Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The No Observed Adverse Effect Level (NOAEL) for parental toxicity to males and females, for exposure to NovaSpec Base Oil by gavage, is considered to be 1000 mg/kg.d.

The NOAEL for reproductive toxic effects to parental males and females, for exposure to NovaSpec Base Oil by gavage, is considered to be 1000 mg/kg.d.

The NOAEL for developmental toxic effects to pups, after parental exposure to NovaSpec Base Oil by gavage, is considered to be 1000 mg/kg.d.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
K1
Additional information

Screening study

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat according to OECD 422 and OECD guidance document 43 was performed in order to obtain information on the possible toxic effect of the test item following repeated (daily) administration by oral gavage to Wistar rats at 3 different dose levels. The study also provided initial information on the possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 4 post-partum.

Twelve Wistar rats per sex/group were assigned to 4 Main study groups.

Additional 5 Wistar rats per sex/group were assigned to a Control and High dose Recovery groups.

Recovery animals scheduled to follow-up observations were not mated but kept without treatment for at least 14 days after the first scheduled euthanasia of dams to detect possible delayed occurrence, or persistence of, or recovery from toxic effects.

The control group was treated with the vehicle only.

 

Analysis of all dose formulations was performed on samples collected on Week 1, Week 3 two occasions, Week 3 and Week 6. The actual concentrations varied between 70% and 105% of the nominal in the Low dose group, 72% and 103% in the Mid dose group, and 79 and 105% in the High dose group. No test item was detected in the control samples. However there was a relatively high individual variations in homogeneity between formulations, the mean concentrations were 45.9 mg/mL (92% of the nominal, n=18, SD = 4.10) for the Low dose group, 139.6 mg/mL (93% of the nominal n=18, SD=12.36 for the Mid dose group, and 458.69 mg/mL (92% of the nominal, n=54, SD=26.95) in the High dose group, therefore considered to be suitable for the study purposes, providing the expected exposure of the animals.

Parameters monitored during the study included signs of morbidity and mortality twice daily, observation of clinical signs, body weight and body weight gain, and food consumption. Five selected males and females from the main group, and all recovery animals were subjected to a clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment in five selected males and females from the main group, and in all recovery animals. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult Main animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals.

 

For the Main adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

 

There was no mortality during the study.

No test item related adverse effects or systemic clinical signs were noted following daily administration of the test item.

Neurological examinations did not reveal any test item related adverse effect.

No test item related adverse effects were noted on body weights or body weight gains.

No effects were observed on the food consumption.

There were no toxicologically relevant effects noted at haematology measurements.

Clinical chemistry measurements did not reveal any test item related adverse effect.

No adverse effects were noted at urinalysis.

There was no effect of treatment noted during evaluation of the reproductive parameters. There were no adverse effects on the F1 offspring viability, clinical signs or development.

No test item related effects were observed at necropsy.

Histopathology examinations on selected tissues did not reveal any test item related effects in the High dose group.

There were no toxicologically significant effects observed in organ weights, however statistically significantly higher than control (+21%) absolute and relative adrenal weight was noted in the High dose group. However, no corresponding effects in clinical pathology or histopathology were noted.

Administration of NovaSpec Base Oil in PEG 400 + Polysorbate 80 via oral gavage to Wistar rats at the dose levels of 100, 300 and 1000 mg/kg bw/day was not associated with any effect on reproduction in the parent males and females, or on development of offspring. Also, no adverse effects were noted in general toxicology parameters. At the dose level of 1000 mg/kg bw/day, elevation of adrenal absolute and relative weights was noted, however considered as not adverse in the lack of any corresponding clinical pathology or histopathology effect.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) of NovaSpec Base Oil for general toxicity, reproduction parameters, and for the development of offspring F1 generation, was considered to be 1000 mg/kg bw/day.

The No Observed Effect Level was determined to be at 1000 mg/kg bw/day in females, and at 300 mg/kg bw/day in males, due to the changes in adrenal absolute and relative weights.

 

2-generation study

This study was conducted in SD rats and all animals were SPF grade. In F0 generation, males and females were exposed to the test item for ten weeks prior to mating, no longer than two weeks of the mating period, and females were exposed continuously during the gestation and lactation period until their pups were weaned. 28 rats/sex/group were selected from these pups as F1 generation. In F1 generation, males and females were exposed to the test item at the same doses with their parents from PND22, including the premating period of at least 10 weeks, mating period of at most 2 weeks, gestation and lactation period (for females).

