Partially hydrogenated β-3,7,11-trimethyldodeca-1,3,6,10-tetraene, reaction products with linear C8-C16 alpha olefin, hydrogenated.

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 24, 2018 to February 15, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study. This study was conducted to support notification in China, and was commissioned as such. It is presented within the EU dossier for completeness purposes.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

OECD Guideline for the Testing of Chemicals, “Prenatal Developmental Toxicity Study” (No.414, adopted on Jan. 22, 2001).

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Partially hydrogenated β-3,7,11-trimethyldodeca-1,3,6,10-tetraene, reaction products with linear C8-C16 alpha olefin, hydrogenated.

EC Number:

939-894-0

Molecular formula:

Variable - substance is a UVCB

IUPAC Name:

Partially hydrogenated β-3,7,11-trimethyldodeca-1,3,6,10-tetraene, reaction products with linear C8-C16 alpha olefin, hydrogenated.

Test material form:

liquid

Details on test material:

Chemical name: Alkenes, C 10-16 a-, reaction products with ( 6E)-7, 11-dimethyl-3-methylene-1 ,6, 1 0-dodecatriene, hydrogenated
Another name: NovaSpec base oil
CAS number: 1481694-12-5
Purity of test item: 100%
Lot number: TS 13788

Physicochemical properties
Vapor pressure: 1.22 Pa (37.8 °C)
Water solubility
Peak 1: 1.45 mg/L
Peak 2: 0.520 mg/L
Peak 3: 0.608 mg/L
[25 °C (Flask method); measured at CERI Kunune. The water solubility of each component is described using the test item concentration without consideration of each constituent ratio.]
Melting point: -39 °C
Boiling point: 216 °C
Appearance: Clear and colorless liquid
Density: 0.822 g/cm3 (20 °C)
Storage conditions: The test sample was stored in a cold and dark storage place.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Species: Rat
Strain: SPRAGUE DAWLEY (SD)
Grade: SPF
Supplier: Beijing Vital River Laboratory Animal Technology Co., Ltd.
Test Animals Quality Certification of supplier: SCXK (Jing) 2016-0006( Certificate number of animals: 11400700318190)
Justification: Rat is the preferred rodent species for prenatal developmental toxicity test and is accredited in OECD Guideline for the Testing of Chemicals, No.414. All females were nulliparous.
Number of animals: One hundred and forty females; sixty males which were spare from G1863B0010 (Certificate number of animals: 11400700318190), the males were used for pairing only.
Animal age on arrival: 84~90 days old
Physical check-up and acclimatization: All animals were checked for health within 24 hours after arrival. The animals meeting the quality requisition were acclimated to the laboratory conditions for six days prior to the experimental start date. All animals were identified with the special animal markers on fur, and were signed on the cage cards at the same time. Clinical observations were performed daily. All animals were weighed on the first day of paring; the body weight range was 225~324g for females and 450~638g for males.

ANIMAL HUSBANDRY
Husbandry:
The test was conducted in the room D109 of the barrier system of this facility. Animal laboratory facility license No.: SYXK (Liao) 2018-0001.
All animals were housed in plastic cages (L46.0 × W31.5 × H20.0cm) on cage racks (L170.0cm × W50.0cm × H160.0cm). There were 5 cages per layer, and 4 layers per rack. There were two rats at most per cage, and mated females were housed individually in cages. During the gestation period, the cage location of females was determined by the mating date.
Environmental conditions: Temperature and humidity in animal room were controlled automatically and daily recorded. The temperature was controlled within 22.0-24.6°C and the relative humidity was controlled within 48-73% in the animal room. Lighting was controlled with the sequence of 12 hours light and 12 hours dark.

Food, Water and Bedding:
The sterilized SPF rodent growth and breeding feed supplied by Beijing Keao Xieli Feed Co., Ltd were used in this test; the lot number of the feed was 18063123. All the nutrition components and contaminants were within the permitted limits described in the national standard. An analysis report of diet was supplied by the supplier.
Drinking water was purified with HT-R01000 purity system and routinely analyzed (at least annually), and all parameters were within the permitted limits described in the national standard.
The corn cob bedding supplied by Beijing Keao Xieli Feed Co., Ltd was used in this test, and the lot number was 18069813. The contaminants of the bedding were within the permitted limits described in the national standard for animal feed. An analysis report of bedding was supplied by the supplier.
Diet and water were available to the animals ad libitum during test. The diet, water and bedding were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Animal Welfare:
Animal use in the whole study complied with national animal welfare laws and regulations (instructive notions with respect to caring for laboratory animals, PRC Ministry of Science, 2006). The animal care and use activities required for conduct of this study were supervised and approved by the testing facility Animal Care and Use Committee (IACUC).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: PEG400 including 0.2% (w/w) TWEEN 80

