Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-SEP-16 to 2022-SEP-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

yes

Remarks:

Deviations did not impact 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)

Version / remarks:

May 2008

Deviations:

yes

Remarks:

Deviations did not impact 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)

Version / remarks:

August 1998

Deviations:

yes

Remarks:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin

EC Number:

941-364-9

Molecular formula:

Not applicable to UVCB substance

IUPAC Name:

Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin

Test material form:

other: Low visocosity, liquid hydrocarbon

Details on test material:

Batch number: 204377961
Physical description: Colourless or pale yellow coloured liquid
Purity: 100% UVCB
Expiry date: 20 August 2022 if kept under storge conditions
Source and site of characterisation: Repsol Refinery, Carretera de la Calzada s/n, Apartado de Correos 12, 13500 Puertollano, Ciudad Real, Spain
Storage conditions: Store under controlled humidity conditions and temperature (18-25 °C) in a sealed container, protecting the product from sunlight (opaque container or in a cabinet)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Shell Global Solutions (Spain); Batch number: 204377961
- Purity, including information on contaminants, isomers, etc.: 100% (UVCB)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable for at least 24 hours at room temperature under normal laboratory light conditions and for at least 8 days in the refrigerator over the concentration range 2 to 800 mg/mL (solutions).

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

OTHER SPECIFICS
- Expiration Date: 2022-Aug-20
- Specific gravity: 0.8319

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: Young adult females, 11-15 weeks old at mating
- Weight at study initiation: 201 - 269 g at onset of treatment
- Fasting period before study: Not specified
- Housing: Individually housed in Makrolon type III polycarbonate cages
- Diet (e.g. ad libitum): Pelleted SM R/M-Z (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) ad libitum
- Water (e.g. ad libitum): Municipal tap water (in water bottles) ad libitum
- Acclimation period:5- 6 days before the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20°C (target: 18 to 24°C)
- Humidity (%): 45-69% (target: 40-70%)
- Air changes (per hr): 10 or more air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2021-SEP-22/24 To: 2021-OCT-15

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Sigma-Aldrich (Steinheim, Germany)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was formulated in the vehicle (corn oil). The formulations were homogenised to visibly acceptable levels at appropriate concentrations to meet dose level requirements. Formulations were prepared at least weekly, filled out in daily portions and stored in a refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Test item 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 (0.92) and test item (0.8319). No correction was made for the purity/composition of the test item as it is a UVCB.

A dose volume of 4 mL/kg body weight was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weight of the animals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on results of the trial formulation, corn oil was selected as vehicle for this study
- Concentration in vehicle: 0, 25, 75, or 250 mg/mL for the 0, 100, 300, and 1000 mg/kg bw/day dose levels, respectively.
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no. (if required): Source: Sigma-Aldrich; Batch Number: MKCN9742
- Purity: Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples (duplicate) were collected for analysis on Week 1 of treatment. Samples for concentration analysis were taken from the middle for Groups 1, 2, 3, and 4 while samples for homogeneity analysis were taken from approximately the top, middle, and bottom for Groups 2, 3, and 4 only.

Analyses were performed using a validated analytical procedure (Test Facility Study No. 20276345).

Concentration and Homogeneity Analysis: at 21°C
For concentration: mean sample concentration results within or equal to ± 10% solutions of theoretical concentration were considered acceptable.

For homogeneity: relative standard deviation (RSD) of concentrations of ≤10% for each group was considered acceptable.

Stability Analysis:
Stability analyses was performed previously in conjunction with the method development and validation study (Test Facility Study No. 20276345). This demonstrated that the test material was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Details on mating procedure:

Impregnation procedure: purchased timed pregnant (females arrived on Day 0 or Day 1 post-coitum)

Duration of treatment / exposure:

Daily from day 6 to 20 post coitum

Frequency of treatment:

Once daily

Duration of test:

From day 0 to 21 post coitum (Caesarean section and necropsy)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1 (Control)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 2 (Low dose)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

Group 3 ( (Intermediate dose)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Group 4 (High dose)

No. of animals per sex per dose:

22 females/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on the results of a 14-day dose range finder and a combined 90-day repeated dose toxicity study with the reproduction / developmental toxicity screening test with oral gavage administration of Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin (EC 941-364-9) in rats (Test Facility StudyNos. 20276348 and 20276349, respectively), and in an attempt to produce graded responses to the test item. For both studies, no toxicity was observed at 300 and 1000 mg/kg/day. Therefore, a maximum dose level of 1000 mg/kg/day was selected for the current study. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. 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: The animals were assigned to groups at random

- Fasting period before blood sampling for (rat) dam thyroid hormones: Not fasted

- Time of day for (rat) dam blood sampling: Sampled between 07.00 and 09.00 from the jugular vein

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Except on days of receipt and necropsy (where frequency was at least once daily), all F0 animals were observed at least twice daily (beginning upon arrival through termination) for mortality. Cage side observations were conducted 0 to 1 hours post-dose at least once daily starting on Day 6 post-coitum up to the day prior to necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observtions were conducted for all animals on Days 2, 6, 15, and 21 post-coitum.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of all animals were recorded on Days 2, 6, 9, 12, 15, 18, and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; over 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; Regular basis throughout the study. Water consumption was monitored by visual inspection of the water bottles. Data were used for health monitoring of the animals only and not reported.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 21 post coitum
- Organs examined: Animals surviving until scheduled euthanasia were anaesthetized by a mixture of carbon dioxide and dioxide (graduate fill to a ratio of 60%/40%) and euthanized by a graduate fill of carbon dioxide on Day 21 post-coitum. The thyroid gland was weighed at necropsy for all animals. Paired organs were weighed together. Organ to body weight ratio (using the body weight on Day 21 post-coitum) was calculated.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Each ovary and uterine horn of all animals were 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:
- The number and distribution of live and dead fetuses
- The sex of each fetus based on the anogenital distance

Blood sampling:

- Plasma: No
- Serum: Yes
- Volume collected : 1.0 mL

Blood of F0-animals was collected on the day of scheduled necropsy (Day 21 post-coitum). Animals were not fasted overnight. Samples were collected between 7:00 and 9:00 a.m. from the jugular vein of all F0 animals. Blood samples were processed for serum, and serum was analyzed for Triiodothyronine (T3), Thyroxine (T4), and Thyroid-Stimulating Hormone (TSH).

