Renewable hydrocarbons (kerosene type fraction)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-05-18 to 2020-10-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read across justification in Section 13

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Deviations did not affect the overall integrity of the study or the interpretation of the study results and conclusions.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Deviations did not affect the overall integrity of the study or the interpretation of the study results and conclusions.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Deviations did not affect 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

Reference substance name:

Renewable hydrocarbons (diesel type fraction)

EC Number:

700-571-2

Cas Number:

928771-01-1

Molecular formula:

C10-20H22-42

IUPAC Name:

Renewable hydrocarbons (diesel type fraction)

Details on test material:

- Name of test material (as cited in study report): NExBTL Renewable diesel
- Description: clear colourless liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Neste Renewable Diesel (Neste Oyj (Keilaranta 21, Espoo, PL95, 00095 Neste, Finland); Batch (Lot) Number: K31/NEXBTL-32
- Expiration date of the lot/batch: 2024-06-12
- Purity test date: 2019-06-12

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under storage conditions: Considered stable for 6 hours at ambient temperature
- Stability under test conditions: Considered stable for 6 hours at ambient temperature
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Stable for at least 6 hours at room temperature under normal laboratory light conditions, for at least 8 days in a refrigerator (2-8°C), and for at least 3 weeks in a freezer (≤ -15°C)

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

OTHER SPECIFICS
- Specific gravity / density: 780.2 kg/m3 at 15°C
- Stability in vehicle: Arachis oil - Stability for at least 6 hours at room temperature under normal laboratory light conditions, for at least 8 days in a refrigerator (2-8°C), and for at least 3 weeks in a freezer (≤ -15°C), has been confirmed over the concentration range 50 to 700 mg/mL (suspensions), project 20223624.

Test animals

Species:

rabbit

Strain:

New Zealand White

Remarks:

time-mated female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 18-20 weeks old
- Weight at study initiation: 3063 and 4348 g at the initiation of dosing
- Fasting period before study: Not specified
- Housing: housed individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles
- Diet (e.g. ad libitum): On the day of arrival, animals received approximately 25 grams of pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Kliba NAFAG Granovit AG, Kaiseraugst, Swizerland). During the remainder of the study, the animals received 140-160 grams food per day, except during designated procedures. In addition, 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 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 21°C
- Humidity (%): 40 to 70%
- 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: 2020-05-06; 2020-05-08 To: 2020-10-01

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The vehicle (arachis oil) was administered as received. An adequate amount of the vehicle was dispensed into daily aliquots. In the low and mid-dose groups, test material dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.

In the high-dose group, the test material (Neste Renewable Diesel) was administered as a solution as received. An adequate amount of the test material was dispensed into daily aliquots, which were stored the same as for the bulk test material until use.

The test material and test material dosing formulations were kept at room temperature 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 vehicle and test material.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle used in the current study was different than vehicle used in the DRF study (Charles River France Safety Assessment SAS Test Facility Study No. 20210639). This was because the test material formulations prepared with propylene glycol at CRL Den Bosch were considered to be not visually homogeneous. Consequently, an additional trial preparation was performed to select an appropriate vehicle for the main study. These trials were not performed as part of this study and these preparations were not used for dosing. Based on the results, arachis oil was selected for use as a vehicle for preparation of the formulations for the current study.
- Concentration in vehicle: 0, 100, 300, or 780.2 mg/mL for the control, low-dose, mid-dose, and high dose group, respectively
- Amount of vehicle (if gavage): 1 mL/Kg for the control, low-dose, and mid-dose groups and 1.28 mL/Kg for the high dose group, respectively.
- Lot/batch no. (if required): Source: Fagron (Capelle aan de IJssel, The Netherlands)
- Purity: Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Method
Analyses were performed by using a validated analytical procedure (Test Facility Study No. 20223624).

- Concentration Analysis
Duplicate sets of middle samples for Groups 1 to 3 (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration.

- Homogeneity Analysis
Duplicate sets of top, middle and bottom samples for groups 2 and 3 (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 Study No. 20223624) 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 Study No. 20223624.