During the test, the clinical observations were made, the body weight and food consumption were weighed; the estrus cycle in F0 and F1 Generation females was evaluated prior to mating; the growth and developmental parameters of pups were evaluated, and the sexual maturation was observed in the pups of F0 generation. The males were killed for sperm analysis and necropsy after mating period, and the females were killed for necropsy after all pups were weaned; one male and one female pup of each litter as possibly were selected for necropsy. All accidental dead parental animals and pups were macroscopically examined for any abnormalities and pathological changes. The histopathology examination was made for the reproductive organs of the parental animals in the control and high-dose groups, and all gross lesions.

 

Results. During the test, no treatment-related clinical signs and death of the parental animals in two generations was found at any dose level.

In two generations, no significant adverse effect on the body weight and food consumption of parental males and females was observed during each period in all dose groups.

For the parental animals of two generations, no treatment-related effect was observed on all reproductive parameters, including the mating index, fertility index of males and females, pregnancy and delivery results and litter related results, etc. Additionally, estrus cyclicity and sperm parameters were not adversely affected by exposure to the test item in both generations.

For the pups of two generations, no treatment-related effect was observed on all developmental parameters, including the live birth index and the viability index of lactation, the mean number and body weight of live pups during lactation period. Additionally, no treatment-related clinical signs and abnormalities were observed in all pups. For the male and female pups of F0 generation selected for mating, the sexual maturation was not adversely affected in all dose groups.

At the necropsy of all parental animals and pups, no treatment-related gross change was observed; in microscopic examination, no toxicologically relevant change was observed in the reproductive organs of the examined parental animals. At the same time, no treatment-related effect was observed on the absolute and relative organ weights of all parental animals and pups selected for necropsy.

 

Based on the test results above, it is concluded that, under the conditions of the two-generation reproduction toxicity study in rats, the No Observed Adverse Effect Level (NOAEL) for parental toxicity to males and females, for exposure to NovaSpec Base Oil by gavage, is considered to be 1000 mg/kg.d.

The NOAEL for reproductive toxic effects to parental males and females, for exposure to NovaSpec Base Oil by gavage, is considered to be 1000 mg/kg.d.

The NOAEL for developmental toxic effects to pups, after parental exposure to NovaSpec Base Oil by gavage, is considered to be 1000 mg/kg.d.

Dermal route:

White oil was used as a vehicle control group in a dermal application study reported by Schreiner et al. (1997) to determine the potential reproductive effects of kerosene. The study employed a sham-treated control and a group in which white oil Squibb (340 SUS) mineral oil was administered at a dose of 1 ml/kg/day (approximately 1000 mg/kg/day). Only the results of the sham-treated control group and the group given white mineral oil are summarised in the report. The study was performed in accordance with OECD guideline 421 with the addition that males were treated for 8 weeks to assess possible effects on the reproductive system. Also females were weighed 7 times during gestation rather than 4, and at necropsy, 7 organs in addition to the reproductive organs were weighed. Ten approximately eight week old male Sprague-Dawley rats (275 to 285 grams) and 10 females of the same age (183 to 187 grams) per test group were treated dermally. Doses were selected on the basis of the results of a preliminary 2-week range finding study. There were two control groups: the vehicle control was given mineral oil only at a rate of 1 ml/kg/day (approximately 1000 mg/kg/day) and, in the sham-treated group, the animals were fitted with collars and were stroked with the tip of a syringe, but no material was applied. Test material or mineral oil was applied daily to the shorn skin of the animals 7 days/week from 14 days pre-mating, during 14 days mating and through 20 days of gestation. Collars were fitted to the animals during the dosing period to prevent ingestion of applied materials. After the final dose, the collars were removed and residual test material was wiped from the skin. Males were treated through gestation until final female sacrifice on days 4 to 6 of lactation.

 

During the mating period the test material remained on the backs of the animals for 6 hours. Prior to pairing, the test material was removed by wiping. Rats were mated overnight on a 1:1 ratio and were separated the following morning. Collars were then applied prior to the next dose being applied. Females were monitored for evidence that mating had taken place. Pregnancy was determined by the presence of a vaginal plug or sperm in a vaginal lavage sample. If observed, the female was considered to be at day 0 of gestation. Any female that did not show evidence of mating was placed with the same male the following evening. Any female that did not show evidence of mating at the end of a 2 week mating period was presumed pregnant (gestation day 0 = last day of co-habitation).