Details on exposure:

Based on information supplied by the sponsor, the following solvents will be selected:
Poly ethylene glycol 400 (PEG400)
CAS No.: 25322-68-3
Physical state: Viscous colorless liquid
Storage: to be stored in the storage room of this facility at room temperature.

TWEEN 80
CAS No.: 9005-65-6
Physical state: Viscous faint yellow liquid
Storage: to be stored in the storage room of this facility at room temperature.

In this test, PEG400 including 0.2% (w/w) TWEEN 80 will be used as vehicle. As the preparation, 0.2g TWEEN 80 will be added in 99.8g PEG400 for per 100g vehicle.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical check of test item concentrations was conducted on the first and final preparation. Formulations of different test-item concentrations and the vehicle control were sampled and analyzed.
To be considered acceptable, the actual analysis results for at least 2/3 samples of each concentration are to be within ± 20% of the nominal concentration and the relative standard deviation are not more than 20%.
The details about the methods used and the results obtained were shown in the analysis report attached in this final report. The results indicate that the concentrations of the prepared test item were within acceptable limits.

Details on mating procedure:

For each mating, two (or one) female rats and one male rat were put together at afternoon and separated in the next morning. The females were examined for presence of the sperms through vaginal smear method. The day of finding the sperms was defined as the day 0 of pregnancy (GD0). The female rats which failed to mate were put together with male rats and examined again until the number of pregnant rats met the requirements of the test. In the test, there were at least twenty-nine mated females in each group.

Duration of treatment / exposure:

All animals were dosed during the day 5~19 of pregnancy (GD5~GD19).

Frequency of treatment:

All animals were dosed daily in the morning

Duration of test:

15 days (GD5~GD20)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Solvent control group

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose level group

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Middle dose level group

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose level group

No. of animals per sex per dose:

30 animals per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

For each mating day, positive females were allocated to four groups based on body weight as snake order. All animals were identifiable by the individual cage card and be marked on fur at the same time.

Examinations

Maternal examinations:

CLINICAL OBSERVATION
All animals were observed daily by cage-side observations until the termination.
As the administration of the animals by gavage begins, the appearance especially of the perineal region was observed. After dosage, dams were observed for morbidity and mortality twice daily (once daily on nonworking day).

BODY WEIGHT
All pregnant rats were weighed on GD0, then were weighed once per 3 days during the dosing period (GD5~19), and on the day of scheduled kill (GD20).

FOOD CONSUMPTION
During the period of administration, all pregnant rats were provided with a known quantity within 200±20g of feed on the day before body-weight determination, and the remaining feed were weighed on the next day within 24h±1.5h. The daily food consumption of each animal was calculated. Computing formula:
Daily food consumption (g) = added food weight (g) -remaining food weight (g)

All rats were euthanized with CO2 on GD20, and the uterus was removed for examination immediately.

Ovaries and uterine content:

The pregnancy status of the animals was ascertained at first. For the pregnant animals, the whole gravid uteri and total placentas were weighed. The number of corpora lutea, absorbed fetuses (early and late separately), dead fetuses (early and late separately) and viable fetuses were counted.

Fetal examinations:

External examination
The sex and body weight of each viable fetus were determined. Each fetus was examined for external alterations, including head, trunk, limbs and tail. After the examination, all live fetuses were killed with CO2.

Examination of soft tissue
One-half live fetuses of each litter were immersed in modified Davidson`s fixative (the recipe for 100 ml of fixative: 14 ml ethanol, 6.25ml glacial acetic acid, 37.5 ml saturated 37% formaldehyde, and 42.25 ml distilled water) for near one week, rinsed twice with tap water, and stored in 70% isopropyl alcohol for soft tissue examination. As examination, limbs and tail of the fetus were cut down firstly. Then four chips were cut to examine the structural alterations of head. Finally, thorax and abdomen of the fetus were opened to examine the size, shape and position of organs.