Details on mating procedure:

Time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France). 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:

From Day 7 to Day 28 post-coitum

Frequency of treatment:

once daily oral gavage 7 days a week from Day 7 to Day 28 post-coitum

Duration of test:

From Day 7 to Day 28 post-coitum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control (Arachis Oil - Group 1)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low Dose (Group 2)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid Dose (Group 3)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High Dose (Group 4)

No. of animals per sex per dose:

22 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test material.

The dose levels were selected based on the results of a dose range finding study (DRF) performed at Charles River France Safety Assessment SAS (Test Facility Study No. 20210639), in an attempt to produce graded responses to the test material. In this DRF, dose levels of 250, 500 and 1000 mg/Kg/day in propylene glycol were tested. No toxicity was observed up to the highest dose level tested and therefore, dose levels of 100, 300 and 1000 mg/Kg/day were selected for use in the main OECD 414 study. These dose levels were also tested in the OECD 414 study in the rat without apparent toxicity up to 1000 mg/Kg/day.

- Rationale for animal assignment (if not random): At arrival, 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: Performed at least once daily, beginning on Day 7 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. Cage debris was examined to detect abortion or premature birth.

BODY WEIGHT: Yes
- Time schedule for examinations:Animals were individually weighed on Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum. In order to monitor the health status, Animal No. 66 was also weighed on Day 8 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was quantitatively measured daily from Day 3 post-coitum onwards.

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
- Sacrifice on gestation 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). No tissues, except the uterus, were weighed.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not for animals found dead or sacrificed before planned necropsy)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
- The number and distribution of live and dead fetuses

Fetal examinations:

- External examinations: Yes: [all per litter]
For late resorptions and recognizable fetuses in development of females found dead, sacrificed before planned necropsy or that delivered on or before the day of scheduled necropsy, a gross external examination was performed.

Litters of females surviving to scheduled necropsy were subjected to detailed external examinations. Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight was determined (not for fetuses of animals found dead or sacrificed before planned necropsy). Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed.

- Soft tissue examinations: Yes: [all per litter]

Litters of females surviving to scheduled necropsy were subjected to detailed visceral examinations. 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 using a technique described by Stuckhardt and Poppe (1984). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar (1972).

The eyes of Fetus A89-03 and livers of Fetus A022-01, A041-02 and A085-08 were collected and fixed in 10% buffered formalin at discretion of the Study Director. 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 (1965). After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. 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]
Litters of females surviving to scheduled necropsy were subjected to detailed skeletal examinations.

All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson (1926).

Subsequently, the skeletal examination was done on all fetuses from all groups. All specimens were archived in glycerin with bronopol as preservative. 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: Not specified

Statistics:

For information on statistics, please see section 'Any other information on materials and methods, incl. tables'.

Indices:

Maternal Variables
1) Body Weight Gains: Calculated against the body weight on Day 7 post-coitum.

2) Corrected Body Weight Gains: Body weight determined on Day 29 post-coitum minus the body weight on Day 7 post-coitum and the weight of gravid uterus.

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

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:

Historical Control Data is presented in Appendix 3 of the final study report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were observed through the study period. Clinical signs that were observed, related to the premature deaths of individual females and have been described in the mortality section below.

Any other clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No treatment-related mortality was observed during the study period.

In total six animals did not survive until scheduled necropsy as detailed below:

- Female No. 66 (300 mg/Kg dose group) was sacrificed in extremis on Day 8 post-coitum due to absent food consumption for a prolonged period in combination with severe body weight loss. As this mainly occurred prior to start of treatment, this was considered unrelated to treatment.

- Female No. 36 (100 mg/kg/day dose group) was sent to necropsy on Day 25 post-coitum after organic material was found on the manure tray, indicative of a possible early delivery. However, at necropsy, 12 dead fetuses and an early resorption were found in utero. Given the incidental nature and in the absence of a dose relationship, this was considered unrelated to treatment.

The deaths of the following four females were considered related to the gavage procedure and unrelated to treatment.