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes, the anogenital distance of each foetus was measured

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:

Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and its weight was determined. The anogenital distance (AGD) was measured for all viable fetuses and normalized to the cube root of the fetal body weight.

Visceral Examinations:

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 and this examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development .

Tissues with variations or malformations were stored in 10% formalin.

Head Examinations:

The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique.

Skeletal Examinations:

All fetuses were eviscerated, followed by fixation in 96% aqueous ethanol, and maceration in potassium hydroxide. Thereafter, they were processed for double staining with Alcian Blue 8GX and Alizarin Red S. Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads).

Statistics:

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

Indices:

Caesarean Section and Necropsy Data:
 
1) Pre-implantation loss (%, group mean): (Number of corpora lutea - Number of implantations / Number of corpora lutea) x100
 
2) Post-implantation loss (%, group mean): (Number of implantations - Number of live foetuses / Number of implantations) x100
 
Foetal Data:
 
1) Sex distribution (%, group mean): (Number of male (female) foetuses / Number of foetuses) x 100
 
2) External abnormalities/litter (%, group mean): (Number of foetuses with abnormality / Number of foetuses) x 100
           
3) Visceral abnormalities/litter (%, group mean): (Number of foetuses with abnormality / Number of examined foetuses) x 100
           
4) Skeletal abnormalities/litter (%, group mean): (Number of foetuses with abnormality / Number of examined foetuses) x 100

Historical control data:

Historical control data is provided below under 'Attachments' in this Robust Study Summary

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were observed through the study period. Salivation was observed in 1/22, 2/22, 14/22, and 19/22 females in the control, 100, 300, and 1000 mg/kg/day dose groups, respectively. This was considered to be a physiological response (rather than a sign of systemic toxicity) and not toxicologically relevant taking into account the nature and minor severity of the effect and time of occurrence (i.e., after dosing).

Other clinical signs observed (incidental abnormal breathing sounds, fur loss, thin fur cover, and skin scabs) were considered to be of no toxicological relevance as they 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 or were observed during a single day in only one female.

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:

effects observed, treatment-related

Description (incidence and severity):

At 100 and 300 mg/kg/day, mean body weight, body weight gain and body weight gain corrected for gravid uterus weight were observed to be comparable to the corresponding controls. Mean gravid uterus weight was considered to be comparable to the control animals for all treatment groups. Mean gravid uterus weight at 100 mg/kg/day was slightly lower than control (13%; not statistically significant). This was mainly based on two females (Nos. 24 and 39) with small litters (3 fetuses each). In the absence of a dose-related effect, this was not considered to be toxicologically relevant.

At 1000 mg/kg/day, mean body weight gain was statistically significantly reduced (p<0.01) between Days 6-9 post-coitum (mean gain 0.9 g vs 9.5 g in the control group). During this period, 7/22 females were observed to have slight body weight loss (up to 3% on Day 9 compared to Day 6 post-coitum). From Day 9 post coitum onwards body weight gain was comparable to control. Mean body weight gain corrected for gravid uterus at 1000 mg/kg/day was statistically significantly lower (p<0.05) than control (18.86 g vs 25.90 g, respectively).

As the body weight gain recovered from Day 12 post-coitum onwards to levels comparable to control, and in the absence of other signs of maternal toxicity, these findings were considered not to represent an adverse effect of the test material.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption at 100 and 300 mg/kg/day was considered to be comparable to the corresponding controls. At 1000 mg/kg/day, mean food consumption was statistically significantly decreased over (p<0.01) Days 6-12 post-coitum (up to 23% lower than control) but recovered to values comparable to control from Day 12 post coitum onwards. As a result of the initial decrease, overall food consumption (Days 6 21 post-coitum) was statistically significantly (p<0.05) lower (8%) than control.

As food consumption recovered from Day 12 post-coitum onwards to levels comparable to control, and in the absence of other signs of maternal toxicity, these findings were considered not to represent an adverse effect of the test material.

Food efficiency:

not specified

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

not specified

Description (incidence and severity):

Water consumption was monitored by visual inspection of the water bottles. Data were used for health monitoring of the animals only and not reported.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Mean serum levels of T3 at 100 mg/kg/day were in the same range as the corresponding control animals. Mean serum levels of Triiodothyronine (T3) were statistically significantly reduced (p<0.01) at 300 and 1000 mg/kg/day (0.75 and 0.63x of control, respectively), and were considered treatment-related. Nonetheless, at 300 mg/kg/day, the mean T3 level remained within the available historical control range. However, at 1000 mg/kg/day, the mean T3 level (and individual serum levels of 16/22 females) were below the lower limit of the available historical control range. The downstream biological consequences of thyroid hormone changes were not assessed within this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed. This finding was not taken into account when determining the maternal NOAEL.

Mean serum levels of total T4 at 100 mg/kg/day were slightly increased (1.20x of control). Mean serum levels of total Thyroxine (T4) were considered unaffected by treatment at 300 and 1000 mg/kg/day and mean T4 serum levels at all dose levels remained within the available historical control range.
.
Mean serum levels of Thyroid Stimulating Hormone (TSH) showed an apparent dose-related trend towards an increase across the dose groups, reaching statistical significance (p<0.01) at 1000 mg/kg/day only (2.38x of control). This effect was considered treatment-related. However, mean TSH levels for all groups remained within the available historical control range.

