Renewable hydrocarbons (diesel type fraction)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-02-25 to 2020-06-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 were considered to not have impacted 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 were considered to not have impacted 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 were considered to not have impacted the overall integrity of the study or the interpretation of the study results and conclusions

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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 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:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Deutschland (Sulzfeld, Germany)
- Age at study initiation: 10-14 weeks old
- Weight at study initiation: 181 - 263 g at the initiation of dosing
- Fasting period before study: Not specified
- Housing: On arrival and following randomization, females were housed individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 22°C
- Humidity (%): 52 to 54%
- 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: 2020-02-18 and 2020-02-20 To: 2020-06-18

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material dosing formulations (w/w) were homogenized by stirring to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 6 hours after removal from the refrigerator.

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): Not specified
- Concentration in vehicle: 0, 50, 150, and 500 mg/mL for the control, low-, mid-, and high-dose groups, respectively.
- Amount of vehicle (if gavage): 2 mL/Kg
- Lot/batch no. (if required): Not specified (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 using a validated analytical procedure (Test Facility Study No. 20223624).

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 ±10% for solutions 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 Study No. 20223624) demonstrated that the test item 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 Wistar Han Rats (Crl:WI(Han)) were received from Charles River Laboratories Deutschland, Sulzfeld, Germany. The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).

Duration of treatment / exposure:

From Day 6 to Day 20 post-coitum, inclusive

Frequency of treatment:

Once daily oral gavage 7 days a week

Duration of test:

Day 6 to Day 20 post-coitum, inclusive

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control (Arachis Oil - Group 1)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low Dose (Group 2)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

Mid Dose (Group 3)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

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 the range finder in the rat (Test Facility Reference No. 20223626) in which 1000 mg/Kg/day was tested. No maternal or developmental toxicity was observed and 0, 100, 300 and 1000 mg/Kg/day were selected as dose levels for the main study. The high-dose level should produce some maternal and/or developmental toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

- 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: Clinical observations were performed at least once daily, beginning on Day 2 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 premature birth.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes. Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-
21 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
- Sacrifice on gestation day # 21 post-coitum
- Organs examined: All animals 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 uterus and thyroid gland were weighed.

OTHER:
- Clinical Pathology: Blood of F0-animals was collected on the day of scheduled necropsy. Animals were not fasted overnight. Samples were collected, between 7:00 and 9:00 a.m., from the jugular vein in the animal facility.

Thyroid Hormone (Immulite)
Blood samples at a target volume of 1.0 mL were collected into tubes without anticoagulant. Blood samples were processed for serum, and serum was analyzed for the parameters listed below:

1) Triiodothyronine (T3)
2) Thyroxine (T4)
3) Thyroid-Stimulating Hormone (TSH)

The values for T3 levels generated using the Immulite were not reported, but kept in study data. Part of the remaining volume was used for measurement of T3 using LC-MS.

Hormone Analysis Triiodothyronine (T3) (LC-MS)
Remaining samples of the Immulite thyroid hormone analysis were analyzed for T3 according to the bioanalytical method validated in Test Facility Study No. 20213516. After receipt, the samples were stored in an ultra-low freezer (≤ -75°C) until analysis.

- Organ Weights – F0-Generation: The thyroid gland and uterus were weighed at necropsy for all animals. Organ weight as a percent of body weight (using the body weight on Day 21 post-coitum) were calculated.

- Histology - F0-Generation
Thyroid glands of all animals were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
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: The sex of each fetus based on the anogenital distance (\not for animals found dead, sacrificed before planned necropsy, or that delivered).

In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.

Fetal examinations:

Live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity using a small metal feeding tube. Litters of females surviving to scheduled necropsy were subjected to detailed external, visceral and skeletal 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 sexed, examined in detail to detect macroscopic visible abnormalities and their weight was determined. The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight. For late resorptions, a gross external examination was performed.

- Soft tissue examinations: Yes: [half per litter]
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were 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).

- Skeletal examinations: Yes: [half per litter]
All fetuses were eviscerated, followed by fixation in 96% aqueous ethanol, and maceration in potassium hydroxide. Thereafter, they were stained with Alizarin Red S by a method similar to that described by Dawson (1926).

Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). As skeletal malformations were suspected for fetus (A014-10), selected for visceral examination, this fetus was also subjected to skeletal examination.

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: Yes: [half per litter]
The heads were removed from one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique (1965).

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 6 post-coitum.

2) Corrected Body Weight Gains: Body weight determined on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus.

