Octadecene, reaction products with hexadecene, hydrogenated

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 May - 25 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

K1 GLP study to current guidelines. This study was conducted to meet the requirements of other jurisdictions such as China.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See "Any other information on materials and methods incl. tables" for details

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Oligomerisation products of alpha-alkenes C16-18 (even numbered), hydrogenated, hydroisomerised

EC Number:

832-827-5

Cas Number:

2241366-04-9

Molecular formula:

Variable - UVCB

IUPAC Name:

Oligomerisation products of alpha-alkenes C16-18 (even numbered), hydrogenated, hydroisomerised

Test material form:

liquid

Details on test material:

Name: SynNova Base Oil
CAS number: 2241366-04-9
Batch/Lot number: TS20371/TS21270
Description: Liquid - water white colorless oil.
Purity: 100%
Expiry date: 25 February 2021/31 July 2021
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to ensure personnel health and safety.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hannover

Details on test animals or test system and environmental conditions:

Species and strain: Han:WIST rats
Source: Toxi-Coop Zrt., H-1122 Budapest, Magyar Jakobinusok tere 4B
Hygienic level at supplier: SPF
Hygienic level during
the study: Standard housing conditions
Justification of strain: The rat is regarded as a suitable rodent species for reproduction studies and the test guideline states it is the preferred rodent species. The Hannover Wistar rat was selected due to experience with this strain in teratology studies.
Number of animals: 89 female animals; 22, 22, 22 and 23 mated female animals in the control, low, mid and high dose groups, respectively,
30 male animals for mating; no study-procedures were carried out on the male animals; untreated, proven breeders from NEXTREAT Laboratories’ spare colony were used.
Age of animals at start: Young adult rats, nulliparous and non-pregnant, 11 -12 weeks old
Body weight at the start: Did not exceed ± 20% of the mean weight at onset of treatment and were in the range of 201-246 g
Acclimatisation period: At least 20 days
Animal health: Only healthy animals were used for the test. The health status was certified by the Veterinarian.
Cage type: T3H polycarbonate
Housing / Enrichment: The animals were housed individually. “SAFE 3/4-S-FASERN” certified wooden chips (batch number: 03027210315, expiry date: 15 March 2024) produced by J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) and “Sizzle pet” nest material (batch number: 491607, expiry date: 05 August 2023) produced by LBS (Serving Biotechnology) Ltd. (Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH, United Kingdom) were available to animals during the study. Fresh bedding was provided for the animals as frequently as appropriate/practical, but at least twice weekly. Copies of the Certificates of Analyses are archived with the raw data.
Cages were arranged in such a way that possible effects due to cage placement are minimised.
Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20 – 24 °C (target: 22 ± 3 °C)
Relative humidity: 39 – 67 % (target: 30-70%)
Ventilation: 15-20 air exchanges/hour

The minimum and maximum temperature and relative humidity values were recorded daily during the study.

Food and water Supply

Animals received standard laboratory rat diet, ad libitum. The food is not considered to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate for the batch used, which is archived with the raw data.

Details of the diet used in the study were as follows:

Name: SM Rat/Mouse, Breeding & Maintenance, 10 mm, autoclavable
Manufacturer: ssniff Spezialdiäten GmbH (D-59494 Soest, Germany)
Batch number: 233 77046
Expiry date: 30 September 2021

Animals received tap water from the municipal supply, as for human consumption, from drinking bottles, ad libitum. Water quality control analysis and microbiological assessment are performed once per year by the laboratory of Veszprém County Government Office, Department of Public Health (Veszprém Megyei Kormányhivatal Népegészségügyi Főosztály, H-8200 Veszprém, József A. u. 36., Hungary). The quality control results are retained in the archives of NEXTREAT Laboratories.


Animal identification
Adult animals were identified by temporary numbers prior to mating. Once the mating has been confirmed, the dams allocated to the study were identified by definitive numbers written with indelible ink on the tail, and cross referenced with the temporary numbers. During necropsy and caesarean section procedures each evaluated dam was given additional number (evaluation number), indicating group number.