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 effects were observed on thyroid gland weight/s.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross necropsy did not reveal any treatment-related effects in the thyroid glands.

Other findings observed amongst the control and/or treated animals were considered to be of no toxicological significance since they remained within the range of biological variation for rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related microscopic observations were seen in the thyroid glands. The recorded microscopic findings in the thyroid gland were within the range of background pathology encountered in rats of this age and strain.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

All females were gravid with viable fetuses, except for one female (No. 38) in the low dose group (100 mg/kg/day), which was non gravid.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The percentage of pre-implantation loss was higher than control in females treated at 100 and 300 mg/kg/day. However, as treatment was only initiated after implantation was completed and in the absence of a dose response, this was considered a chance finding.

The percentage of post-implantation loss in the control and test groups was 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 pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The mean number of corpora lutea and implantation sites in the control and test groups were similar and in the range of normal biological variation. All females were gravid with viable fetuses, except for one female (No. 38) in the low dose group (100 mg/kg/day), which was non gravid.

Other effects:

not examined

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:

effects observed, non-treatment-related

Description (incidence and severity):

Mean fetal weights (male, female and combined) in the control and test groups were considered similar and unaffected by treatment. Higher fetal weights observed at 100 mg/kg/day (males, females and combined; only statistically significant for females) were considered a chance finding as this occurred in the absence of a dose response.

Reduction in number of live offspring:

no effects observed

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:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on litter size were observed in any group. The mean number of fetuses per litter was slightly lower at 100 mg/kg/day (10.0 vs 11.7 in the control group). In the absence of a dose-related effect or statistically significant difference, this was considered unrelated to treatment.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was unaffected by treatment up to 1000 mg/kg/day.

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus (No. 28 L1) in the low dose group (100 mg/kg/day) presented with an external malformation (polydactyly of a hindpaw) which was confirmed at skeletal examination. This single occurrence of a malformation in a low-dose fetus did not suggest any treatment-related effect and was therefore considered a chance finding. No external variations were observed through the study period.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations occurred in one fetus each in the low- and high-dose groups. The fetus (No. 28-L1) in the low dose group (100 mg/kg/day) presented extra hindpaw phalanges, consistent with polydactyly observed in this fetus at external examination while the fetus (No. 71-L1) in the high dose group (1000 mg/kg/day) had an extra lumbar vertebra. Due to the single occurrences of these malformations, they were deemed chance findings. All skeletal variations observed occurred in the absence of a dose-related incidence trend, infrequently and/or in control fetuses only. Therefore, they were not considered treatment-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations were observed in two fetuses, one in the control group and the other in the high dose group. The fetus in the control group (Fetus No. 21-L2) had fused lung lobes while the fetus in the high dose group (1000 mg/kg/day) presented situs inversus and fused lung lobes. Given the single occurrences of these malformations and/or occurrence in the control group, they were deemed chance findings.

Visceral variations were limited to supernumerary liver lobes, absent renal papilla, and convoluted or dilated ureters. The low incidences, group distribution, and occurrence in control fetuses ruled out any relationship to treatment with the test material.

Other effects:

not examined

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:

effects observed, non-treatment-related

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Formulation Analysis:

 

Concentration analysis:

The concentrations analyzed in the formulation of Groups 2, 3, and 4 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 90-110% of target concentration) and no test material was detected in the Group 1 formulation.

 

Homogeneity:

The formulation of Groups 2, 3, and 4 were homogeneous (i.e., coefficient of variation ≤ 10%).

Table 2. Results of Formulation Analysis
Date of analysis	Concentration (mg/mL)			Recovery (%)
	Target	Nominal	Analyzed	Individual	Mean
2021-SEP-27	25	22.6	24.3	108	104
		24.8	24.8	100
	250	247	254	103	102
		255	255	100

Table 3. Summary of Body Weights: Gestation (F0: Female)
Group		Day(s) Relative to Mating (Litter: A)
		2	6	9	12	15	18	21
Group 1 Control 0 mg/kg/day	Mean	220.0	232.7	242.1	256.5	270.5	303.6	340.3
	SD	13.5	14.6	15.4	15.3	17.5	21.8	27.3
	N	22	22	22	22	22	22	20
Group 2 100 mg/kg/day	Mean	215.4	228.8	237.7	251.8	264.9	293.8	331.3
	SD	11.3	12.0	12.4	12.3	13.8	17.3	22.3
	N	21	21	21	21	21	21	20
	% Diff	-2.1	-1.7	-1.8	-1.9	-2.1	-3.3	-2.6
Group 3 300 mg/kg/day	Mean	220.7	234.1	242.0	257.3	270.6	303.2	340.9
	SD	12.1	13.0	12.5	15.6	16.8	18.9	23.6
	N	22	22	22	22	22	22	21
	% Diff	0.3	0.6	-0.1	0.3	0.0	-0.1	0.2
Group 4 1000 mg/kg/day	Mean	218.6	231.8	232.7	246.5	261.2	294.2	326.7
	SD	13.2	14.7	13.8	15.5	17.8	20.9	28.8
	N	22	22	22	22	22	22	20
	% Diff	-0.7	-0.4	-3.9	-3.9	-3.4	-3.1	-4.0