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

4) Organ Weight Relative to Body Weight: Calculated against the body weight on Day 21 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:

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 noted during the observation period.

Salivation observed after dosing, which showed a dose-related incidence trend, was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity, likely related to the taste of the test material.

Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats 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 with the test imaterial.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was observed through the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was similar to the control level over the study period.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum levels of total triiodothyronine (T3) were considered unaffected by treatment.

2 females in the control group (Nos. 9 and 17) had serum levels of thyroid stimulating hormone (TSH) well above the upper limit of the historical control range. Consequently, decreased TSH serum levels were noted in all treatment groups, (0.66x, 0.71x and 0.61x of controls for the 100, 300 and 1000 mg/Kg/day groups, respectively). The difference was not statistically significant and all mean values remained within the normal range. There was no dose-response relationship as well and therefore, this finding was not considered to be treatment-related.

At 1000 mg/Kg/day, serum levels of total thyroxine (Total T4) were slightly decreased (0.90x of controls). The effect was considered minimal and no statistical significance was reached. As the mean values remained well within the historical control range, this was also not considered to be treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related thyroid gland weight changes were observed through the study period.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross necropsy did not reveal any remarkable findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic findings in the thyroid glands.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre-implantation loss in the control and test 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

Dead fetuses:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

At scheduled necropsy, two females at 1000 mg/Kg/day were not pregnant (Nos. 73 and 87). All other females were pregnant with viable fetuses. Given that treatment in this type of study starts after implantation, these cases of non-pregnancy were considered incidental and unrelated to treatment.

Other effects:

no effects observed

Description (incidence and severity):

The numbers of corpora lutea and implantation sites in the control and test 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

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):

Fetal weights (male, female and combined) were comparable to the controls for all treatment groups.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

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.

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological examination were 240 (22), 232 (22), 233 (22) and 207 (20) in the control, 100, 300 and 1000 mg/Kg/day groups, respectively.

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

Two fetuses were externally malformed. One fetus (A078-08) in the 1000 mg/Kg bw/day dose group had a small lower jaw and no eye bulges, and one control group fetus (A014-10) missed a digit. Skeletal findings substantiated these malformations, but as they occurred in a control fetus, singly and/or occurred previously in historical control fetuses, they were considered chance findings. No external variations were observed in any dose group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on skeletal morphology following treatment up to 1000 mg/Kg/day.

Skeletal malformations occurred in 2 (2), 2 (2), 0 (0) and 3 (3) fetuses (litters) in the control, 100, 300 and 100 mg/kg/day groups, respectively.

At the high dose, one fetus (A078-08) exhibited a small lower jaw, missing eye bulges, and its underlying anomalies also had a vertebral anomaly. This latter malformation also occurred in the control fetus with ectrodactyly (A014-10) and in a 100 mg/Kg bw/day dose group fetus (A027-10) (together a with small orbit). The single occurrence and group distribution of this anomaly does not indicate a relationship with treatment with the test material and therefore was considered a chance
finding.

Other malformations observed at 1000 mg/Kg bw/day were sternoschisis (fetus A076-08) and bent limb bones (fetus A084-04). Both these findings were also observed in control and/or the 100 mg/Kg/day dose group fetuses. Namely, bent limb bones in fetus A010-04 and A024-02 of the control and 100 mg/Kg groups, respectively, and sternoschisis in the above mentioned control fetus (A014-10- that additionally missed a tibia). Due to the single occurrence and group distribution of bent limb bones and sternoschisis, both were considered chance findings.

Among the variations, 7th cervical full ribs were only observed at 1000 mg/Kg bw/day. Three fetuses from three litters had this supernumerary rib at the cervical-thoracic border leading to an incidence of 3.1% per litter. This was above the historical control maximum value (2.7%). Findings related to a 7th cervical full rib are a decreased litter incidence of 14th full ribs, 14th rudimentary ribs, or caudal shift of the pelvic girdle or an increased litter incidence of 7th cervical ossification sites. However, these parameters were considered unaffected by treatment in this study.

The other variations occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within or near the range of available historical control data. Therefore, they were not considered treatment-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to 1000 mg/Kg/day.

The only viscerally malformed fetus in this study was a control fetus with ectrodactyly (A014-10). This fetus also had a right-sided aortic arch, diaphragmatic hernia, and its intestines were missing. All these malformations were spontaneous in origin as they accumulated in one control fetus.

Two visceral variations that were observed (small supernumerary liver lobes and convoluted ureter) occurred at low incidences and at frequencies that were within the range of available historical control data. Therefore, they were not considered test treatment-related.

