Registration Dossier
Registration Dossier
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EC number: 941-364-9 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
Reproductive Toxicity NOAEL (Rat): ≥1000 mg/kg/day (OECD 422)
Additionally, gonadal histopathology
and/or sperm parameters (counts; morphology) were among endpoints
included in a subchronic study with aerosol inhalation exposure to
diesel fuel. No effects on reproductive organs were observed and the
NOAEC was 1710 mg/m3 (for aerosolised diesel fuel).
An extended one generation reproduction toxicity test (OECD 443) based on Read-Across substance EC 265 -059 -9 is proposed to fulfil the data requirement but the need for this study for this is questioned based on the fact that there were no effects observed on reproductive organs in repeated dose toxicity tests or in prenatal developmental tests. Overall it is considered unlikely that exposure will affect reproductive performance.
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 06-APRIL-2021 to 28-DEC-2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- July 2016
- Deviations:
- yes
- Remarks:
- Deviations did not impact the overall integrity of the study or the interpretation of the study results and conclusions.
- Qualifier:
- according to guideline
- Guideline:
- other: EPA Health Effects Test Guideline OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.
- Version / remarks:
- July 2000
- Deviations:
- yes
- Remarks:
- Deviations did not impact the overall integrity of the study or the interpretation of the study results and conclusions.
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- yes
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Co-processed Refinery Products REACH Consortium; Batch (lot) No. 204377961
- Purity, including information on contaminants, isomers, etc.: Information about the purity and composition of the test item is not available since the test item is an UVCB (Substance of Unknown or Variable composition, Complex Reaction Products or Biological Materials): 100% w/w
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20276345) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Stability in corn oil for at least 24 hours at room temperature under normal laboratory light conditions and for at least 8 days in the refrigerator is confirmed over the concentration range 2 to 800 mg/mL (solutions).
FORM AS APPLIED IN THE TEST (if different from that of starting material): Colorless to pale yellow liquid
OTHER:
- Specific gravity / density: 0.8319 - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Crl: WI(Han)
- Details on species / strain selection:
- The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories Deutschland (Sulzfeld, Germany)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) males: 6-7 weeks old, females: 6-7 weeks old
- Weight at study initiation: (P) males: 161 - 216 g at onset of treatment, females: 121 - 170 g at onset of treatment
- Fasting period before study: Not specified
- Housing:
On arrival and during the pretest (females only) and pre-mating period: Group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm).
Mating phase: males and females cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
Post-mating phase: Males: home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage; Females: individually housed in Makrolon plastic cages (MIII type, height 18 cm).
Lactation phase: Females: Makrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams.
Locomotor activity monitoring: F0-animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water
- Diet (e.g. ad libitum): Pelleted SM R/M-Z (SSNIFFF® Spezialdiäten GmbH (Soest, Germany)) ad libitum except during designated procedures. During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): Municipal tap water (in water bottles) ad libitum. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period: 9 days prior to start of the pretreatment period
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21°C (target: 18 to 24°C)
- Humidity (%): 44 to 76% (target: 40-70%)
- Air changes (per hr): 10 or more air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
IN-LIFE DATES: From: 07-APRIL-2021 To: 19-AUG-2021 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Remarks:
- Sigma-Aldrich (Steinheim, Germany)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test material dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.
Test material dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/compos
ition of the test material.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil (Sigma-Aldrich, Steinheim, Germany); selected based on trial preparations performed to select a suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 0, 25, 75, or 250 mg/mL for the control, 100, 300, and 1000 mg/kg/day dose groups, respectively
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required): Not specified
- Purity: Not specified - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: maximum 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- Further matings after two unsuccessful attempts: not specified; Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: No - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Concentration:
Dose formulation samples for all groups were collected for concentration analysis during Weeks 1, 6, and 12 (2x ~500 mg) and samples were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility. Analyses were performed using a validated analytical procedure (Test Facility Study No. 20276345) where the temperature was set to maintain 18-22°C. Acceptance criteria for mean sample concentration results were set as within or equal to ± 10% of theoretical concentration.
Homogeneity:
Dose formulation samples for Groups 2, 3, and 4 were collected for homogeneity analysis during Weeks 1, 6, and 12 (2x ~500 mg) and samples were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility. Analyses were performed using a validated analytical procedure (Test Facility Study No. 20276345) where the temperature was set to maintain 18-22°C. Acceptance criteria for homogeneity results was a coefficient of variation (CV) of concentrations of ≤ 10% for each group.