Anova & Dunnett

Table 4. Summary of Body Weight Gains (g): Gestation (F0: Females)
Group		Day(s) Relative to Mating (Litter: A)
		6 → 9 [G]	9 → 12 [G1]	12 → 15 [G1]	15 → 18 [G1]	18 → 21 [G1]	6 → 21 [G1]
Group 1 Control 0 mg/kg/day	Mean	9.5	14.5	13.9	33.1	37.7	108.2
	SD	3.5	3.2	4.8	6.6	6.5	15.7
	N	22	22	22	22	20	20
Group 2 100 mg/kg/day	Mean	8.9	14.1	13.1	28.9	36.1	101.6
	SD	2.4	4.5	4.2	6.3	9.2	19.2
	N	21	21	21	21	20	20
Group 3 300 mg/kg/day	Mean	7.9	15.3	13.3	32.6	36.3	106.2
	SD	4.4	5.7	4.7	5.2	6.9	14.7
	N	22	22	22	22	21	21
Group 4 1000 mg/kg/day	Mean	0.9**	13.8	14.7	33.0	33.7	95.8
	SD	5.2	4.5	5.3	5.8	10.0	18.2
	N	22	22	22	22	20	20

[G] - Kruskal-Wallis & Dunn: ** = p ≤ 0.01

[G1] - Anova & Dunnett

Table 5. Summary of Gravid Uterine Weights and Gravid Uterus Adjusted Body Weights: Gestation (F0: Female)
Day(s) Relative to Mating (Litter: A)		Group 1 Control 0 mg/kg/day	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1000 mg/kg/day
Bodyweight on Day 6 (g) [G]	Mean	232.7	228.8	234.1	231.8
	SD	14.6	12.0	13.0	14.7
	N	22	21	22	22
	% Diff	-	-1.7	0.6	-0.4
Terminal Body Weight (g) [G]	Mean	340.3	331.3	340.9	326.7
	SD	27.3	22.3	23.6	28.8
	N	20	20	21	20
	% Diff	-	-2.6	0.2	-4.0
Gravid Uterus Weight (g) [G]	Mean	82.10	71.70	76.12	78.13
	SD	15.9	19.10	13.09	16.33
	N	22	21	22	22
	% Diff	-	-12.67	-7.29	-4.84
Adjusted BWG (6-abw) (g) [G]	Mean	25.90	29.01	28.80	18.86*
	SD	9.15	8.48	8.25	10.74
	N	20	20	21	20
	% Diff	-	12.03	11.22	-27.19

[G] - Anova & Dunnett: * = p ≤ 0.05

Table 6. Summary of Food Consumption: Gestation (F0: Females)
Group		Day(s) Relative to Mating (Litter: A)
		6 → 9	9 → 12	12 → 15	15 → 18	18 → 21	6 → 21
Group 1 Control 0 mg/kg/day	Mean	20.50	19.94	20.59	21.83	21.18	20.81
	SD	2.11	1.61	1.81	1.82	1.76	1.51
	N	22	22	22	22	22	22
Group 2 100 mg/kg/day	Mean	21.35	19.83	20.92	21.97	21.57	21.13
	SD	2.46	2.73	2.11	2.37	2.23	1.89
	N	21	21	21	21	21	21
	% Diff	4.14	-0.57	1.60	0.62	1.84	1.53
Group 3 300 mg/kg/day	Mean	20.24	19.09	20.50	22.41	21.71	20.79
	SD	3.98	2.61	3.05	2.81	2.74	2.40
	N	22	22	22	22	22	22
	% Diff	-1.26	-4.26	-0.44	2.64	2.50	-0.09
Group 4 1000 mg/kg/day	Mean	15.88**	16.97**	19.03	22.59	20.95	19.08*
	SD	2.94	2.60	2.33	1.85	2.88	1.91
	N	22	22	22	22	22	22
	% Diff	-22.54	-14.89	-7.58	3.47	-1.07	-8.29

Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

Table 7. Summary of Thyroid Hormone Values (F0 Female)
Day: 21 Relative to Mating (Litter: A)		Reporting Special Chemistry
Group		T3 (ng/mL) [G]	T4 (ng/mL) [G]	TSH (ng/mL) [G]
Group 1 Control 0 mg/kg/day	Mean	0.386	20.01	0.2425
	SD	0.051	3.11	0.1698
	N	22	22	22
Group 2 100 mg/kg/day	Mean	0.382	24.03**	0.3191
	SD	0.079	4.91	0.1531
	N	21	21	21
	tCtrl	0.99	1.20	1.32
Group 3 300 mg/kg/day	Mean	0.291**	21.23	0.3443
	SD	0.057	3.85	0.2992
	N	22	22	22
	tCtrl	0.75	1.06	1.42
Group 4 1000 mg/kg/day	Mean	0.244**	18.16	0.5770**
	SD	0.074	3.63	0.3300
	N	22	22	22
	tCtrl	0.63	0.91	2.38

[G] - Anova & Dunnett: ** = p ≤ 0.01

Table 8. Summary of Organ Weights (F0: Female)
Day(s) Relative to Mating (Litter: A)		Group 1 Control 0 mg/kg/day	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1000 mg/kg/day
Terminal Body Weight (g) [G]	Mean	340.3	331.3	340.9	326.7
	SD	27.3	22.3	23.6	28.8
	N	20	20	21	20
	% Diff	-	-2.6	0.2	-4.0
Gland, Thyroid Weight (g) - [G]	Mean	0.01362	0.01404	0.01423	0.01473
	SD	0.00268	0.00272	0.00264	0.00236
	N	22	21	22	22
	% Diff	-	3.04908	4.47114	8.14147
Gland, Thyroid (%bw) - [G]	Mean	0.00394	0.00427	0.00421	0.00449
	SD	0.00079	0.00077	0.00071	0.00076
	N	20	20	21	20