Examination of skeleton
The other half of each litter was prepared and examined for skeletal alterations. After removal of the skin and soft tissue, the remainder of each fetus was stained by using alizarin red staining method, and stored in 70% glycerol for examination. This procedure included the skull, vertebra, sternum, ribs, limb bones and pelvis. At the same time, the number of sternal ossification points, namely the number of ossified sternal segments, was recorded.

Statistics:

Calculate the average and standard deviation of the data for each group including body weight, food consumption, body weight change corrected for gravid uterine weight, number of corpora lutea and implantations, number of viable fetuses and body weight of fetuses, etc. All of these data were statistic analyzed by decision tree. The data about abnormal clinical sign, ratio of absorptive fetus, dead fetus and viable fetus, and incidence of individual anomalies were evaluated by the chi-square test. All statistic analysis was done in SPSS 17.0.

Indices:

Maternal Data:
-Number of animals at test start, number of animals surviving, number of pregnant animals, number of animals with total intrauterine mortality
-Clinical signs (by gestation day)
-Mortality (by gestation day), if any
-Body weight and body weight gain: mean ± S.D.
-Corrected body weight on GD20 (body weight minus gravid uterine weight) and corrected body weight gain: mean ± S.D.
-Net body weight change (body weight on GD20 minus body weight on GD5 minus gravid uterine weight): mean ± S.D.
-Food consumption: mean ± S.D.
-Gravid uterine weight

Caesarean Section and Necropsy Data:
-Number of corpora lutea: mean ± S.D.
-Number of implantations: mean ± S.D.
-Number of viable fetuses: mean ± S.D.
-Percent of viable fetuses
-Number and percent of intrauterine mortality:
sub-divided into
pre-implantation loss,
post-implantation loss,
early and late embryonic as well as fetal death.
-Fetal body weight (accuracy 0.001 g): mean * S.D.

Historical control data:

Not specified

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No death and any symptom were found for all animals in all groups in the test

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No death and any symptom were found for all animals in all groups in the test

Body weight and weight changes:

no effects observed

Description (incidence and severity):

During the administration period, only the mean bodyweight gain of pregnant rats in high dose-group during GD14~17 had a significant increase compared with the control group(P≤0.01), the body weight and body weight change of pregnant rats in other dose groups had no significant difference compared with the control group (P>0.05). Similarly, the mean corrected body-weight gain on GD20, the mean corrected body-weight gain during the gestation period and the mean maternal net body-weight gain from the day of treatment (GD5) to the termination (GD20) in the pregnant rats in all dose groups had no significant difference compared with the control group (P>0.05).
According to the results, no adverse effect for the bodyweight of the pregnant rats in all dose groups was observed during the administration period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

During the administration period, the food consumption of the pregnant rat in high-dose group on GD17 had a significant increase compared with the control group (P≤0.01), no statistically significant difference in food consumption of the pregnant rat during the treated period was observed in other dose groups as compared with the vehicle control group (P>0.05).
According to the results, no adverse effect for the food consumption of the pregnant rats in all dose groups was observed during the administration period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No statistically significant difference was observed in the pre-implantation loss compared with the control group in all dose groups(P>0.05); In the mid- and high-dose groups, no statistically significant difference was observed in the post-implantation loss compared with the control group (P>0.05), there was a statistically significant decrease of the post-implantation loss in low-dose group compared with the control group(P≤0.01), but without toxicological significance.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The percent of absorbed fetuses in all dose groups had a statistically significant increase compared with the control group (P≤0.05 or P≤0.01), but that was considered without toxicological significance.

Early or late resorptions:

not specified

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant increase of the percent of fetal death in mid- and high-dose groups compared with the control group(P≤0.05 or P≤0.01), but the death fetuses focused mainly on single litter, and no dose correlation, that was considered caused by individual difference not an adverse effect of the treatment of the test item, no statistically significant difference was observed in the percent of death fetuses in low-dose group(P>0.05).