- Female No. 78 (1000 mg/kg/day dose group) was killed in extremis on Day 16 post coitum. Prior to dosing she was found pale and showed hunched posture. After dosing her posture became abnormal and it was decided to sacrifice this female for animal welfare reasons. At necropsy, foamy contents was observed in the trachea and dark red discoloration and foci were noted in the lungs.

- Female No. 45 (300 mg/kg/day dose group) was killed in extremis on Day 16 post-coitum, shortly after the dosing procedure. Blood was noted on the dosing tube and the female had laboured respiration and rales after dosing. At necropsy, foamy contents was observed in the trachea and a dark red discoloration of the lungs on the left side.

- Female No. 56 (300 mg/kg/day dose group) died shortly after dosing on Day 17 post-coitum. Blood was noted on the dosing tube. At necropsy, foamy contents was observed in the trachea and many dark red foci were noted in the lungs.

- Female No. 2 (Control group) was found dead on Day 18 post-coitum. On Day 17 post-coitum, blood was noted on the dosing tube and laboured respiration was noted. This female remained on study, but was found dead the next day. At necropsy, irregular surface and black/brown discoloration of the right lung was noted. The thoracic cavity contained watery clear fluid. For this female, dosing was omitted on Day 7 post-coitum as blood emerged from the nose on the first day of dosing.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment with the test material. From Day 15 post-coitum onwards, body weight gain was slightly increased at 1000 mg/Kg/day compared to concurrent controls, statistically significant on Day 15 and 21 post-coitum only. Given the direction of change and the minimal size of the effect it was not considered to be treatment-related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes in food consumption before or after correction for body weight were recorded.

Between Days 12 and 20 post-coitum, food consumption in the control group was slightly reduced compared to previous periods, whereas for the treatment groups food consumption remained similar throughout this part of the treatment period. This resulted in incidental statistically significantly increased food consumption at 300 and 1000 mg/Kg/day. Given the direction of change and the minimal size of the effect it was not considered to be treatment-related.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross necropsy did not reveal any remarkable findings. Macroscopic findings related to the premature deaths of individual females have been described in the mortality section above.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre- and post-implantation loss in the control and treatment groups were similar and in the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

4 females at 1000 mg/Kg/day were not pregnant at scheduled necropsy compared to 1 in the control and 100 mg/Kg/day groups, and 2 in the 300 mg/Kg/day group. As treatment started after implantation, this was considered a spurious finding and unrelated to treatment with the test material.

Other effects:

no effects observed

Description (incidence and severity):

The numbers of corpora lutea and implantation sites in the control and treatment groups were similar and in the range of normal biological variation.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: Systemic Toxicity

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on fetal body weights (both sexes) were observed up to 1000 mg/Kg/day. Notably, mean fetal weights (male, female and combined) in concurrent controls were slightly lower compared to mean weights in the treatment groups and at the lower end of the range of the historical control data. However, the mean fetal weights were comparable in all treatment groups and were comparable to the historical control mean.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The numbers of fetuses (litters) available for fetal morphological examination were 179 (20), 179 (20), 144 (17) and 166 (17) in the control, 100, 300 and 1000 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 1000 mg/Kg/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed on litter size of any group.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on external morphology following treatment with the test material up to 1000 mg/Kg/day.

External malformations occurred in three fetuses from three litters each at the control and high dose level and in one fetus each at 100 and 300 mg/Kg/day.

Malformations observed at the high dose were omphalocele, gastroschisis and distended abdomen (Fetus A074-11, A083-09 and A085-08, respectively). Omphalocele also occurred at 300 mg/Kg/day in one fetus (A052-12) and a distended abdomen was observed for two control fetuses (A019-08 and A020-10). Both omphalocele and distended abdomen have been observed in historical control fetuses Together with the single occurrence of these malformations in the treatment groups, they were considered unrelated to treatment. The fetus with gastroschisis (A083-09) was also malformed viscerally (abnormal liver lobation and opacity of the eyes) and skeletally (vertebral anomaly and sternoschisis) and was as such considered a spurious finding. The malformed fetus at 100 mg/Kg/day (A026-10) had arhinencephaly and a short tail, both with matching skeletal findings. Due to the single occurrence in a low dose fetus, these malformations were considered chance findings.