Stability Analysis:
Stability analyses was not performed in the current study but previously in conjunction with the method development and validation study (Test Facility Study No. 20276345). This demonstrated that the test material was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. - Duration of treatment / exposure:
- Males: 7 days a week for a minimum of 90 days, including at least 10 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy.
Females: 7 days a week for at least 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. - Frequency of treatment:
- Once daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Group 1 (Control - corn oil)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- Group 2 (Low dose)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Remarks:
- Group 3 (Intermediate dose)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- Group 4 (High dose)
- No. of animals per sex per dose:
- 10/sex/dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
The dose levels were selected based on the results of a 14 Day Dose Range Finder (DRF) with oral administration of Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin (EC 941-364-9) in rats (Test Facility Reference No. 20276348), and in an attempt to produce graded responses to the test material.
- Rationale for animal assignment (if not random): Animals were randomly assigned to groups at arrival. Males and females were randomized separately.
- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling but water was available. F0-females were not fasted overnight. - Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, beginning during the first administration of the test item and lasting throughout the Dosing Periods up to the day prior to necropsy. During the Dosing Period, observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals.
- Arena Observations: Once before the first administration of the test item and weekly during the Treatment Period.
BODY WEIGHT: Yes
- Time schedule: On Day 1 of treatment (prior to dosing) and weekly thereafter.
Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Quantitatively measured per cage weekly, except for males and females which are housed together for mating and for females without evidence of mating; Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule: Regular basis throughout the study (Monitored by visual inspection of the water bottles. No quantitative investigation was conducted as no effect was suspected).
OTHER:
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule and frequncy:
Pretreatment Period: Once for all main study animals once (including spare animals).
Dosing Period: During Week 13 for all Group 1 and 4 animals.
The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol, tropicamide 5 mg/mL solution). Since treatment-related effects were not observed in Group 1 and 4 ani mals, the ophthalmic examinations were not performed for the other animals.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals: First five F0-males per group and of all F0-females (except for Female No. 61 (300 mg/kg/day), which was found dead)
- Parameters checked in table [No. 2] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals: First five F0-males per group and of all F0-females (except for Female No. 61 (300 mg/kg/day), which was found dead)
- Parameters checked in table [No. 3] were examined.
SERUM HORMONES: Yes
- Time of blood sample collection: On the day of scheduled necropsy
- Animals fasted: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No
- How many animals: All animals
- Parameters checked in table [No. 4] were examined.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Males: during Week 13 of treatment; Females: during the last week of lactation (i.e. PND 6-13)
- Dose groups that were examined: all dose groups
- Battery of functions tested: Hearing ability ; Pupillary reflex; Static righting reflex; Fore- and hind-limb grip strength; Locomotor activity
URINALYSIS: No
IMMUNOLOGY: No
GENERAL REPRODUCTION DATA:
From the mating period onwards, for each female, male number paired with, mating date, confirmation of pregnancy and delivery day were recorded. Females were allowed to litter with Postnatal Day (PND) 1 defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 was considered to be the day when the female started to deliver and was defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care. - Oestrous cyclicity (parental animals):
- Estrous cycles were evaluated by examining the vaginal cytology obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment, and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females, except the ones that died spontaneously.
- Sperm parameters (parental animals):
- For the testes of all male rats in Groups 1 (0 mg/kg/day) and 4 (1000 mg/kg/day), and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution
(if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups; Blood serum was analysed for total Triiodothyronine (T3) , Thyroxine (T4), and Thyroid stimulating hormone (TSH).
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals; after a minimum of 28 days of test material administration
- Maternal animals: All surviving animals; PND 14-16, or after total litter loss, or failure to deliver
GROSS NECROPSY: Yes (see Table 5).
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
Unscheduled Deaths/Euthanasia:
A necropsy was conducted for animals that died on study, and specified tissues were saved
Scheduled Euthanasia:
Animals surviving until scheduled euthanasia were weighed, and deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies were conducted as follows:
- Males (which sired or failed to sire): Following completion of the mating period (a minimum of 13 weeks of administration)
- Females which delivered: PND 14-16
- Females which failed to deliver (Female Nos. 43 and 53): With evidence of mating: Post-coitum Day 27
All F0 males surviving to scheduled necropsy were fasted overnight (water was available) - maximum of 24 hours before necropsy while F0 females were not fasted.
ORGAN WEIGHTS:
The organs identified in table 5 were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.
