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Toxicological information

Developmental toxicity / teratogenicity

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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

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 / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1 (Control)
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 3
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

   13thRudimentary Rib(s)

22

-

-

17

15

-

-

10

   13thFull 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.