The remaining control fetus with an external malformation (A012-05) had malrotated hindlimbs without skeletal origin. External variations were not observed in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on skeletal morphology were observed following treatment up to 1000 mg/Kg/day.

Besides the anomalies associated with external malformations in fetuses, five different malformations were observed.

The malformations observed at 1000 mg/Kg/day were a costal cartilage anomaly (Fetus A069-07), sternoschisis and vertebral anomaly (both in Fetus A083-09, considered a spurious finding due to multiple anomalies).

A costal cartilage anomaly was also observed in a 100 mg/Kg/day fetus (A030-02) and vertebral anomalies were also noted in a 300 mg/Kg/day (Fetus A047-11) and in five fetuses at 100 mg/Kg/day (A034-01, -11, A038-08, A040-05 and -12).

The two remaining malformations were caudal vertebral anomaly and ectrodactyly with both occurring in 100 mg/Kg fetuses (A026-11 and A028-02, respectively). The high number of vertebral anomalies observed at 100 mg/Kg/day led to a mean litter incidence that was higher than the historical control maximum value (2.2% versus 1.7% per litter, respectively). However, as this was observed in the low dose group, it was considered unrelated to treatment with the test material. Other malformations in this study occurred singly and in the absence of a dose-related response and were therefore considered chance findings.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment with the test material up to 1000 mg/Kg/day. At 1000 mg/Kg/day, besides the multiple malformed fetus A083-09 described earlier, Fetus A079-01 had tetralogy of Fallot. This heart anomaly was also observed in Fetus A052-03 in the 300 mg/Kg/day dose group. Due to the single occurrence and because it is seen previously in historical control fetuses, this finding was considered a chance finding.

The other two malformed fetuses at 300 mg/Kg/day were from Female No. A047. Both fetuses (A047-02 and -06) had abnormal lung lobation and the latter one also had diaphragmatic hernia. The fact that abnormal lung lobulation occurred in only one litter at the mid dose does not indicate a treatment relationship and as both malformations were listed in historical control data, they were considered unrelated to treatment with the test material. The anomalous fetus at 100 mg/Kg/day (A038-06) had malpositioned kidneys that due to the single occurrence was considered a chance finding.

In the control group, one of the fetuses was observed with a distended abdomen (A020-10) and also appeared to have a large atrium. The incidences for absent or small gallbladder at 300 and 1000 mg/Kg/day were significantly increased (statistically) compared to concurrent control. No clear dose response was observed and incidences at 300 and 1000 mg/Kg/day were 2.8 and 2.9% per litter, respectively. The mean values remained within the historical control maximum value (3.1% per litter). Therefore, the increased incidence of absent or small gallbladders was considered unrelated to treatment.

There were increased mean litter incidences of retrocaval ureter in the 300 and 1000 mg/Kg/day groups, but not statistically significant. Retrocaval ureters were observed in 0.5%, 2.6%, 6.5% and 4.9%, per litter in the control, 100, 300 and 1000 mg/kg/day groups, respectively. As values were above the historical control upper limit (4.1% per litter) at 300 and 1000 mg/Kg/day, a treatment-related effect could not be excluded. It should be noted, that this variation is the most commonly seen variation in the historical control data. All other variations noted were considered unrelated to treatment 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:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental Toxicity

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Dose Formulation Analyses

Accuracy

The concentrations analyzed in the formulations of Group 2 and Group 3 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). A small response at the retention time of the test material was observed in the chromatograms of the Group 1 formulation. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks. .

 

Homogeneity

The formulations of Group 2 and Group 3 were homogeneous (i.e. coefficient of variation ≤ 10%).