The housing cages were identified with individual identity cards, with information about study code, sex, dose group, cage number, animal number, date of mating and caesarean section/necropsy date.

The litters were identified at necropsy with litter numbers. The flasks used for fixation and all sheets used for recording data were identified only by the litter numbers up to the end of fetal examinations. The fetuses were identified during the caesarean section with individual numbers according to their implantation sites. For visceral examination they were identified by digit-clipping; for skeletal examination the fetuses were identified by means of a water-proof plastic ribbon tied around their neck.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

A constant volume of 1.5 mL/kg body weight was administered to all dose groups, including the control. The individual volume of the treatment was based on the most recent individual body weight of the animals.

The control or test item formulations were administered to mated, sperm positive assumed pregnant female rats daily by oral gavage on a 7 days/week basis, approximately at similar times with minor variations as practical, from GD6 to GD19.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the test item formulations for concentration and homogeneity was performed at the Test Facility. Top, middle and bottom duplicate samples were taken from the test item formulations two times during the study (during the first and last weeks of the treatment period), one set to analyze and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

The formulation analyses were conducted under the control of the Analyst. Acceptance criteria of the concentration analysis was 100 ± 15% of the nominal concentration. Acceptance criteria of the homogeneity was that the RSD% of replicates (top, middle and bottom of test item formulations) should be less than 10%.

Details on mating procedure:

Mating procedure, Randomization

The oestrus cycle of female animals was examined a day before start of pairing. After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2 hours (1 male : 1 female) until at least 22 sperm positive females/group were attained. The mating of siblings was avoided and the mating partners of the females were randomly chosen. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of sperm in the vaginal smear was considered as evidence of copulation (GD0). Sperm positive females were separated and caged individually.

The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that group averages of the body weight were as similar as possible. Females inseminated by the same male were evenly distributed across groups.

Duration of treatment / exposure:

Daily

Frequency of treatment:

7days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

89 female animals; 22, 22, 22 and 23 mated female animals in the control, low, mid and high dose groups, respectively.

Control animals:

yes, concurrent no treatment

Details on study design:

The objective of the study was to assess the effects of the test item on pregnant female rats and on the developing conceptuses and provide general information concerning the effects of prenatal exposure on the pregnant test animals and on the developing organism [1,2]; this may include assessment of maternal effects as well as death, structural abnormalities, or altered growth in the fetus. One control and 3 test item-treated groups of Hannover Wistar rats were treated daily by oral (gavage) administration.

The test item was administered daily to pregnant animals from implantation to one day prior to the day of scheduled kill, which was as close as possible to the normal day of delivery without risking loss of data resulting from early delivery. The experiment was not intended to examine solely the period of organogenesis (e.g. gestation days 5-15 in the rodent), but also effects through the entire period of gestation to the day before caesarean section. The day of mating (when the sperm-positive vaginal smear was identified) was regarded as gestation day 0 (GD0). Treatment was performed daily by oral (gavage) administration, between GD6 to GD19. Caesarean section and necropsy with macroscopic examination were performed on GD20.


Justification of the doses and route of administration
The dose levels were selected by the Sponsor in consultation with the Study Director, based on the results from an OECD 422 reproductive toxicity study [3] with the aim of inducing toxic effects but no death or suffering at the highest dose, and to obtain a No Adverse Effect Dose Level (NOAEL) at the lowest dose.

The oral route was selected since it is one of the routes of administration requested by the regulatory authorities, and it is considered suitable to provide the exposure required for this developmental toxicology study.

ANIMAL WELFARE
Procedures involving the care and use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). During the study, the care and use of animals were conducted in accordance with the relevant principles currently used with regard to animal welfare.

This study was performed with vertebrate animals as no in vitro alternative was available. The study was designed such that the minimum number of animals was used.
 