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not examined

Other effects:

not specified

Details on maternal toxic effects:

No death and any symptom were found for all animals in all groups in the test.
During the administration period, only the mean bodyweight gain of pregnant rats in high dose-group during GD14~17 had a significant increase compared with the control group(P≤0.01), the body weight and body weight change of pregnant rats in other dose groups had no significant difference compared with the control group (P>0.05). Similarly, the mean corrected body-weight gain on GD20, the mean corrected body-weight gain during the gestation period and the mean maternal net body-weight gain from the day of treatment (GD5) to the termination (GD20) in the pregnant rats in all dose groups had no significant difference compared with the control group (P>0.05).
During the administration period, the food consumption of the pregnant rat in high-dose group on GD17 had a significant increase compared with the control group (P≤0.01), no statistically significant difference in food consumption of the pregnant rat during the treated period was observed in other dose groups as compared with the vehicle control group (P>0.05).
According to the results above, no adverse effect for the bodyweight and food consumption of the pregnant rats in all dose groups was observed during the administration period.

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: No effects noted.

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No statistically significant difference in the body weight of live fetuses was observed in all dose groups compared with the vehicle control group (P>0.05).

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No statistically significant difference in the sex distribution of live fetuses was observed in all dose groups compared with the vehicle control group (P>0.05).

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No adverse effect attributable to treatment was observed across all groups with respect to external malformations or variations.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Two fetuses in the control group showed signs of incomplete ossification of skull bone, but the frequency of these abnormalities in the dose groups had no significant difference compared with the control group (P>0.05) and which were considered as incidental findings. No adverse effect attributable to treatment was observed across all groups with respect to skeletal malformations or variations; some examined fetuses had less than six sternal ossification points, and the mean number of sternal ossification points in mid-dose group had a statistically significant increase compared with the control group (P≤0.01), but without toxicological significance, there was no difference in the mean number of sternal ossification points in low- and high-dose groups (P>0.05).

Visceral malformations:

no effects observed

Description (incidence and severity):

One, 3, 1 and 2 fetuses in the control group, low-, mid- and high-dose groups showed signs of pyelectasis in one or double kidneys, two fetuses in the low-dose group showed signs of hydrocephalus. No adverse effect attributable to treatment was observed across all groups with respect to soft-tissue malformations or variations.

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Developmental endpoints: Within the high- and mid-dose groups, no statistically significant difference was observed in the percent of live fetuses compared with the vehicle control group (P>0.05), the percent of live fetuses in low-dose group had a statistically significant increase compared with the control group (P≤0.01), and the percent of absorbed fetuses in all dose groups had a statistically significant increase compared with the control group (P≤0.05 or P≤0.01), but that was considered without toxicological significance; there was a statistically significant increase of the percent of fetal death in mid- and high-dose groups compared with the control group(P≤0.05 or P≤0.01), but the death fetuses focused mainly on single litter, and no dose correlation, that was considered caused by individual difference not an adverse effect of the treatment of the test item, no statistically significant difference was observed in the percent of death fetuses in low-dose group(P>0.05); no statistically significant difference was observed in the pre-implantation loss compared with the control group in all dose groups(P>0.05); In the mid- and high-dose groups, no statistically significant difference was observed in the post-implantation loss compared with the control group (P>0.05), there was a statistically significant decrease of the post-implantation loss in low-dose group compared with the control group(P≤0.01), but without toxicological significance.
Besides, there was a statistically significant increase of the gravid-uterine weight in all dose groups compared with the control group (P≤0.01), but no obvious abnormity was observed for pregnant rats and fetuses, that was considered there was no adverse effect of the treatment of the test item; no statistically significant difference in total placenta weight was observed in all dose groups compared with the vehicle control group (P>0.05).

Fetal examination: No statistically significant difference in the body weight and the sex distribution of live fetuses was observed in all dose groups compared with the vehicle control group (P>0.05).
One, 3, 1 and 2 fetuses in the control group, low-, mid- and high-dose groups showed signs of pyelectasis in one or double kidneys, two fetuses in the low-dose group showed signs of hydrocephalus, two fetuses in the control group showed signs of incomplete ossification of skull bone, but the frequency of these abnormalities in the dose groups had no significant difference compared with the control group (P>0.05) and which were considered as incidental findings. No adverse effect attributable to treatment was observed across all groups with respect to external, soft-tissue and skeletal malformations or variations; some examined fetuses had less than six sternal ossification points, and the mean number of sternal ossification points in mid-dose group had a statistically significant increase compared with the control group (P≤0.01), but without toxicological significance, there was no difference in the mean number of sternal ossification points in low- and high-dose groups (P>0.05).

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: No effects noted.