Table 2. Summary of Body Weights (F0 Generation)
Sex: Female		Post Coitum (Day)
		0	7	9	12	15	18	21	24	27	29
Group 1 0 mg/Kg/day	Mean	3743	3670	3710	3780	3864	3897	3905	3952	3990	4022
	SD	326.6	302.2	301.7	315.8	303.8	280.4	326.2	327.0	313.4	298.6
	N	21	21	21	21	21	20	20	20	20	20
Group 2 100 mg/Kg/day	Mean	3618	3579	3619	3681	3785	3828	3840	3872	3933	3988
	SD	225.2	201.4	209.9	208.2	224.5	232.0	231.1	236.6	229.3	222.0
	N	21	21	21	21	21	21	21	21	20	20
Group 3 300 mg/Kg/day	Mean	3595	3548	3578	3650	3753	3830	3834	3894	3952	3989
	SD	304.1	256.1	276.3	283.2	287.6	286.7	267.8	254.0	243.9	259.8
	N	20	20	19	19	19	17	17	17	17	17
Group 4 1000 mg/Kg/day	Mean	3588	3573	3629	3703	3848	3887	3905	3952	3981	4012
	SD	305.2	274.1	281.8	388.8	305.2	289.6	274.9	243.9	268.1	266.1
	N	18	18	18	18	18	17	17	17	17	17

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

 

Table 3. Summary of Body Weight Gain (%) - F0 Generation
Sex: Female		Post Coitum (Day)
		7	9	12	15	18	21	24	27	29
Group 1 0 mg/Kg/day	Mean	0	1	3	5	6	6	8	9	10
	SD	0.0	0.9	1.5	2.6	3.9	3.7	3.7	4.6	5.4
	N	21	21	21	21	21	20	20	20	20
Group 2 100 mg/Kg/day	Mean	0	1	3	6	7	7	8	10	12
	SD	0.0	1.3	1.7	2.6	2.8	2.9	3.2	3.1	3.1
	N	21	21	21	21	21	21	21	20	20
Group 3 300 mg/Kg/day	Mean	0	0	2	5	7	7	8	10	11
	SD	0.0	2.1	1.9	2.2	2.3	2.6	2.8	3.2	3.5
	N	20	19	19	19	19	17	17	17	17
Group 4 1000 mg/Kg/day	Mean	0	2	4	8**	8	9*	10	11	12
	SD	0.0	0.9	0.9	1.8	2.1	2.3	3.4	4.3	4.9
	N	18	18	18	18	18	17	17	17	17

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

 