Examinations

Maternal examinations:

Clinical signs
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). The principles and criteria summarized in the OECD Humane Endpoints Guidance Document No. 19 were taken into consideration [5].

General clinical observations were made daily, preferably at the same time(s) each day after treatment and once before necropsy.

Detailed clinical observations were made on all animals at the onset of treatment (GD6) then on GD13 and at necropsy (GD20). Observation was performed on the skin, fur, eyes and mucous membranes, occurrence of secretions and excretions, autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as presence of clonic or tonic movements, stereotypes, bizarre behaviour was also observed. Special attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

On GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

Body weight
Body weight of each animal was recorded with precision of ±1 g on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Body weight gain of pregnant females was calculated for each interval, including
GD 0-6, GD 6-20 and GD 0-20.

Food consumption
Food consumption was measured with precision of ±1 g on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption of pregnant females was calculated for each interval, including GD0-6, GD6-20 and GD0-20.

Ovaries and uterine content:

Before expected delivery, on GD20, Caesarean section was performed on each treated dam. Sodium pentobarbital (Euthanimal 40% (400 mg/mL pentobarbital sodium); Manufacturer: Alfasan Nederland BV; Batch number: 2001004-06; Expiry date: 31 January 2023) was administered by intramuscular injection for euthanasia, followed by exsanguination.

The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes. All gross findings are retained in 10% buffered formalin solution.

The weight of the thyroid gland with parathyroid glands for all dams was measured with a precision of 0.001 g. As a paired organ, it was weighed individually, but reported together. Absolute organ weights were measured, and relative paired organ weights to the body weights were calculated and reported. The organs are retained in 10% buffered formalin solution.

The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. If no implantation sites were evident but corpora lutea were present, the uterus was stretched and hold in front of a light source to clearly identify the implantation sites. Uteri that appeared non-gravid were further examined to confirm the non-pregnant status by Salewski staining method.

The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percent of pre- and post-implantation losses was calculated. The degree of resorption (early, late) was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.

Animals were checked for early delivery or abortion, but no signs of these phenomena were noted.

Blood sampling:

For thyroid hormone analysis, blood samples were taken by cardiac puncture into two tubes, one containing lithium heparin as anticoagulant (to be processed for plasma) and one containing no anticoagulant (to be processed for serum) at study termination (at least 1 mL each). The samples were taken before 11:30 AM each day, to avoid the diurnal variation of the hormone concentration among animals. Blood samples from non-pregnant females were not pooled with pregnant dams.

The processed plasma was divided into two aliquots and the serum into three aliquots. The first plasma aliquots were assessed for T4, the first and second serum aliquots for T3 and TSH, respectively.

Thyroxine (T4)

The T4 levels were determined from the plasma samples by IDEXX Catalyst One Chemistry Analyzer (Catalyst Total T4 Test slide, batch: 708096, exp.date: 01 June 2022
measurable range: 6.4-257.4 nmol/L).
 

Triiodothyronine (T3)

Total Triiodothyronine (T3) ELISA kit (Cat. No.: RCD025R, produced by BioVendor – Laboratorní medicína a.s., batch number: X21-090, exp.date: 28 February 2022, (Limit of detection (LOD): 0.049 ng/mL / limit of quantification (LOQ): 0.164 ng/mL) was used for T3 level determination from the serum samples. BMG FLUOstar Omega Microplate Reader was used for the optical density measurements.

Thyroid-stimulating hormone (TSH)

Rat Thyroid Stimulating Hormone (TSH) ELISA kit (Cat. No.: abx156194, produced by Abbexa Ltd., batch number: E2108560W, exp.date: 31 March 2022, (Limit of detection (LOD):
29 pg/mL / limit of quantification (LOQ): 97 pg/mL) was used for TSH level determination from the serum samples. BMG FLUOstar Omega Microplate Reader was used for the optical density measurements.

The samples were analysed on the day of sampling or stored in a freezer until analysis.

The remaining plasma and serum aliquots are stored as backups for confirmatory analyses. Any samples not required for analysis will be discarded following finalization of the report.