HISTOPATHOLOGY: Yes (see Table 6)
Representative samples of the tissues identified in the table 6 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
- All Group 1 and 4 animals and unscheduled deaths (found dead): Tissues identified in Text Table 6 (except animal identification, nasopharynx, clitoral gland, harderian gland, lacrimal gland, preputial gland, salivary gland (parotid), larynx, and tongue).
- Males that failed to sire and females that failed to deliver pups (Group 2 and 3): Gross lesions/masses, cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- Remaining animals of Group 2 and 3: Gross lesions/masses, thyroid gland, liver (both sexes), pituitary gland, lungs, kidneys (males), adrenal gland and vagina (females).
Alpha 2μ-globulin staining:
Paraffin blocks of all male kidneys were analyzed using HRP-polymer based chromogenic immunostaining of alpha-2u-globulin.
Microscopic Evaluation:
For the testes of all males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. - Postmortem examinations (offspring):
- SACRIFICE
Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection, were euthanized by an intraperitoneal injection of sodium pentobarbital. The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.
Unscheduled Deaths – F1-Generation
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally). Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Scheduled Euthanasia – F1-Generation
On PND 4, the surplus pups were euthanized by decapitation. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. Additionally, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. If possible, the pups selected for blood sampling were the same pups as selected for thyroid preservation.
HISTOPATHOLOGY / ORGAN WEIGTHS
Not evaluated - Statistics:
- Please see 'Any other information on materials and methods incl. tables' for information on statistics.
- Reproductive indices:
- Reproductive indices
1. Mating index (%): (Number of females mated / Number of females paired) x 100
2. Fertility index (%): (Number of pregnant females / Number of females mated) x 100 - Offspring viability indices:
- 1. Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
2. Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100
3. Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
4. Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check /Number of live pups at First Litter Check) x 100
5. Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check /Number of live pups at First Litter Check) x100
6. Viability index 1 (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
7. Viability index 2 (%): (Number of live offspring on Day 7 after littering / Number live offspring on Day 4 (after culling)) x 100
8. Viability index 3 (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 7 after littering) x 100
9. Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100
10. Cumulative survival index: ((Number of live offspring on Day 13 / Number of live offspring on Day 4 (after culling)) x (Number of live offspring on Day 4 (before culling) / Total number of offspring born)) x 100 - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no clinical signs of toxicity observed during the daily clinical or weekly arena observations. Slight salivation (dose-related) was observed post-dosing in treated animals through most of the treatment period. Taking into account the nature and minor severity of the effect and its time of occurrence (i.e., after dosing), this was considered to be a physiological response rather than a sign of treatment-related systemic toxicity.
Any other clinical signs observed during the treatment period occurred within the range of background findings and the incidence observed, were considered unrelated to treatment. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One female rat (No. 61) in the 300 mg/kg/day dose group was found dead on Day 23 post coitum (before scheduled necropsy). The cause of death could not be determined during histopathologic evaluation of the tissues and twelve intact, normally developed fetuses in the uterus (six left, six right) were observed. Red contents were noted in the stomach, which may indicate cannibalization of one or more pups. Based on the time of death and on the observed macroscopic findings, this death was considered most-likely related to labor difficulties. Additionally, given the single occurrence and in the absence of a dose-response relationship, this death was not considered to be treatment-related.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment-related changes were in body weights or body weight gain were observed through the study period.
In the absence of a dose-related trend, increases in body weights and body weight gain observed in female rats were considered unrelated to treatment. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment-related changes in food consumption before or after correction for body weight were recorded.
In the absence of a dose-related trend, the observed increases in absolute food consumption in female rats were considered unrelated to treatment. Additionally, food consumption values after correcting for body weight were found to be within the control range. - Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not examined
- Description (incidence and severity):
- No quantitative investigation was conducted as no effect was suspected.