[G] - Anova & Dunnett

Table 9. Summary of Macroscopic Pathology: Incidence (F0: Female)
Incidence	Group 1 Control 0 mg/kg/day	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1000 mg/kg/day
Number of Animals	22	22	22	22
GLAND, SALIVARY, MANDIBULAR
Submitted	1	0	0	0
Focus, dark; red	1	-	-	-
GLAND, THYROID
Submitted	22	22	22	22
No Visible Lesions	21	22	22	21
Focus, dark; red	1	0	0	0
Enlargement	0	0	0	1
Small	0	0	0	1
SKIN
Submitted	1	1	0	0
Scab; cervical, dorsal	1	0	-	-
Thin hair coat	1	0	-	-
Thin hair coat; forelimb,left	0	1	-	-

Table 10. Summary of Microscopic Pathology: Incidence (F0: Female)
Incidence	Group 1 Control 0 mg/kg/day	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1000 mg/kg/day
Number of Animals	22	22	22	22
GLAND, THYROID
Examined	22	22	22	22
No Visible Lesions	22	21	21	19
Hypertrophy; follicular cell	0	1	1	2
.... minimal	0	1	1	2
Ectopia; thymic corpuscle	0	0	0	1

Table 11. Summary of Maternal Performance and Mortality (F0: Female)
Day(s) Relative to Mating (Litter: A)		Group 1 Control 0 mg/kg/day	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1000 mg/kg/day
Group Size - Females		22	22	22	22
Number of Females Pregnant [f]	N+ve	22	21	22	22
	%	100.0	95.5	100.0	100.0
Female with Live Fetuses [f]	N+ve	22	21	22	22
	%	100.0	100.0	100.0	100.0
Total Resorptions [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Female with all Nonviable [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Terminal Euthanasia [f]	N+ve	22	22	22	22
	%	100.0	100.0	100.0	100.0
Unscheduled Death/Euthanasia [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0

[f] - Fisher's Exact

Table 12. Summary of Ovarian and Uterine Examinations and Litter Observations (F0: Female)
Day(s) Relative to Mating (Litter: A)		Group 1 Control 0 mg/kg/day	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1000 mg/kg/day
Female with Live Fetuses	N+ve	22	21	22	22
	%	100.0	100.0	100.0	100.0
Number of Corpora Lutea [k]	Mean	13.6	12.7	13.6	13.0
	SD	1.5	1.3	1.3	2.4
	N	22	21	22	22
	% Diff	-	-6.8	0.0	-4.7
Number of Implantations [k]	Mean	12.6	10.8	11.8	12.0
	SD	1.5	3.0	1.8	2.9
	N	22	21	22	22
	% Diff	-	-14.5	-6.1	-4.3
Pre-implantation Loss (%) [k]	Mean	7.50	15.97	12.84	7.69
	SD	7.52	21.14	13.52	12.32
	N	22	21	22	22
	% Diff	-	112.98	71.19	2.48
Total Number of Fetuses [k]	Mean	11.7	10.0	11.2	11.6
	SD	2.2	3.0	2.0	2.8
	N	22	21	22	22
	% Diff	-	-14.3	-4.7	-0.8
Number of Live Fetuses [k]	Mean	11.7	10.0	11.2	11.6
	SD	2.2	3.0	2.0	2.8
	N	22	21	22	22
	% Diff	-	-14.3	-4.7	-0.8
Number of Dead Fetuses [k]	Mean	0.0	0.0	0.0	0.0
	SD	0.0	0.0	0.0	0.0
	N	22	21	22	22
	% Diff	-	-	-	-
Number of Early Resorptions [k]	Mean	0.9	0.7	0.6	0.4
	SD	1.2	1.1	0.9	0.6
	N	22	21	22	22
	% Diff	-	-17.3	-26.3	-52.6
Number of Late Resorptions [k]	Mean	0.0	0.0	0.0	0.0
	SD	0.0	0.0	0.0	0.0
	N	22	21	22	22
	% Diff	-	-	-	-
Total Number of Resorptions [k]	Mean	0.9	0.7	0.6	0.4
	SD	1.2	1.1	0.9	0.6
	N	22	21	22	22
	% Diff	-	-17.3	-26.3	-52.6
Post-implantation Loss (%) [k]	Mean	7.40	6.89	5.61	3.52
	SD	11.16	11.13	7.66	5.52
	N	22	21	22	22
	% Diff	-	-7.00	-24.27	-52.44
Number of Live Male Fetuses [k]	Mean	6.0	5.2	4.9	5.3
	SD	2.3	2.1	2.4	1.7
	N	22	21	22	22
	% Diff	-	-12.0	-17.6	-11.5
Number of Live Female Fetuses [k]	Mean	5.8	4.8	6.3	6.4
	SD	2.1	2.5	2.2	2.1
	N	22	21	22	22
	% Diff	-	-16.7	8.7	10.2
Live Male Fetus/Litter (%) [k]	Mean	50.37	53.71	43.02	45.30
	SD	15.55	16.94	19.10	11.51
	N	22	21	22	22
	% Diff	-	6.64	-14.58	-10.06
Live Female Fetuses/Litter (%) [k]	Mean	49.63	46.29	56.98	54.70
	SD	15.55	16.94	19.10	11.51
	N	22	21	22	22
	% Diff	-	-6.73	14.79	10.21
Mean Fetal Weight males (g) [G]	Mean	5.328	5.434	5.253	5.211
	SD	0.181	0.261	0.296	0.246
	N	22	21	21	22
	% Diff	-	1.985	-1.398	-2.195
Mean Fetal Weight females (g) [G]	Mean	5.052	5.212*	5.004	4.973
	SD	0.217	0.265	0.206	0.157
	N	22	21	22	22
	% Diff	-	3.172	-0.941	-1.558
Mean Fetal Weight all (g) [G]	Mean	5.193	5.333	5.105	5.086
	SD	0.176	0.242	0.216	0.192
	N	22	21	22	22
	% Diff	-	2.696	-1.696	-2.072
Mean Fetal AGD males (mm) [G1]	Mean	2.92	2.93	2.79	2.74
	SD	0.27	0.23	0.25	0.32
	N	22	21	21	22
	% Diff	-	0.25	-4.51	-6.24
Mean Fetal AGD females (mm) [G1]	Mean	1.39	1.33	1.32	1.39
	SD	0.29	0.32	0.26	0.34
	N	22	21	22	22
	% Diff	-	-3.90	-5.19	-0.10
Mean Normalized Fetal AGD m [G]	Mean	1.672	1.667	1.604	1.579
	SD	0.146	0.137	0.130	0.176
	N	22	21	21	22
	% Diff	-	-0.301	-4.042	-5.573
Mean Normalized Fetal AGD f [G]	Mean	0.810	0.771	0.770	0.813
	SD	0.168	0.186	0.152	0.202
	N	22	21	22	22
	% Diff	-	-4.850	-4.938	0.423