Fetal examinations:

After ensuring humane death, each fetus was weighed individually (accuracy ±0.01 g) and subjected to external examination. The sex of each fetus was determined and the anogenital distance was determined of all live fetuses. Thereafter, the fetuses were individually identified; approximately half of each litter was subjected to detailed visceral examination, and the other half was processed for skeletal examination.

Particular attention was paid to the reproductive tract which was examined for signs of altered development. External fetal sex (as determined by gross examination) was compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue malformations). In addition, indication of incomplete testicular descent/cryptorchidism was noted in male fetuses. 

For the fetuses subjected to visceral examination, the abdominal and thoracic regions were opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomiya mixture, then after fixation the body was micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

For the fetuses subjected to skeletal examination, the abdominal region was opened and the viscera and skin of fetuses were removed and the cadaver was fixed in alcian-blue - acetic acid - ethanol mixture. After fixation in isopropanol, the skeletons were stained by KOH-Alizarin
red-S method and examined by means of a dissecting microscope.

All abnormalities (variations, malformations and retardations) found during the fetal examinations were recorded.

Statistics:

See "Any other information on materials and methods incl. tables"

Historical control data:

As per lab historical data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in the study.

Mortality:

no mortality observed

Description (incidence):

There was no mortality during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effect was observed on body weight and body weight gain in the low, mid and high dose groups (100, 300 and 1000 mg/kg bw/day) when compared to the control.

The statistically significant increase in mean body weight gain in the high dose group between GD12 and GD 14 (p˂0.05) was considered not test item related, and was rather a result of the relatively higher number of females (3 out of 21 dams) with less than 6 fetuses in the control dose group in comparison with the treated animals.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no toxicologically relevant effect noted in food consumption in any of the treated groups.

Compared to the control, statistically significant higher group mean food consumption was noted at the high dose group between GD12 and GD 14 (p˂0.05). The cumulative food consumption GD6 – GD20 and GD0-20 was also statistically significantly higher (p˂0.05, respectively) in this group. This can also be explained with the lower number of fetuses in the control dose group, resulting in a less intensive appetite and body weight gain of the pregnant females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

No test item-related difference was observed in thyroid and parathyroid gland weights between the control group and treated groups (100, 300 and 1000 mg/kg bw/day).

Statistically significantly (p˂0.05) higher than control relative thyroid weight (adjusted to body weight) group mean was noted in the low dose group. As the mean value was within the historical control range, and no dose response was shown, the change was considered to be incidental, and not test item related.

The mean TSH, T4 and T3 level was comparable in the control and test item treated groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item related changes were observed in gravid uterine weight of the low, mid and high dose group animals (100, 300 and 1000 mg/kg bw/day) when compared to control data.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item related macroscopic findings were noted at any dose level (100, 300 and
1000 mg/kg bw/day) at necropsy.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No structure/morphology effect was noted in the thyroid and parathyroid glands during histopathological evaluation.

No test item-related difference was observed in thyroid and parathyroid gland weights between the control group and treated groups (100, 300 and 1000 mg/kg bw/day).

Statistically significantly (p˂0.05) higher than control relative thyroid weight (adjusted to body weight) group mean was noted in the low dose group. As the mean value was within the historical control range, and no dose response was shown, the change was considered to be incidental, and not test item related.

The mean TSH, T4 and T3 level was comparable in the control and test item treated groups.

Other effects:

no effects observed

Description (incidence and severity):

No test item related changes were observed in gravid uterine weight of the low, mid and high dose group animals (100, 300 and 1000 mg/kg bw/day) when compared to control data.

Maternal developmental toxicity

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Eighty-nine sperm positive females were included in the study (22, 22, 22 and 23 in the control, low, mid and high dose group, respectively). The number of confirmed pregnant, evaluated dams in the dose groups treated at 0, 100, 300 and 1000 mg/kg/day was 21, 21, 21 and 22, respectively.