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Observation results of animal symptoms (symptoms/animal number)

Gestation day	Vehicle control 0 mg/(kg.d)	Low-dose 100 mg/(kg.d)	Mid-dose 300 mg/(kg.d)	High-dose 1000 mg/(kg.d)
Number#	29	29	29	30
GD0~4	-	-	-	-
GD5	-	-	-	-
GD6	-	-	-	-
GD7	-	-	-	-
GD8	-	-	-	-
GD9	-	-	-	-
GD10	-	-	-	-
GD11	-	-	-	-
GD12	-	-	-	-
GD13	-	-	-	-
GD14	-	-	-	-
GD15	-	-	-	-
GD16	-	-	-	-
GD17	-	-	-	-
GD18	-	-	-	-
GD19	-	-	-	-
GD20	-	-	-	-

Note: # Number of all tested rats including non-pregnant females;

GD5~19 are dosing period; GD20 is the day of scheduled kill; -: No symptom

 

Body weight and body-weight gain of pregnant rats (g)

Gestation day	Vehicle control 0 mg/(kg.d)	Low-dose 100 mg/(kg.d)	Mid-dose 300 mg/(kg.d)	High-dose 1000 mg/(kg.d)
Number of pregnant rats	26	28	27	28
GD0	281.8±21.2	281.6±21.6	282.2±21.2	283.5±25.1
GD5	306.5±27.8	313.7±23.0	308.8±21.4	312.7±26.6
GD8	317.8±22.1	322.0±25.0	319.3±21.1	321.9±27.0
GD11	332.3±20.2	338.0±26.3	336.1±21.6	339.4±29.0
GD14	346.2±20.5	352.7±23.7	351.8±20.1	354.6±28.2
GD17	271.0±21.4	381.2±29.7	380.7±22.8	387.6±34.9
GD20	410.6±31.3	425.5±37.0	425.0±25.9	430.3±42.2
GD5>8	11.4±16.0	8.2±6.7	10.5±6.5	9.2±6.9
GD8>11	14.5±6.0	16.1±8.4	16.8±6.4	17.5±8.8
GD11>14	13.9±6.6	14.6±6.7	15.7±9.1	15.2±9.5
GD14>17	24.8±8.3	28.6±11.4	28.9±9.5	33.0±11.1**
GD17>20	39.6±15.2	44.2±11.3	44.3±8.7	42.7±12.2
GD5>20	104.2±28.2	111.7±23.9	116.2±15.3	117.6±24.2
Corrected body-weight on GD20	340.1±25.2	340.3±28.4	339.5±20.7	343.9±28.2
Corrected body-weight gain	58.4±14.4	58.7±18.2	57.3±13.2	60.4±13.7
Net body-weight change	33.7±18.7	26.6±13.5	30.7±14.3	31.3±11.3

Note: Data expressed as mean ± SD;

Statistical analysis by ANOVA, followed by Duncan test as p≤0.05 in SPSS17.0; Compared with the control group, **-p≤0.01

Corrected body weight on GD20 = Body weight on GD20 minus Gravid uterine weight;

Corrected body-weight gain = Corrected body weight on GD20 minus Body weight on GD0

Net body-weight change = Body weight on GD20 minus Body weight on GD5 minus Gravid-uterine weight

 

Food consumption of animals (g/day)

Gestation day	Vehicle control 0 mg/(kg.d)	Low-dose 100 mg/(kg.d)	Mid-dose 300 mg/(kg.d)	High-dose 1000 mg/(kg.d)
Number of pregnant rats	26	28	27	28
GD7>8	21.9±2.6	23.3±3.2	23.7±3.7	22.4±3.3
GD10>11	23.8±2.2^K	25.1±2.9	25.6±4.2	25.6±3.2
GD13>14	23.5±3.4	23.6±3.5	25.1±3.2	25.4±4.2
GD16>17	25.6±2.4^A	25.9±5.0	26.9±4.0	28.5±3.4**
GD19>20	24.6±4.5	24.8±3.9	26.0±3.8	25.1±3.7

Note: Data expressed as mean ± SD;

Statistical analysis by ANOVA, followed by Duncan test as p≤0.05 in SPSS17.0.

^K– Statistical analysis by Kruskal-Wallis, followed by Mann-Whitney test as p≤0.056 in SPSS17.0;

^A– Statistical analysis by ANOVA, followed by Duncan test as p≤0.05 in SPSS17.0;

Compared with the control group, **-p≤0.01.