[G] - Anova & Dunnett: * = p ≤ 0.05

[G1] - Anova & Dunnett

[k] - Kruskal-Wallis & Dunn

Table 13. Summary of Fetal Abnormalities by Finding (F1: External)
		Group 1 Control 0 mg/kg/day	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1000 mg/kg/day
Number of Fetuses Examined:		258	211	246	256
Number of Fetuses Evaluated:		258	211	246	256
Number of Litters Examined:		22	21	22	22
Number of Litters Evaluated:		22	21	22	22
Paw/Digit
Hindpaw, Left, Polydactyly - Malformation	Fetuses N(%)	0 (0.00)	1 (0.37)	0 (0.00)	0 (0.00)
	Litters N(%)	0 (0.0)	1 (4.8)	0 (0.0)	0 (0.0)

 

Table 14. Summary of Fetal Abnormalities by Finding (F1: FreshVisBody)
		Group 1 Control 0 mg/kg/day	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1000 mg/kg/day
Number of Fetuses Examined:		127	104	121	127
Number of Fetuses Evaluated:		258	211	246	256
Number of Litters Examined:		22	21	22	22
Number of Litters Evaluated:		22	21	22	22
General
General, Situs inversus - Malformation	Fetuses N(%)	0 (0.00)	0 (0.00)	0 (0.00)	1 (0.65)
	Litters N(%)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.5)
Kidney
Renal papilla, Right, Absent - Variation	Fetuses N(%)	0 (0.00)	1 (0.68)	0 (0.00)	0 (0.00)
	Litters N(%)	0 (0.0)	1 (4.8)	0 (0.0)	0 (0.0)
Liver
Lobe, Left medial, Supernumerary - Variation	Fetuses N(%)	3 (2.06)	1 (0.79)	1 (0.76)	2 (1.41)
	Litters N(%)	3 (13.6)	1 (4.8)	1 (4.5)	2 (9.1)
Lobe, Right medial, Supernumerary - Variation	Fetuses N(%)	0 (0.00)	2 (1.90)	2 (1.41)	0 (0.00)
	Litters N(%)	0 (0.0)	1 (4.8)	2 (9.1)	0 (0.0)
Lung
Lobe, Left cranial, Fused - Malformation	Fetuses N(%)	0 (0.00)	0 (0.00)	0 (0.00)	1 (0.65)
	Litters N(%)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.5)
Lobe, Right medial, Fused - Malformation	Fetuses N(%)	1 (0.65)	0 (0.00)	0 (0.00)	0 (0.00)
	Litters N(%)	1 (4.5)	0 (0.0)	0 (0.0)	0 (0.0)
Ureter
Ureter, Left, Convoluted - Variation	Fetuses N(%)	1 (0.76)	1 (0.79)	1 (0.76)	0 (0.00)
	Litters N(%)	1 (4.5)	1 (4.8)	1 (4.5)	0 (0.0)
Ureter, Right, Convoluted - Variation	Fetuses N(%)	1 (0.65)	2 (1.63)	0 (0.00)	0 (0.00)
	Litters N(%)	1 (4.5)	2 (9.5)	0 (0.0)	0 (0.0)
Ureter, Right, Dilatation, Minimal - Variation	Fetuses N(%)	0 (0.00)	1 (0.68)	0 (0.00)	0 (0.00)
	Litters N(%)	0 (0.0)	1 (4.8)	0 (0.0)	0 (0.0)

[Fetuses %] - Kruskal-Wallis & Dunn

FetusesN(%) N=Group Fetal Incidence;(%)=Mean Litter % of Fetuses with the Abnormality