There were no test item-related differences in the number of corpora lutea, in pre-implantation loss, in number of implantations, in early embryonic loss and in late embryonic loss between the control group and the treated groups (100, 300 and 1000 mg/kg bw/day).

There was no statistically significant difference in fetal death and post-implantation loss in any test item treated group compared to the control.

The total intrauterine mortality was comparable with the control, no statistically significant difference was observed.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no test item-related differences in the number of corpora lutea, in pre-implantation loss, in number of implantations, in early embryonic loss and in late embryonic loss between the control group and the treated groups (100, 300 and 1000 mg/kg bw/day).

There was no statistically significant difference in fetal death and post-implantation loss in any test item treated group compared to the control.

The total intrauterine mortality was comparable with the control, no statistically significant difference was observed.

Other effects:

no effects observed

Description (incidence and severity):

No macroscopic abnormalities were observed in the placentas in any of the experimental groups in the study.

Details on maternal toxic effects:

No toxic effects were noted.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No effects at the highest dose tested

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The weight of fetuses in the test item-treated dose groups was comparable with the control.

No test item-related differences were observed in the total number of body weight retarded fetuses (evaluated as external variation) in any of the test item treated groups.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no toxicologically significant difference in the sex distribution of fetuses between of the control and treatment groups.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

No test item effect was observed on anogenital distance of the fetuses.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no fetal external, visceral or skeletal abnormalities ascribed to the test item administration to pregnant female rats from GD6 to 19, at dose levels up to and including 1000 mg/kg bw/day. The number and percentages of the abnormalities recorded in test item treated groups was comparable or lower than control.

Two fetuses showed skeletal malformations (split vertebrae and hemivertebra) in the mid dose group, with no dose response therefore are regarded as incidental.

Minor structural changes (variations) occurred, including thymic cord, 2 or less incomplete ossification of the skull bones, 3 or more unossified sternal bodies, supernumerary ribs (14th, short unilateral or bilateral) slight wavy/wavy ribs, unossified vertebrae, bipartite and/or unilateral ossification of vertebrae, less than 3 ossified metacarpal bones, or less than 4 ossified metatarsal bones observed with low incidence throughout all groups including controls, or with lower incidence in the treated groups than in the controls or without a dose response and/or without attaining statistical significance. These variations are common background changes in this strain of rats were not considered toxicologically significant, or related to test item administration.

Visceral malformations:

no effects observed

Other effects:

no effects observed

Effect levels (fetuses)

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects at the highest dose tested

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Overall status of the fetuses

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of evaluated litters	21	21	21	22
Examined fetuses, total	213	234	214	238
Overall intact fetuses, mean	9.14	10.38	9.14	10.00	NS
Overall intact fetuses (%), mean	88.1	93.2	89.2	92.7	NS
Overall intact fetuses, total	192	218	192	220	NS
Fetuses with overall variation, mean	1.00	0.76	0.95	0.82	NS
Fetuses with overall variation, (%), mean	11.9	6.8	10.0	7.3	NS
Fetuses with overall variation, total	21	16	20	18	NS
Fetuses with overall malformation, mean	0.0	0.0	0.1	0.0	NS
Fetuses with overall malformation (%), mean	0.0	0.0	0.9	0.0	NS
Fetuses with overall malformation, total	0	0	2	0	NS

NS: Statistically not significant compared to control

 

Summary of Body weight, litter weight and sex distribution of fetuses

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of evaluated litters	21	21	21	22
Examined fetuses, total	213	234	214	238
Mean fetal weight (g)	3.317	3.284	3.373	3.375	NS
Mean body weight of male fetuses (g)	3.407	3.395	3.461	3.469	NS
Mean body weight of female fetuses (g)	3.246	3.176	3.299	3.292	NS
Mean litter weight (g)	33.69	36.49	34.38	36.60	NS
Fetuses with retarded body weight (%), (runts)	7.05	2.90	3.24	0.86	NS
Number of fetuses with retarded body weight	10	7	7	2	NS
Anogenital distance of male fetuses (mm), mean	2.238	2.253	2.250	2.264	NS
Anogenital distance of female fetuses (mm), mean	1.259	1.271	1.263	1.267	NS
Ratio of male fetuses (%)	39.7	46.0	48.9	46.3	NS
Ratio of female fetuses (%)	60.3	54.0	51.1	53.7	NS