Other effects:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on fetal anogenital distance (both sexes) observed after treatment up to 1000 mg/Kg/day.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

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 analysed in the formulations of Group 2 (100 mg/Kg bw/day), Group 3 (300 mg/Kg bw/day), and Group 4 (1000 mg/Kg bw/day) 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 (Control) formulation prepared for use in Week 1. 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 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Table 2. Summary of Body Weights (g) – 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
Day 2	Mean	212	212	209	205
	SD	14.5	16.4	15.4	16.5
	N	22	22	22	20
Day 6	Mean	228	228	225	221
	SD	16.9	16.2	16.9	16.7
	N	22	22	22	20
Day 9	Mean	235	236	232	231
	SD	16.5	16.9	17.4	17.5
	N	22	22	22	20
Day 12	Mean	250	252	248	247
	SD	17.0	17.5	17.8	17.8
	N	22	22	22	20
Day 15	Mean	268	268	265	263
	SD	19.0	17.9	19.1	19.2
	N	22	22	22	20
Day 18	Mean	298	298	295	292
	SD	23.1	19.6	22.5	21.5
	N	22	22	22	20
Day 21	Mean	335	333	331	324
	SD	28.3	25.0	28.0	27.8
	N	22	22	22	20

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

Table 3. 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 2-6	Mean	89	85	86	88
	SD	7.1	6.3	7.1	7.2
	N	22	22	22	20
Days 6-9	Mean	81	80	82	85
	SD	7.6	7.1	20.1	5.9
	N	22	22	22	20
Days 9-12	Mean	80	81	82	86*
	SD	10.4	7.8	5.9	7.0
	N	22	22	22	20
Days 12-15	Mean	82	77	81	83
	SD	6.1	11.2	7.3	7.2
	N	22	22	22	20
Days 15-18	Mean	78	76	78	81
	SD	6.2	8.0	5.5	7.4
	N	22	22	22	20
Days 18-21	Mean	70	66	70	70
	SD	6.0	5.8	5.5	7.6
	N	22	22	22	20
Mean of Means		80	77	80	82

 

Table 4. Summary of Clinical Biochemistry Parameters Evaluated – F0 Generation
Parameter		Group 1 0 mg/Kg/day	Group 2 100 mg/Kg/day	Group 3 300 mg/Kg/day	Group 4 1000 mg/Kg/day
TSH uIU/mL	Mean	0.339	0.225	0.241	0.206
	SD	0.302	0.141	0.144	0.130
	N	22	22	22	20
Total T3 ng/dL	Mean	42.0	40.6	38.4	39.1
	SD	10.4	10.2	6.8	9.5
	N	22	22	22	20
Total T4 ug/dL	Mean	2.10	2.06	2.20	1.89
	SD	0.58	0.50	0.52	0.54
	N	22	22	22	20

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

 

Table 5. Summary of Macroscopic Findings – F0 Generation
	Group 1 0 mg/Kg/day	Group 2 100 mg/Kg/day	Group 3 300 mg/Kg/day	Group 4 1000 mg/Kg/day
END OF TREATMENT
Animals examined	22	22	22	22
Animals without findings	21	22	21	21
Animals affected	1	0	1	1
Liver       Right median lobe accessory lobe	1	0	0	0
Thymus       Focus/foci	0	0	0	1
Spleen       Alopecia	0	0	1	0

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

 

Table 6. Summary of Organ Weights – F0 Generation
Parameter		Group 1 0 mg/Kg/day	Group 2 100 mg/Kg/day	Group 3 300 mg/Kg/day	Group 4 1000 mg/Kg/day
END OF TREATMENT
Body Weight (g)	Mean	335	333	331	324
	SD	28	25	28	28
	N	22	22	22	20
Thyroids (g)	Mean	0.0147	0.0151	0.0145	0.0142
	SD	0.0024	0.0027	0.0018	0.0022
	N	22	22	21	20

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

 

Table 7. Summary of Organ/Body Weight Ratios – F0 Generation
Parameter		Group 1 0 mg/Kg/day	Group 2 100 mg/Kg/day	Group 3 300 mg/Kg/day	Group 4 1000 mg/Kg/day
END OF TREATMENT
Body Weight (g)	Mean	335	333	331	324
	SD	28	25	28	28
	N	22	22	22	20
Thyroids (%)	Mean	0.0044	0.0046	0.0044	0.0044
	SD	0.0007	0.0008	0.0006	0.0007
	N	22	22	21	20

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

 