Table 4. Summary of Relative Food Consumption (g/Kg Body Weight/Day) – F0 Generation
Post-Coitum		Group 1 0 mg/Kg/day	Group 2 100 mg/Kg/day	Group 3 300 mg/Kg/day	Group 4 1000 mg/Kg/day
Days 3-4	Mean	39	36	36	40
	SD	7.5	12.4	13.6	7.9
	N	21	21	20	18
Days 4-5	Mean	42	40	40	43
	SD	4.3	9.6	10.6	2.8
	N	21	21	20	18
Days 5-6	Mean	42	42	40	44
	SD	6.0	4.1	10.4	2.9
	N	21	21	20	18
Days 6-7	Mean	41	42	41	44
	SD	4.2	3.5	10.1	3.5
	N	21	21	20	18
Days 7-8	Mean	40	40	37	43
	SD	6.7	6.6	11.5	2.8
	N	21	21	20	18
Days 8-9	Mean	36	38	35	41
	SD	9.5	7.3	13.2	4.5
	N	21	21	19	18
Days 9-10	Mean	37	39	36	42
	SD	7.2	5.8	12.0	4.0
	N	21	21	19	18
Days 10-11	Mean	38	38	36	41
	SD	4.6	6.2	8.0	3.8
	N	21	21	19	18
Days 11-12	Mean	37	36	36	40
	SD	6.0	8.2	8.9	6.2
	N	21	21	19	18
Days 12-13	Mean	33	34	37	39
	SD	10.6	10.1	7.2	6.7
	N	21	21	19	18
Days 13-14	Mean	29	32	29	36
	SD	10.9	9.8	11.7	7.2
	N	21	21	19	18
Days 14-15	Mean	25	32	27	35**
	SD	11.5	8.8	12.5	6.0
	N	21	21	19	18
Days 15-16	Mean	26	32	29	32
	SD	12.4	8.8	11.4	11.6
	N	21	21	19	18
Days 16-17	Mean	28	31	34	35*
	SD	11.9	10.6	6.6	7.9
	N	20	21	17	17
Days 17-18	Mean	29	33	35	35
	SD	12.9	7.3	6.8	7.5
	N	20	21	17	17
Days 18-19	Mean	27	30	35*	35*
	SD	12.0	8.3	5.7	7.7
	N	20	21	17	17
Days 19-20	Mean	28	31	34	34
	SD	10.3	6.8	6.3	9.3
	N	20	21	17	17
Days 20-21	Mean	31	31	32	33
	SD	6.9	7.7	7.4	7.8
	N	20	21	17	17
Days 21-22	Mean	32	30	32	30
	SD	7.2	6.5	7.3	7.6
	N	20	21	17	17
Days 22-23	Mean	28	30	30	29
	SD	7.8	6.7	6.7	7.2
	N	20	21	17	17
Days 23-24	Mean	26	29	28	27
	SD	7.5	5.2	7.1	8.1
	N	20	21	17	17
Days 24-25	Mean	24	27	27	25
	SD	8.6	8.8	6.7	7.8
	N	20	21	17	17
Days 25-26	Mean	24	28	25	25
	SD	8.0	8.5	10.4	8.4
	N	20	20	17	17
Days 26-27	Mean	23	28	27	25
	SD	7.7	7.2	9.0	7.7
	N	20	20	17	17
Days 27-28	Mean	22	28*	28	25
	SD	8.8	8.5	6.9	9.6
	N	20	20	17	17
Days 28-29	Mean	23	28	27	27
	SD	8.5	6.3	10.0	9.0
	N	20	20	17	17
Mean of Means		31	33	33	35

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

 

Table 5. Summary of Maternal Survival and Pregnancy Status
Dose Group	Group 1 0 mg/Kg/day		Group 2 100 mg/Kg/day		Group 3 300 mg/Kg/day		Group 4 1000 mg/Kg/day
	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	1	4.5	0	0.0	1	4.5	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	1	100.0	0	0.0	1	100.0	0	0.0
Females that were euthanized	0	0.0	1	4.5	2	9.1	1	4.5
Non gravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	1	100.0	2	100.0	1	100.0
Females examined at scheduled necropsy	21	95.5	21	95.5	19	86.4	21	95.5
Non gravid	1	4.8	1	4.8	2	10.5	4	19.0
Gravid	20	95.2	20	95.2	17	89.5	17	81.0
With Resorptions only	0	0.0	0	0.0	0	0.0	0	0.0
With Viable fetuses	20	100.0	20	100.0	17	100.0	17	100.0
Totals Females Gravid	21	95.5	21	95.5	20	90.9	18	81.8

 

Table 6. Summary of Fetal Data at Scheduled 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
		Male	Female			Early	Late
Group 1 0 mg/Kg/day	Total	93	86	179	1	6	13	20	199	227	28	NA	20
	Mean	4.7	4.3	9.0	0.1	0.3	0.7	1.0	10.0	11.4	1.4	37.5
	S.D.	2.35	2.13	2.21	0.22	0.73	1.27	1.62	2.58	1.79	2.19	5.27
Group 2 100 mg/Kg/day	Total	84	95	179	1	10	10	21	200	237	37	NA	20
	Mean	4.2	4.8	9.0	0.1	0.5	0.5	1.1	10.0	11.9	1.9	38.8
	S.D.	1.54	1.62	2.37	0.22	0.95	0.95	1.61	2.00	2.32	1.87	4.09
Group 3 300 mg/Kg/day	Total	71	73	144	0	6	28	34	178	191	13	NA	17
	Mean	4.2	4.3	8.5	0.0	0.4	1.6	2.0	10.5	11.2	0.8	38.6
	S.D.	1.70	1.90	2.62	0.00	0.61	2.34	2.50	1.62	1.25	0.97	4.79
Group 4 1000 mg/Kg/day	Total	76	90	166	0	8	6	14	180	191	11	NA	17
	Mean	4.5	5.3	9.8	0.0	0.5	0.4	0.8	10.6	11.2	0.6	38.8
	S.D.	1.66	1.45	1.44	0.00	0.62	0.70	1.24	1.46	1.68	0.86	4.00