NS: Statistically not significant compared to control

 

Summary of Body weight, litter weight and sex distribution of fetuses

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of evaluated litters	21	21	21	22
Examined fetuses, total	213	234	214	238
Mean fetal weight (g)	3.317	3.284	3.373	3.375	NS
Mean body weight of male fetuses (g)	3.407	3.395	3.461	3.469	NS
Mean body weight of female fetuses (g)	3.246	3.176	3.299	3.292	NS
Mean litter weight (g)	33.69	36.49	34.38	36.60	NS
Fetuses with retarded body weight (%), (runts)	7.05	2.90	3.24	0.86	NS
Number of fetuses with retarded body weight	10	7	7	2	NS
Anogenital distance of male fetuses (mm), mean	2.238	2.253	2.250	2.264	NS
Anogenital distance of female fetuses (mm), mean	1.259	1.271	1.263	1.267	NS
Ratio of male fetuses (%)	39.7	46.0	48.9	46.3	NS
Ratio of female fetuses (%)	60.3	54.0	51.1	53.7	NS

NS: Statistically not significant compared to control

 

Summary of pregnancy data

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of mated females	22	22	22	23
Number of non-pregnant females	1	1	1	1
Number of evaluated females on GD20 (Caesarean section)	21	21	21	22

 

DOSE FORMULATION ANALYSIS

 

Concentration and homogeneity of the dose formulations were determined twice during the study (03 and 22 June 2021).

 

Based on the results, all test item formulations were shown to be homogeneous and were found to be in the range of 94 to 106% of nominal concentrations, as detailed in the table below. No test item was detected in the negative (vehicle) control sample. Based on these results, formulations were considered suitable for the study purposes.

 

Analytical results

Nominal concentration (mg/mL)	Measured concentration ± 95% confidence interval (mg/mL)	RSD (%)	Percentage of the nominal concentration (%)
1^st Analytical sampling (Sampling: 03 June 2021)
Control	not detectable	-	-
66.7	70.78±0.95	1	106
200	188.38±7.77	4	94
666.7	630.98±11.29	2	95
2^nd Analytical sampling (Sampling: 22 June 2021)
Control	not detectable	-	-
66.7	68.96±1.21	2	103
200	188.04±2.28	1	94
666.7	651.72±13.42	2	98

 

HISTOPATHOLOGICAL OBSERVATIONS

EVALUATED ANIMALS

TEST ITEM: SynNova® Base Oil					DOSE: 100 mg/kg bw/day (low dose)							SEX: Female								STUDY CODE: N21001-414
OBSERVATIONS	ANIMAL NUMBERS:		2501	2502		2503	2504	2505	2506	2507	2508		2509	2510	2511	2512	2513	2514	2515		2516	2517	2518	2519	2520	2521
THYROID GLAND (a):		lesion	-	-		-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-
THYROID GLAND (b):		lesion	-	-		-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-

 

NON PREGNANT / NON EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil

DOSE: 100 mg/kg bw/day (low dose)

SEX: Female

STUDY CODE: N21001-414

OBSERVATIONS

ANIMAL NUMBERS:

# 2522 NP

THYROID GLAND (a):

lesion

-

THYROID GLAND (b):

lesion

-

 

 

 

 

EVALUATED ANIMALS

TEST ITEM: SynNova® Base Oil					DOSE: 100 mg/kg bw/day (low dose)							SEX: Female								STUDY CODE: N21001-414
OBSERVATIONS	ANIMAL NUMBERS:		2501	2502		2503	2504	2505	2506	2507	2508		2509	2510	2511	2512	2513	2514	2515		2516	2517	2518	2519	2520	2521
THYROID GLAND (a):		lesion	-	-		-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-
THYROID GLAND (b):		lesion	-	-		-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-