 

The developmental endpoint results

Parameter	Vehicle control 0 mg/(kg.d)	Low-dose 100 mg/(kg.d)	Mid-dose 300 mg/(kg.d)	High-dose 1000 mg/(kg.d)
Mated female (n)	29	29	29	30
Pregnant female (n)	26	28	27	28
Corpora lutea
Total number	366	444	436	465
Mean±SD	14.1±3.8	15.9±3.4	16.1±2.6	16.6±3.0
Implantation
Total number	347	422	413	441
Mean±SD	13.3±3.8	15.1±3.2	15.3±2.3	15.7±3.1
Gravid uterine weight (g) Mean±SD	70.5±2.4^A	85.2±21.2**	85.5±18.2**	86.4±24.5**
Total placental weight (g) Mean±SD	9.4±2.5^A	10.7±2.5	10.7±2.3	10.7±3.1
Live fetus
Number of litters	25	28	25	28
Total number	302	397	371	401
Mean±SD	12.1±3.6	14.2±3.5	14.3±2.8	14.3±4.5
Percent (%)	87.0(302/347)^C	94.1(397/422)**	789.8(371/413)	90.9(401/441)
Absored fetus
Number of litters	13	11	15	10
Total number^1	45	24	31	33
Percent (%)	13.0(45/347)^C	5.7(24/422)**	7.5(31/413)*	7.5(33/441)*
Dead fetus
Number of litters	0	1	1	2
Total number^2	0	1	11	7
Percent (%)	0(0/347)^C	0.2(1/422)	2.7(11/413)**	1.6(7/441)*
Pre-implantation loss (%)	5.2(19/366)^C	5.0(22/444)	5.3(23/436)	5.2(24/465)
Post-implantation loss (%)	13.0(45/347)^C	5.9(25/422)**	10.2(42/413)	9.1(40/441)

Note:¹Including early absorbed fetuses and late absorbed fetuses;²Including early dead fetuses and late dead fetuses;

^C– Statistical analysis by Chi-square test in SPSS17.0;

^A– Statistical analysis by ANOVA, followed by Duncan test as p≤0.05 in SPSS17.0;

Compared with the control group, *-p≤0.05, **-p≤0.01

 

Fetal examination results

Parameter	Vehicle control 0 mg/(kg.d)	Low-dose 100 mg/(kg.d)	Mid-dose 300 mg/(kg.d)	High-dose 1000 mg/(kg.d)
Body weight of fetus (g) Mean±SD	3.863±0.466A	3.833±0.457	3.893±0.172	3.809±0.310
Sex^b
Number of male fetuses	146	187	186	202
Sex distribution [%] (males/total)	48.3(146/302)	47.1(187/397)	50.1(186/371)	50.4(202/401)
External exam
Number of examined fetuses/litter	302/25	397/28	371/26	401/28
Number of external normal fetuses	302	397	371	400
Fetuses with visceral abnormality (%)[number]	0^C	0	0	0.2(1/401)
Fetuses with visceral abnormality (%)[litter]	0^C	0	0	3.6(1/28)
Fetuses with anury [%]	0^C	0	0	0.2(1/401)
Soft tissue exam
Number of examined fetuses/litter	158/25	205/28	190/26	208/28
Visceral normal fetuses [number]	157	202	189	206
Fetuses with visceral abnormality [number/litter]	1/1	3/2	1/1	2/1
Fetuses with visceral abnormality (%)[number]	0.6(1/158)^C	1.5(3/206)	0.5(1/190)	1.0(2/208)
Fetuses with visceral abnormality (%)[litter]	4.0(1/25)^C	7.1(2/28)	3.8(1*26)	3.6(1/28)
Fetuses with pyelectasis [%]	0.6(1/158)^C	1.5(3/206)	0.5(1/190)	1.0(2/208)
Fetuses with hydrocephalus [%]	0^C	1.0(2/206)	0	0
Skeleton exam
Number of examined fetuses/litter	144/24	191/28	181/26	193/28
Skeletal normal fetuses [number]	142	191	181	193
Fetuses with skeletal abnormality [number/litter]	2/1	0/0	0/0	0/0
Fetuses with skeletal abnormality (%) [number]	1.4(2/144)^C	0	0	0
Fetuses with skeletal abnormality (%) [litter]	4.2(1/24)^C	0	0	0
Fetuses with incomplete ossification of skull bone [%]	1.4(2/144)^C	0	0	0
Sternal ossification points Mean±SD	5.5±0.5^A	5.5±0.6	5.8±0.3**	5.7±0.3

