxicity. Increased liver-to-body weight ratios were observed in animals exposed to the test material at 300 and 600 mg/Kg/day but were considered to be of no toxicological relevance as the magnitude of change was minimal. Remaining maternal parameters evaluated such as macroscopic examination, remaining organ weights, remaining clinical chemistry parameters and haematology parameters were unaffected by treatment with no toxicologically significant changes observed through the study period.

 

Slightly lower fetal body weights and placental weights were observed in male and female fetuses at 600 mg/Kg/day, with a more pronounced effect in females. Although no statistical significance was reached and values remained within the available historical control data, lower placental weights were observed for female fetuses that presented with a slightly lower body weight. Based on the small magnitude of change, this effect was considered to be non-adverse. An increased incidence for unossified tarsals was observed in fetuses at 600 mg/kg/day when compared to concurrent controls, caused by four affected fetuses originating from three different litters. These fetuses were observed with body weights below their mean litter weights and the mean group litter weight and therefore, this was considered to be a consequence of low fetal weights rather than being a direct treatment-related effect. Remaining developmental parameters evaluated such as litter size, pre- and post-implantation loss, sex ratio, external, visceral and skeletal malformations and/or developmental variations were unaffected by treatment with no toxicologically significant changes observed through the study period.

 

Based on the lack of adverse treatment-related effects observed at the highest dose tested in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Tall Oil in rabbits was determined to be 600 mg/Kg/day.