None significantly different from control groupNA = 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 7. Summary of Fetal at Scheduled Necropsy (% per Litter)
Group		Group 1 0 mg/Kg/day	Group 2 100 mg/Kg/day	Group 3 300 mg/Kg/day	Group 4 1000 mg/Kg/day
Corpora Lutea	Mean	11.4	11.9	11.2	11.2
	S.D.	1.79	2.32	1.25	1.68
	N	20	20	17	17
Implantation Sites	Mean	10.0	10.0	10.5	10.6
	S.D.	2.58	2.00	1.62	1.46
	N	20	20	17	17
Viable Fetuses (%)	Mean	91.3	89.4	81.2	92.7
	S.D.	13.26	15.87	23.73	10.18
	N	20	20	17	17
Dead Fetuses (%)	Mean	0.5	0.3	0.0	0.0
	S.D.	2.03	1.50	0.00	0.00
	N	20	20	17	17
Early Resorptions (%)	Mean	2.5	4.8	3.6	4.4
	S.D.	5.64	8.84	6.41	5.65
	N	20	20	17	17
Late Resorptions (%)	Mean	5.7	5.5	15.2	2.9
	S.D.	10.99	11.22	22.64	5.65
	N	20	20	17	17
Total Resorptions (%)	Mean	8.2	10.3	18.8	7.3
	S.D.	12.73	15.74	23.73	10.18
	N	20	20	17	17
Pre-implantation Loss (%)	Mean	11.9	14.8	6.9	5.4
	S.D.	19.16	13.08	8.68	6.63
	N	20	20	17	17
Post-implantation Loss (%)	Mean	8.7	10.6	18.8	7.3
	S.D.	13.26	15.87	23.73	10.18
	N	20	20	17	17
Males (%)	Mean	52.0	46.9	50.1	45.4
	S.D.	21.08	13.28	16.55	14.04
	N	20	20	17	17
Females (%)	Mean	48.0	53.1	49.9	54.6
	S.D.	21.08	13.28	16.55	14.04
	N	20	20	17	17
Male Fetal Weights (g)	Mean	38.5	39.6	38.7	39.9
	S.D.	4.53	5.42	5.72	5.69
	N	20	20	17	17
Female Fetal Weights (g)	Mean	37.5	38.7	38.5	38.1
	S.D.	6.24	4.01	4.80	3.79
	N	20	20	16	17
Combined Fetal Weights (g)	Mean	37.5	38.8	38.6	38.8
	S.D.	5.27	4.09	4.79	4.00
	N	20	20	17	17

PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

None significantly different from control group

 

Table 8. Overview of Litter Incidence (absolute numbers and % per litter) of External Malformations Observed
Findings	Group 1 0 mg/Kg/day	Group 2 100 mg/Kg/day	Group 3 300 mg/Kg/day	Group 4 1000 mg/Kg/day	Historical Control Data (HCD)
Omphalocele	-	-	1 fetus; 1 litter 0.5 % per litter	1 fetus; 1 litter 0.5 % per litter	Mean = 0.2 Max = 3.0 % per litter
Gastroschisis	-	-	-	1 fetus; 1 litter 0.7% per litter	Not Present
Distended abdomen	2 fetuses; 2 litters 0.9% per litter	-	-	1 fetus; 1 litter 0.7% per litter	Mean = 0.0 Max = 0.6 % per litter
Arhinencephaly	-	1 fetus; 1 litter 0.5 % per litter	-	-	Not Present
Short tail	-	1 fetus; 1 litter 0.5 % per litter	-	-	Mean = 0.0 Max = 0.4 % per litter

- = not observed in this group

 