Note:^C– Statistical analysis by Chi-square test in SPSS17.0;

^A– Statistical analysis by ANOVA in SPSS17.0;

Compared with the control group, **-p≤0.01

Applicant's summary and conclusion

Conclusions:

The exposure of gestating rats to different doses of NovaSpec Base Oil by gavage during GD5~GD19 did not result in any death or significant specific toxicity at the highest dose of 1000 mg/ (kg.d); at the same time, no treatment related effect on the body weight gain and food consumption of pregnant rats was observed. Based on these results, it is concluded that the test item had no maternal toxicity at this dose level.
The fetal examination showed that no adverse effect in the survival, body weight and sex distribution of live fetuses was observed in all dose groups, additionally, no adverse effect attributable to treatment was observed across all groups with respect to external, soft-tissue and skeletal malformations or variations. Based on these results, it is concluded that the test item had no developmental toxicity and teratogenicity at the dose levels.
In conclusion, under the conditions of this study, NovaSpec Base Oil administered to pregnant SD rats by gavage in PEG400 including 0.2%(w/w) TWEEN 80, had not produced any maternal toxicity, or teratogenic and embyo-/fetotoxicity at the dose of 1000 mg/(kg.d).
Based on these findings, the following effect levels, No Observed Adverse Effect Level (NOAEL) were derived:
NOAELmaternal toxicity: 1000 mg/ (kg.d)
NOAELembryo-/fetotoxicity: 1000 mg/ (kg.d)
NOAELteratogenicity: 1000 mg/kg/ (kg.d).

Executive summary:

Introduction. This study is conducted to detect maternal and developmental toxicity during the “critical” phase of organogenesis, in particular focused on structural abnormalities in and altered growth of the fetus caused by NovaSpec Base Oil after prenatal exposure. The method of this test was designed to be compatible with OECD Guideline for the Testing of Chemicals, “Prenatal Developmental Toxicity Study” (No.414, adopted in Jan. 22, 2001).

 

Method. This study was conducted in SD rats and all animals were SPF grade. Based on the results of a preliminary range finding test, the animals were treated in the main study with test item at doses of 100, 300 and 1000mg/ (kg.d). A concurrent vehicle control group was included in the study. There were twenty-nine or thirty mated female rats in each group, which resulted in 26, 28, 27 and 28 pregnant rats in the vehicle control group and the low-, mid- and high-, respectively. In the test, all animals were administered the test item by gavage daily during the day 5~19 of pregnancy (GD5~GD19), and were euthanized with CO2 on GD20. After that, removed the uterus of each animal immediately, ascertained the pregnancy status, weighed gravid uterine and total placenta, counted the number of corpora lutea, absorptive fetuses, dead fetuses and viable fetuses for each pregnant animal. Each fetus was examined for external alterations, and the sex and body weight of each fetus were determined at the same time. The fetus was examined for soft tissue or skeleton after being prepared.  

 

Results. No deaths or treatment-related clinical toxiciy were observed in the course of this study.

During the administration period, the mean body weights and body weight change of pregnant rats in all dosed groups had no statistically significant difference compared with the control group. At the same time, no significant effect in food consumption of the pregnant rat during the treated period was observed in all dose groups.

In all dose groups, no adverse effect was observed in all prenatal reproductive parameters.

The fetal examination showed that no adverse effect in body weight and sex distribution of live fetuses was observed in all dosed groups. No adverse effect attributable to treatment was observed across all groups with respect to external, soft-tissue and skeletal malformations or variations, some examined fetuses had less than six sternal ossification points, but there was no adverse effect attributable to treatment.

Conclusion. In conclusion, under the conditions of this study, NovaSpec Base Oil administered to pregnant SD rats by gavage in PEG400 including 0.2%(w/w) TWEEN 80, had not produced any maternal toxicity, or teratogenic and embyo-/fetotoxicity at the dose of 1000 mg/(kg.d).

 

Based on these findings, the following effect levels, No Observed Adverse Effect Level (NOAEL) were derived:

NOAELmaternal toxicity: 1000 mg/ (kg.d)

NOAELembryo-/fetotoxicity: 1000 mg/ (kg.d)

NOAELteratogenicity: 1000 mg/ (kg.d)