Table 8. 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	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	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 examined at scheduled necropsy	22	100.0	22	100.0	22	100.0	22	100.0
Non gravid	0	0.0	0	0.0	0	0.0	2	9.1
Gravid	22	100.0	22	100.0	22	100.0	20	90.9
With Resorptions only	0	0.0	0	0.0	0	0.0	0	0.0
With Viable fetuses	22	100.0	22	100.0	22	100.0	20	100.0
Totals Females Gravid	22	100.0	22	100.0	22	100.0	20	90.9

 

Table 9. 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	113	127	240	0	16	0	16	256	262	6	NA	22
	Mean	5.1	5.8	10.9	0.0	0.7	0.0	0.7	11.6	11.9	0.3	5.3
	S.D.	1.91	2.79	2.43	0.00	0.88	0.00	0.88	2.36	2.39	0.55	0.27
Group 2 100 mg/Kg/day	Total	114	118	232	0	17	1	18	250	268	18	NA	22
	Mean	5.2	5.4	10.5	0.0	0.8	0.0	0.8	11.4	12.2	0.8	5.4
	S.D.	2.30	1.99	3.05	0.00	1.19	0.21	1.18	2.98	2.34	1.37	0.50
Group 3 300 mg/Kg/day	Total	109	124	233	0	9	0	9	242	252	10	NA	22
	Mean	5.0	5.6	10.6	0.0	0.4	0.0	0.4	11.0	11.5	0.5	5.3
	S.D.	1.33	1.53	1.65	0.00	0.67	0.00	0.67	1.75	1.79	0.51	0.27
Group 4 1000 mg/Kg/day	Total	102	105	207	0	9	0	9	216	221	5	NA	20
	Mean	5.1	5.3	10.4	0.0	0.5	0.0	0.5	10.8	11.1	0.3	5.3
	S.D.	1.92	1.80	1.95	0.00	0.76	0.00	0.76	1.61	1.43	0.55	0.21

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 10. 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	11.9	12.2	11.5	11.1
	S.D.	2.39	2.34	1.79	1.43
	N	22	22	22	20
Implantation Sites	Mean	11.6	11.4	11.0	10.8
	S.D.	2.36	2.98	1.75	1.61
	N	22	22	22	20
Viable Fetuses (%)	Mean	93.9	92.0	96.5	95.5
	S.D.	7.43	11.16	5.59	8.09
	N	22	22	22	20
Dead Fetuses (%)	Mean	0.0	0.0	0.0	0.0
	S.D.	0.00	0.00	0.00	0.00
	N	22	22	22	20
Early Resorptions (%)	Mean	6.1	7.5	3.5	4.5
	S.D.	7.43	11.28	5.59	8.09
	N	22	22	22	20
Late Resorptions (%)	Mean	0.0	0.5	0.0	0.0
	S.D.	0.00	2.13	0.00	0.00
	N	22	22	22	20
Total Resorptions (%)	Mean	6.1	8.0	3.5	4.5
	S.D.	7.43	11.16	5.59	8.09
	N	22	22	22	20
Pre-implantation Loss (%)	Mean	2.2	7.8	3.9	2.4
	S.D.	4.26	14.85	4.58	5.33
	N	22	22	22	20
Post-implantation Loss (%)	Mean	6.1	8.0	3.5	4.5
	S.D.	7.43	11.16	5.59	8.09
	N	22	22	22	20
Males (%)	Mean	49.8	48.9	46.9	49.7
	S.D.	21.61	13.44	11.41	18.05
	N	22	22	22	20
Females (%)	Mean	50.2	51.1	53.1	50.3
	S.D.	21.61	13.44	11.41	18.05
	N	22	22	22	20
Male Fetal Weights (g)	Mean	5.5	5.5	5.4	5.5
	S.D.	0.28	0.53	0.30	0.21
	N	22	22	22	20
Female Fetal Weights (g)	Mean	5.2	5.2	5.1	5.2
	S.D.	0.25	0.45	0.24	0.23
	N	21	22	22	19
Combined Fetal Weights (g)	Mean	5.3	5.4	5.3	5.3
	S.D.	0.27	0.50	0.27	0.21
	N	22	22	22	20
Male AGD (mm)	Mean	2.77	2.77	2.75	2.82
	S.D.	0.284	0.275	0.153	0.235
	N	22	22	22	20
Female AGD (mm)	Mean	1.29	1.30	1.24	1.33
	S.D.	0.315	0.318	0.306	0.300
	N	21	22	22	19