 

NON PREGNANT / NON EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil

DOSE: 100 mg/kg bw/day (low dose)

SEX: Female

STUDY CODE: N21001-414

OBSERVATIONS

ANIMAL NUMBERS:

# 2522 NP

THYROID GLAND (a):

lesion

-

THYROID GLAND (b):

lesion

-

 

EVALUATED ANIMALS

TEST ITEM: SynNova® Base Oil					DOSE: 300 mg/kg bw/day (mid dose)							SEX: Female								STUDY CODE: N21001-414
OBSERVATIONS	ANIMAL NUMBERS:		3501	3502		3503	3504	3505	3506	3507	3508		3509	3510	3511	3512	3513	3514	3515		3516	3517	3518	3519	3520	3521
THYROID GLAND (a):		lesion	-	-		-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-
THYROID GLAND (b):		lesion	-	-		-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-

 

NON PREGNANT / NON EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil

DOSE: 300 mg/kg bw/day (mid dose)

SEX: Female

STUDY CODE: N21001-414

OBSERVATIONS

ANIMAL NUMBERS:

# 3522 NP

THYROID GLAND (a):

lesion

-

THYROID GLAND (b):

lesion

-

 

EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil					DOSE: 1000 mg/kg bw/day (high dose)								SEX: Female								STUDY CODE: N21001-414
OBSERVATIONS	ANIMAL NUMBERS:		4501	4502		4503	4504	4505	4506	4507	4508	4509		4510	4511	4512	4513	4514	4515	4516		4517	4518	4519	4520	4521	4522
THYROID GLAND (a):		lesion	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-
THYROID GLAND (b):		lesion	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-	-		-	-	-	-	-	-

 

 

 

NON PREGNANT / NON EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil				DOSE: 1000 mg/kg bw/day (high dose)		SEX: Female	STUDY CODE: N21001-414
OBSERVATIONS	ANIMAL NUMBERS:		# 4523 NP
THYROID GLAND (a):		lesion	-
THYROID GLAND (b):		lesion	-

 

EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil			TEST SYSTEM: Han:Wist rat			STUDY CODE: N21001-414
HISTOPATHOLOGICAL OBSERVATIONS		GROUPS (mg/kg bw/day)		0	100		300	1000
THYROID GLAND (a):	lesion			0/21	0/21		0/21	0/22
THYROID GLAND (b):	lesion			0/21	0/21		0/21	0/22

 

 

NON PREGNANT / NON EVALUATED ANIMALS

 

TEST ITEM: SynNova® Base Oil			TEST SYSTEM: Han:Wist rat			STUDY CODE: N21001-414
HISTOPATHOLOGICAL OBSERVATIONS		GROUPS (mg/kg bw/day)		0	100		300	1000
THYROID GLAND (a):	lesion			0/1	0/1		0/1	0/1
THYROID GLAND (b):	lesion			0/1	0/1		0/1	0/1

 

 

 

COMMENT:                                                                 NOT PRESENT                              =    -                                                                                                       ABBREVATIONS: M    =MORIBUND                         R= RECOVERY

                                                                                     PRESENT                                      =    +                                                                D    =DIED                                                                                     a = one side                                 b = other side

                                                                                     NO DATA                                      =    /                                                                 Æ    =MISSING ORGAN

                                                                                     GRADE OF ALTERATION:           1     =    MINIMAL CHANGE                            NAFC    =NO ABNORMAL FINDING

                                                                                                                                           2     =    MILD CHANGE                                   TO CORRELATE WITH NECROPSY OBSERVATION

                                                                                                                                           3     =    MODERATE CHANGE                        X     =NOT EXAMINED

                                                                                                                                           4     =    SEVERE (MARKED) CHANGE           NP  = NON PREGNANT

 

Applicant's summary and conclusion

Conclusions:

In summary, daily administration of SynNova® Base Oil by oral gavage to pregnant Hannover Wistar rats from gestation day 6 (GD6) to gestation day 19 (GD19) at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any mortality or clinical signs of the dams under the conditions of this study.