Table 9. Overview of Litter Incidence (absolute numbers and % per litter) of Visceral Malformations Observed
Findings	Group 1 0 mg/Kg/day	Group 2 100 mg/Kg/day	Group 3 300 mg/Kg/day	Group 4 1000 mg/Kg/day	Historical Control Data (HCD)
Abnormal lobulation lung	-	-	2 fetuses; 1 litter 1.3% per litter	-	Mean = 0.1 Max = 2.7 % per litter
Diaphragmatic hernia	-	-	1 fetus; 1 litter 0.7 % per litter	-	Mean = 0.0 Max = 0.6 % per litter
Tetralogy of fallot	-	-	1 fetus; 1 litter 0.5% per litter	1 fetus; 1 litter 0.7% per litter	Mean = 0.1 Max = 0.8 % per litter
Malpositioned kidneys	-	1 fetus; 1 litter 0.5 % per litter	-	-	Mean = 0.1 Max = 0.9 % per litter
Abnormal lobulation liver	-	-	-	1 fetus; 1 litter 0.7 % per litter	Mean = 0.1 Max = 0.6 % per litter
Eye opacity	-	-	-	1 fetus; 1 litter 0.7 % per litter	Not Present

- = not observed in this group

 

Table 10. Overview of Litter Incidence (absolute numbers and % per litter) of Skeletal Malformations Observed
Findings	Group 1 0 mg/Kg/day	Group 2 100 mg/Kg/day	Group 3 300 mg/Kg/day	Group 4 1000 mg/Kg/day	Historical Control Data (HCD)
Vertebral anomaly	-	5 fetuses; 3 litters 2.2% per litter	1 fetus; 1 litter 0.7 % per litter	1 fetus; 1 litter 0.7 % per litter	Mean = 0.5 Max = 1.7 % per litter
Costal cartilage anomaly	-	1 fetus; 1 litter 0.7 % per litter	-	1 fetus; 1 litter 0.7 % per litter	Mean = 0.0 Max = 0.6 % per litter
Caudal vertebral anomaly	-	1 fetus; 1 litter 0.5 % per litter	-	-	Mean = 0.1 Max = 0.7 % per litter
Ectrodactyly	-	1 fetus; 1 litter 0.4 % per litter	-	-	Not Present
Sternoschisis	-	-	-	1 fetus; 1 litter 0.7 % per litter	Not Present

- = not observed in this group

Applicant's summary and conclusion

Conclusions:

Based on the lack of adverse treatment-related effects observed in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Neste Renewable Diesel in rabbits was determined to be at least 1000 mg/Kg/day.

Executive summary:

A key Guideline OECD 414 developmental toxicity study was conducted to determine the potential of the test material (Neste Renewable Diesel; CAS# 928771-01-1) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits. The test material and vehicle (arachis oil) were administered once daily via oral gavage, 7 days a week at doses of 0, 100, 300, 1000 mg/Kg/day from Day 7 to Day 28 post-coitum, inclusive.

 

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

 

No treatment-related mortality or changes in any of the maternal parameters investigated (i.e. clinical appearance, body weight, food consumption and macroscopic examination) were observed through the study period.

 

Six females did not survive until scheduled necropsy (one in the control group, one at 100 mg/Kg/day, three at 300 mg/Kg/day, and one at 1000 mg/Kg/day). As these premature deaths were considered to have occurred either as a result of the gavage procedure, symptoms occurring prior to start of the treatment, or were considered incidental, they were determined not to be treatment-related.

 

Increased mean litter incidences of retrocaval ureter were observed in the 300 and 1000 mg/Kg/day dose groups. The incidences for retrocaval ureter were not significantly increased (statistically) and had no apparent dose-response. However, as values were above the historical control maximum value at 300 and 1000 mg/Kg/day, a treatment-related effect could not be excluded. As this concerns a variation with no detrimental effects on development, it was considered to be non-adverse.

 

No treatment-related changes were observed in any of the remaining developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights, external, visceral, and skeletal malformations and developmental variations).

 

Based on the lack of adverse treatment-related effects observed in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Neste Renewable Diesel in rabbits was determined to be at least 1000 mg/Kg/day.