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

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

None significantly different from control group

Table 11. Summary of Fetuses and Litters with Malformations (Absolute Nos)
	Fetuses				Litters
	Group 1 0 mg/Kg/day	Group 2 100 mg/Kg/day	Group 3 300 mg/Kg/day	Group 4 1000 mg/Kg/day	Group 1 0 mg/Kg/day	Group 2 100 mg/Kg/day	Group 3 300 mg/Kg/day	Group 4 1000 mg/Kg/day
Number Examined Externally	240	232	233	207	22	22	22	20
Ectrodactyly	1	0	0	0	1	0	0	0
Lower Jaw – Absent or Small	0	0	0	1	0	0	0	1
Eye – Bulge Absent and/or Small	0	0	0	1	0	0	0	1
Number Examined Viscerally	120	116	118	103	22	22	22	20
Aortic Arch – Right Sided	1	0	0	0	1	0	0	0
Diaphragmatic Hernia	1	0	0	0	1	0	0	0
Intestine - Absent	1	0	0	0	1	0	0	0
Number Examined Skeletally	121	116	115	104	22	22	22	20
Bent Limb Bones	1	1	0	1	1	1	0	1
Vertebral Anomaly with or without Rib  Anomaly	1	1	0	1	1	1	0	1
Orbit(s) – Small	0	1	0	0	0	1	0	0
Sternochisis	1	0	0	1	1	0	0	1
Limb bones – Absent	1	0	0	0	1	0	0	0
Total Number with Malformations
External	1	0	0	1	1	0	0	1
Soft Tissue	1	0	0	0	1	0	0	0
Skeletal	2	2	0	3	2	2	0	3
Combined	2	2	0	3	2	2	0	3

 

Table 12. Summary of Fetuses and Litters with Variations (Absolute Nos)
	Fetuses				Litters
	Group 1 0 mg/Kg/day	Group 2 100 mg/Kg/day	Group 3 300 mg/Kg/day	Group 4 1000 mg/Kg/day	Group 1 0 mg/Kg/day	Group 2 100 mg/Kg/day	Group 3 300 mg/Kg/day	Group 4 1000 mg/Kg/day
Number Examined Externally	240	232	233	207	22	22	22	20
Number with Findings	0	0	0	0	0	0	0	0
Number Examined Viscerally	120	116	118	103	22	22	22	20
Liver – Small Supernumerary Lobe(s)	1	0	4	2	1	0	4	1
Ureter(s) – Convoluted	1	1	0	1	1	1	0	1
Number Examined Skeletally	121	116	115	104	22	22	22	20
14^thRudimentary Rib(s)	56	66	70	58	19	21	21	17
Reduced Ossification of the Skull	23	7	12	17	12	5	8	10
Bent Rib(s)	26	22	9	21	12	10	5	12
Sternebra(e) – Malaligned	17	12	25	14	11	12	16	9
Pelvic Girdle – Caudal Shift	6	7	9	11	6	6	6	8
Metacarpal(s) and / or Metatarsal(s) Malpositioned	1	0	0	0	1	0	0	0
14^thFull Rib(s)	10	8	10	4	8	6	8	2
Metacarpal(s) and / or Metatarsal(s)  Unossified	3	2	4	2	3	2	4	2
7^thCervical Ossification Site(s)	4	2	3	1	3	2	3	1
Vertebral Centra – Reduced Ossification	2	3	4	0	2	3	3	0
7^thCervical – Full Rib(s)	0	0	0	3	0	0	0	3
Vertebral Arches – Reduced Ossification	0	1	0	1	0	1	0	1
Scapula(e) - Bent	0	0	0	1	0	0	0	1
Sternebra(e) #5 and/or #6 Unossified	0	0	0	1	0	0	0	1

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 rats 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 Wistar Han rats (22/dose). 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 6 to 20 post-coitum, inclusive.

 

Maternal parameters and endpoints evaluated in this study included mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), gross necropsy findings, organ weights (thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and post-implantation loss. Fetal (F1-generation) parameters determined included the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, 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, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), organ weights (thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and post-implantation loss) were observed through the study period.

 

At 1000 mg/Kg/day, 3 fetuses of 3 litters were noted with a 7^thfull rib. This finding was observed in this high dose group only and the incidence exceeded available historical control data. Therefore, it was considered related to treatment with the test material. However, as this concerns a variation with no known detrimental effects on development, it was considered non-adverse. No other treatment-related changes were observed in any of the developmental parameters investigated in the study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, 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 rats was determined to be at least 1000 mg/Kg/day.