 

Animals were checked twice daily for morbidity and mortality during weekdays but only once daily at weekends. Animals were also observed immediately prior to dosing and after the last animal had been dosed for appearance, behaviour and motor activity, respiratory function, central nervous system function, excretory function and biological discharges. Effects of test material on the skin were assessed and scored weekly using Draize scales for erythema and oedema and for chronic deterioration. Males were weighed on the first day of dosing, then weekly and on the day of sacrifice. Females were also weighed on the first day of dosing, then weekly until mating was confirmed and thereafter on gestation days 0, 3, 6, 10, 13, 16 and 20 and on post partum days 0 and 4. Food consumption was also monitored on a similar schedule except through the mating period. Each presumed-pregnant female was observed daily from gestation day 20 for parturition; evidence of dystocia was noted. The day of delivery was designated postpartum day 0. Maternal behaviour and appearance were monitored daily until sacrifice. Each litter was examined as soon as possible after birth to establish the number and sex of pups, stillbirths, live births and the presence of gross abnormalities. Pups were examined daily for presence of milk in their stomachs. Any pup found dead was examined externally and unusual findings were recorded. The body weight of each viable offspring was individually measured and recorded on post partum days 1 and 4.

 

Adult females that did not deliver were sacrificed on day 25 of gestation. Dams that delivered and maintained their litters until post partum day 4 were sacrificed with their offspring on post-partum days 4 to 6. All males were sacrificed after the females had been killed. All animals were examined macroscopically for structural anomalies and pathological changes, with emphasis on the reproductive organs. The numbers of implantation sites and corpora lutea of each adult female were recorded. No tissues from offspring were retained. The liver, kidneys, adrenals, thymus, spleen, brain and heart of all parental animals were weighed. In addition the testes and epidymides of parental males were weighed. Skin from treated sites, ovaries and testes and epididymides were prepared for histological examination. Pathological evaluation was performed on reproductive organs from all males and pregnant females in both control groups and the high dose group and on treated skin from all groups.

 

No animals died or were prematurely sacrificed and no clinical signs of toxicity were observed. Skin irritation among males varied from slight to moderate with increasing dose and was most severe in the high dose group. Mild to moderate skin irritation was observed in females at the highest concentration. At terminal sacrifice, no findings were reported except for those on the skin. Microscopic changes were found in the skin of vehicle control and all kerosine-treated groups in the males. In females changes were only observed in the high dose group animals. A mean dermal irritation score of 1.3 (maximum of 3) was observed.Body weights were unaffected by treatment. Reproductive/fertilitywas unaffected by treatment. No test-material-related microscopic changes were observed in the testes or epididymides of adult male rats or in the ovaries of adult female rats.

Squibb (340 SUS) mineral oil (a white mineral oil) caused no reproductive or developmental toxicity with 1 mL/kg/day (i.e., 1000 mg/kg/day) in an OECD 421 guideline study, but did cause mild to moderate skin irritation. Therefore, the reproductive/developmental NOAEL for this study is greater than or equal to 1000 mg/kg/day and no LOAEL was determined.

Effects on developmental toxicity

Description of key information

NOAELmaternal toxicity: 1000 mg/ (kg.d)

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

NOAELteratogenicity: 1000 mg/ (kg.d)


Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure
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.
Qualifier:
according to
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
Specific details on test material used for the study:
No further details specified in the study report.
Species:
rat
Strain:
Sprague-Dawley
Details on test animals 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).
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)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Solvent control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose level group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Middle dose level group
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.
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
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
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.
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.
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).
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.
Key result
Developmental effects observed:
no

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±2.2K

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±2.4A

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±2.4A

85.2±21.2**

85.5±18.2**

86.4±24.5**

Total placental weight (g)

Mean±SD

9.4±2.5A

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 number1

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 number2

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

Sexb

 

 

 

 

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]

0C

0

0

0.2(1/401)

Fetuses with visceral abnormality (%)[litter]

0C

0

0

3.6(1/28)

Fetuses with anury [%]

0C

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 [%]

0C

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±0.5A

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

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)


Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
K1
Additional information

Oral route

This study was 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.

This study was conducted in SD rats and all animals were SPF grade. 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 toxicity were observed in the course of this study.

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.

 

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)

 

On the basis of the lack of effects observed, it is proposed that a study in a second species is not applicable. It is unlikely, taking the data set as a whole, that other adverse effects in a second species would be observed. On the basis of the evidence, and taking account of animal welfare, a second study is not required to augment hazard assessment of this non-hazardous substance.

Justification for classification or non-classification

No classification is applicable.