Table 15. Summary of Fetal Abnormalities by Finding (F1: Skeletal)
		Group 1 Control 0 mg/kg/day	Group 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1000 mg/kg/day
Number of Fetuses Examined:		131	107	125	129
Number of Fetuses Evaluated:		258	211	246	256
Number of Litters Examined:		22	21	22	22
Number of Litters Evaluated:		22	21	22	22
Forelimb
Forepaw phalanges, 1 or more, Unossified - Variation	Fetuses N(%)	0 (0.00)	0 (0.00)	1 (0.65)	0 (0.00)
	Litters N(%)	0 (0.0)	0 (0.0)	1 (4.5)	0 (0.0)
Hindlimb
Hindpaw phalanges, 1 or more, Supernumerary - Malformation	Fetuses N(%)	0 (0.00)	1 (0.68)	0 (0.00)	0 (0.00)
	Litters N(%)	0 (0.0)	1 (4.8)	0 (0.0)	0 (0.0)
Metatarsal, 1 or more, Malpositioned - Variation	Fetuses N(%)	0 (0.00)	1 (0.79)	0 (0.00)	0 (0.00)
	Litters N(%)	0 (0.0)	1 (4.8)	0 (0.0)	0 (0.0)
Pelvic Girdle
Ilium, Both, Misaligned - Variation	Fetuses N(%)	10 (7.03)	4 (4.05)	3 (1.95)	11 (9.22)
	Litters N(%)	6 (27.3)	4 (19.0)	2 (9.1)	9 (40.9)
Rib
Costal cartilage, 1 or more, Fused - Variation	Fetuses N(%)	2 (1.41)	1 (0.79)	0 (0.00)	0 (0.00)
	Litters N(%)	2 (9.1)	1 (4.8)	0 (0.0)	0 (0.0)
Rib, 1 or more, Wavy rib - Variation	Fetuses N(%)	12 (8.87)	10 (8.95)	2 (1.41)	3 (2.32)
	Litters N(%)	7 (31.8)	6 (28.6)	2 (9.1)	2 (13.6)
Skull
Frontal, Left, Incomplete ossification - Variation	Fetuses N(%)	0 (0.00)	1 (1.19)	0 (0.00)	0 (0.00)
	Litters N(%)	0 (0.0)	1 (4.8)	0 (0.0)	0 (0.0)
Interparietal, Incomplete ossification - Variation	Fetuses N(%)	3 (2.16)	5 (4.97)	1 (0.76)	0 (0.00)
	Litters N(%)	3 (13.6)	4 (19.0)	1 (4.5)	0 (0.0)
Parietal, Both, Incomplete ossification - Variation	Fetuses N(%)	2 (1.52)	1 (1.19)	1 (0.76)	0 (0.00)
	Litters N(%)	2 (9.1)	1 (4.8)	1 (4.5)	0 (0.0)
Parietal, Right, Incomplete ossification - Variation	Fetuses N(%)	0 (0.00)	1 (1.19)	0 (0.00)	0 (0.00)
	Litters N(%)	0 (0.0)	1 (4.8)	0 (0.0)	0 (0.0)
Squamosal, Both, Incomplete ossification - Variation	Fetuses N(%)	1 (0.76)	1 (1.19)	2 (1.52)	0 (0.00)
	Litters N(%)	1 (4.5)	1 (4.8)	1 (4.5)	0 (0.0)
Supraoccipital, Incomplete ossification - Variation	Fetuses N(%)	1 (0.76)	1 (0.95)	0 (0.00)	0 (0.00)
	Litters N(%)	1 (4.5)	1 (4.8)	0 (0.0)	0 (0.0)
Zygomatic arch, Both, Incomplete ossification - Variation	Fetuses N(%)	1 (0.76)	0 (0.00)	3 (2.27)	0 (0.00)
	Litters N(%)	1 (4.5)	0 (0.0)	1 (4.5)	0 (0.0)
Zygomatic arch, Left, Incomplete ossification - Variation	Fetuses N(%)	0 (0.00)	1 (1.19)	0 (0.00)	0 (0.00)
	Litters N(%)	0 (0.0)	1 (4.8)	0 (0.0)	0 (0.0)
Zygomatic arch, Right, Incomplete ossification - Variation	Fetuses N(%)	0 (0.00)	2 (2.38)	0 (0.00)	0 (0.00)
	Litters N(%)	0 (0.0)	1 (4.8)	0 (0.0)	0 (0.0)
Sternebra
Sternebra, 1 or more, Branched - Variation	Fetuses N(%)	1 (0.76)	0 (0.00)	1 (0.65)	0 (0.00)
	Litters N(%)	1 (4.5)	0 (0.0)	1 (4.5)	0 (0.0)
Sternebra, 1 or more, Misaligned - Variation	Fetuses N(%)	3 (2.32)	1 (4.76)	2 (1.56)	1 (0.76)
	Litters N(%)	3 (13.6)	1 (4.8)	2 (9.1)	1 (4.5)
Sternebra, 1 or more, Unossified - Variation	Fetuses N(%)	0 (0.00)	1 (4.76)	0 (0.00)	0 (0.00)
	Litters N(%)	0 (0.0)	1 (4.8)	0 (0.0)	0 (0.0)
Sternebra, 1 or more, Incomplete ossification - Variation	Fetuses N(%)	0 (0.00)	1 (4.76)	2 (1.41)	2 (1.41)
	Litters N(%)	0 (0.0)	1 (4.8)	2 (9.1)	2 (9.1)
Supernumerary rib
Cervical, 1 or more, Full - Variation	Fetuses N(%)	2 (1.41)	0 (0.00)	0 (0.00)	0 (0.00)
	Litters N(%)	2 (9.1)	0 (0.0)	0 (0.0)	0 (0.0)
Cervical, 1 or more, Short - Variation	Fetuses N(%)	5 (4.39)	4 (7.30)	5 (4.13)	6 (4.11)
	Litters N(%)	4 (18.2)	3 (14.3)	5 (22.7)	5 (22.7)
Thoracolumbar, 1 or more, Full - Variation	Fetuses N(%)	15 (10.46)	5 (4.29)	9 (7.49)	17 (11.96)
	Litters N(%)	10 (45.5)	3 (14.3)	6 (27.3)	9 (40.9)
Thoracolumbar, 1 or more, Short - Variation	Fetuses N(%)	83 (64.09)	53 (49.56)	81 (63.82)	80 (63.40)
	Litters N(%)	20 (90.9)	20 (95.2)	22 (100.0)	21 (95.5)
Vertebra
Cervical arch, 1 or more, Small - Variation	Fetuses N(%)	0 (0.00)	0 (0.00)	1 (0.91)	0 (0.00)
	Litters N(%)	0 (0.0)	0 (0.0)	1 (4.5)	0 (0.0)
Lumbar vertebra, 1 or more, Supernumerary - Malformation	Fetuses N(%)	0 (0.00)	0 (0.00)	0 (0.00)	1 (0.76)
	Litters N(%)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.5)
Thoracic centrum, 1 or more, Incomplete ossification - Variation	Fetuses N(%)	0 (0.00)	0 (0.00)	1 (0.91)	0 (0.00)
	Litters N(%)	0 (0.0)	0 (0.0)	1 (4.5)	0 (0.0)

[Fetuses %] - Kruskal-Wallis & Dunn

FetusesN(%) N=Group Fetal Incidence;(%)=Mean Litter % of Fetuses with the Abnormality

Applicant's summary and conclusion

Conclusions:

Based on the effects observed, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Petroleum Gas Oil Fraction, Co-Processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) was determined to be ≥1000 mg/kg/day in the rat.