There was no test item-related effect on maternal body weight, body weight gain and food consumption in any of the test item-treated groups.

No remarkable internal or external observations related to the test item treatment were recorded for any of the pregnant animals during necropsy.

No remarkable abnormalities were observed on the placentas in any examined groups.

There were no test item related changes in the examined parameters of the intrauterine development up to and including 1000 mg/kg bw/day.

No test item-related effect was observed on the fetal body weight and the number of viable fetuses in any of the test item-treated groups, as well as their sex distribution were comparable with the control.

Based on the results of thyroid hormones analysis, thyroid weights, histopathological evaluation of thyroids and the measurement of anogenital distance of fetuses, no endocrine disruptor effect was observed in the study.

There were no statistically significant effects of the test item on the external, visceral and/or skeletal development of fetuses in the study.

The No Observed Effect Level (NOEL) under the conditions of this study for maternal and fetal developmental toxicity is considered to be 1000 mg/kg bw/day.

Executive summary:

The purpose of this Prenatal Developmental Toxicity Study was to assess the effects of the test item on pregnant female rats and on the developing conceptuses and provide general information concerning the effects of prenatal exposure on the pregnant test animals and on the developing organism according to the OECD 414 test guideline.

 

Animals (one control and 3 test item treated groups) were treated daily by oral gavage administration, from Gestation Day 6 (GD6) up to and including GD19 (sperm positive day = day 0 of pregnancy, GD0). Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

 

The doses administered were 100, 300 and 1000 mg/kg bodyweight (bw)/day. The dose levels were selected by the Sponsor in consultation with the Study Director based on the results from an OECD 422 reproductive toxicity study [3] with the aim of inducing toxic effects (developmental and/or maternal toxicity) but no death or suffering at the highest dose.

 

Treatment solutions were analysed for test item concentration twice during the treatment period.

 

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Gross macroscopic examination was performed at necropsy. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentae were examined macroscopically. For the purpose of assessing the endocrine disrupter potential of the test item, the thyroid glands were processed for histopathological examination following the measurement of the organ weight. In addition, TSH, T3 and T4 plasma/serum concentrations were measured.

 

Eighty-nine sperm positive females were included in the study, twenty-two in the control, low dose and mid dose groups and twenty-three in the high dose group.

 

All test item formulations were within the range of 94 to 106% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control sample. Based on these results, test item formulations were considered suitable for the study purposes.

 

In summary, daily administration of SynNova® Base Oil by oral gavage to pregnant Hannover Wistar rats from gestation day 6 (GD6) to gestation day 19 (GD19) at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any mortality or clinical signs of the dams under the conditions of this study.

 

There was no test item-related effect on maternal body weight, body weight gain and food consumption in any of the test item-treated groups.

 

No remarkable internal or external observations related to the test item treatment were recorded for any of the pregnant animals during necropsy.

 

No remarkable abnormalities were observed on the placentas in any examined groups.

 

 

 

There were no test item related changes in the examined parameters of the intrauterine development up to and including 1000 mg/kg bw/day.

 

No test item-related effect was observed on the fetal body weight and the number of viable fetuses in any of the test item-treated groups, as well as their sex distribution were comparable with the control.

 

Based on the results of thyroid hormones analysis, thyroid weights, histopathological evaluation of thyroids and the measurement of anogenital distance of fetuses, no endocrine disruptor effect was observed in the study.

 

There were no statistically significant effects of the test item on the external, visceral and/or skeletal development of fetuses in the study.

 

The No Observed Effect Level (NOEL) under the conditions of this study for maternal and fetal developmental toxicity is considered to be 1000 mg/kg bw/day.