Executive summary:

A key OECD Guideline 414 pre-natal developmental toxicity study was conducted to evaluate the potential of the test material (Petroleum Gas Oil Fraction, Co Processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9)) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested.

 

The test material was administered to time-mated female Wistar Han rats (22/dose) once daily via oral gavage in a corn oil vehicle at doses of 0, 100, 300, or 1000 mg/kg/day from Days 6 to 20 post-coitum, inclusive. The parameters evaluated for the maternal animals (F0 generation) included mortality / moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), macroscopic examination, organ weights (thyroid gland), uterine contents, microscopic examination (thyroid gland), corpora lutea, implantation sites and pre- and post-implantation loss. Additionally, the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations were evaluated for the pups (F1 generation).

 

No mortality or clinical signs of toxicity were observed through the study period. At 1000 mg/kg/day, mean food consumption was decreased over Days 6-12 post-coitum and mean body weight gain was reduced between Day 6-9 post-coitum. As a result, body weight gain corrected for gravid uterus weight was also slightly reduced. Food consumption and body weight gain recovered from Day 12 post-coitum onwards to levels comparable to control and in the absence of other signs of maternal toxicity, these findings were not considered to represent an adverse treatment-related effect.

Mean serum levels of T3 at 100 mg/kg/day were in the same range as the corresponding control animals. Mean serum levels of Triiodothyronine (T3) were statistically significantly reduced (p<0.01) at 300 and 1000 mg/kg/day (0.75 and 0.63x of control, respectively), and were considered treatment-related. Nonetheless, at 300 mg/kg/day, the mean T3 level remained within the available historical control range. However, at 1000 mg/kg/day, the mean T3 level (and individual serum levels of 16/22 females) were below the lower limit of the available historical control range. The downstream biological consequences of thyroid hormone changes are not assessed in this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed and this finding was not taken into account when determining the maternal NOAEL. 

 

Mean serum levels of total T4 at 100 mg/kg/day were slightly increased (1.20x of control). Mean serum levels of total Thyroxine (T4) were considered unaffected by treatment at 300 and 1000 mg/kg/day and mean T4 serum levels at all dose levels remained within the available historical control range.

Mean serum levels of Thyroid Stimulating Hormone (TSH) showed an apparent dose-related trend towards an increase across the dose groups, reaching statistical significance (p<0.01) at 1000 mg/kg/day only (2.38x of control). This effect was considered treatment-related. However, mean TSH levels for all groups remained within the available historical control range. No treatment-related effects were observed on thyroid gland weight/s and gross necropsy as well as histopathology did not reveal any treatment-related effects in the thyroid glands.

 

All females were gravid with viable fetuses, except for one female (No. 38) in the low dose group (100 mg/kg/day), which was non gravid. The percentage of pre-implantation loss was higher than control in females treated at 100 and 300 mg/kg/day. However, as treatment was only initiated after implantation was completed and in the absence of a dose response, this was considered a chance finding. The percentage of post-implantation loss in the control and test groups was similar and in the range of normal biological variation. The mean number of corpora lutea and implantation sites in the control and test groups were similar and in the range of normal biological variation.

 

Mean fetal weights (male, female and combined) in the control and test groups were considered similar and unaffected by treatment. The sex ratio was also unaffected by treatment up to 1000 mg/kg/day and no treatment-related effects on litter size were observed in any group. Anogenital distance (absolute and normalized for body weight) in male and female pups was unaffected by treatment up to 1000 mg/kg/day.

One fetus (No. 28 L1) in the low dose group (100 mg/kg/day) presented with an external malformation (polydactyly of a hindpaw) which was confirmed at skeletal examination. This single occurrence of a malformation in a low-dose fetus did not suggest a treatment-related effect and was therefore considered a chance finding. No external variations were observed through the study period.

 

Visceral malformations were observed in two fetuses, one in the control group and the other in the high dose group. The fetus in the control group (Fetus No. 21-L2) had fused lung lobes while the fetus in the high dose group (1000 mg/kg/day) presented situs inversus and fused lung lobes. Given the single occurrences of these malformations and/or occurrence in the control group, they were deemed chance findings.

 

Visceral variations were limited to supernumerary liver lobes, absent renal papilla, and convoluted or dilated ureters. The low incidences, group distribution, and occurrence in control fetuses ruled out any relationship to treatment with the test material.

 

Skeletal malformations occurred in one fetus each in the low- and high-dose groups. The fetus (No. 28-L1) in the low dose group (100 mg/kg/day) presented extra hindpaw phalanges, consistent with polydactyly observed in this fetus at external examination while the fetus (No. 71-L1) in the high dose group (1000 mg/kg/day) had an extra lumbar vertebra. Due to the single occurrences of these malformations, they were deemed chance findings. All skeletal variations observed occurred in the absence of a dose-related incidence trend, infrequently and/or in control fetuses only. Therefore, they were not considered treatment-related.

 

Based on the effects observed, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Petroleum Gas Oil Fraction, Co-Processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) was determined to be≥1000 mg/kg/day in the rat.