- Ophthalmological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment-related ophthalmology findings were observed through the study period. The nature and incidence of ophthalmology findings observed during the Pre-treatment Period and in Week 13 were similar among the treated and control groups; occurred within the normal range for rats of this age and strain; and were therefore not considered to be treatment-related.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Hematological parameters were unaffected by treatment up to 300 mg/kg/day in male rats and up to 100 mg/kg/day in female rats. Increased white blood cell and lymphocyte counts were observed in male rats at 1000 mg/kg/day and decreased eosinophil counts were observed in female rats at 300 and 1000 mg/kg/day. Additionally, decreased red blood cell counts, along with decreased hemoglobin and hematocrit levels, were observed in both sexes at 1000 mg/kg/day. Mean corpuscular volume was increased in males at 1000 mg/kg/day. The changes observed were small and occurred in the absence of correlating morphological findings and were therefore considered to be non-adverse. The findings are presented in Tables 11 and 12. Coagulation parameters in male and female rats were unaffected by treatment up to 1000 mg/kg/day.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Clinical chemistry effects observed included increased total bilirubin levels in both sexes at 1000 mg/kg/day and decreased bile acids in male rats at all dose levels. Additionally, potassium and inorganic phosphate levels were increased in male rats at 1000 mg/kg/day only. However, the changes observed were small and occurred in the absence of correlating morphological findings and were therefore considered to be non-adverse. Increased urea and creatinine levels in males at 300 and 1000 mg/kg/day correlated to morphologic changes in the kidneys of these males (nephropathy) and were considered adverse. The findings are presented in Tables 13, 14, and 15.
- Endocrine findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Serum levels of total thyroxine (T4) in female rats were unaffected by treatment. Serum T4 levels were increased in males at 100 and 300 mg/kg/day (1.30 and 1.20x to control, respectively, not reaching statistical significance at 300 mg/kg/day). Mean values remained within historical control range .
Serum levels of total triiodothyronine (T3) were decreased in male rats from 300 mg/kg/day onwards and in female rats at all dose levels. Total T3 levels in males were 0.75 and 0.76x of control at 300 and 1000 mg/kg/day, respectively, and in females 0.73, 0.83, and 0.72x of control, respectively (not statistically significant at 300 mg/kg/day). Mean values remained within the historical control range for both sexes.
Serum levels of thyroid-stimulating hormone (TSH) in both sexes were considered unaffected by treatment.
The findings are presented in Tables 14 and 15. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Hearing ability, pupillary reflex, static righting reflex and grip strength were unaffected by treatment.
Motor activity was considered unaffected by treatment in male rats up to 300 mg/kg/day and in females at all dose levels. At 1000 mg/kg/day, the number of total movements in male rats was decreased (0.72x of control). All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. In the absence of a dose-response trend, any changes in functional observations were considered unrelated to treatment or considered to derive from a relatively high mean control value (fore grip strength in males at 1000 mg/kg/day). The findings are presented in Tables 16 and 17. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment-related findings at histopathology were observed in the liver and thyroid gland of male and female rats; in the pituitary gland and kidney of male rats; and in the adrenal gland and vagina of female rats.
- Thyroid gland (males and females):
An increased incidence and/or severity of diffuse follicular cell hypertrophy (up to slight degree) and/ or colloid alteration (up to slight degree) was observed in male and female rats starting at 100 mg/kg/day. The colloid alteration was characterized by basophilic granular or clumped deposits in the colloid-filled follicles of the thyroid gland. The low incidence of minimal follicular cell hypertrophy recorded in the males and females of the control group was within background for rats of this age and strain.
- Liver (males and females):
Centrilobular hepatocellular hypertrophy at minimal degree was observed in 3/10 males and females at 1000 mg/kg/day. In females at 1000 mg/kg/day, an increased incidence of cytoplasmic rarefaction at minimal degree was recorded (9/10 females).
- Pituitary gland (males only):
Minimal multifocal hypertrophy of adenohypophyseal cells was recorded in 3/10 males at 1000 mg/kg/day. This alteration was characterized by prominent, large, pale cells (multifocal) in the adenohypophysis.
- Kidney (males only):
A combination of microscopic findings was observed in the kidney of male rats at 300 and 1000 mg/kg/day as indicated below:
a) Hyaline droplet accumulation was recorded in all male rats at 300 mg/kg/day (minimal-moderate) and at 1000 mg/kg/day (slight-marked). The minimal or slight degree recorded in males of the control group and at 100 mg/kg/day were considered to be within background.
b) Granular casts (in the tubules of the medullary area) were observed in most male rats at 300 mg/kg/day (minimal) and in all male rats at 1000 mg/kg/day (minimal-marked).
c) Tubular basophilia was recorded at an increased incidence and severity and seen as a bilateral finding in the treatment groups. At 300 mg/kg/day it was observed at a minimal-slight degree and at 1000 mg/kg/day at slight to marked degree. Tubular basophilia at moderate of marked degree was recorded as bilateral change and characterized by large and multifocal areas of the cortex with basophilic tubular epithelium. The basophilia represented degeneration and regeneration of tubular epithelium. Tubular basophilia at low degrees was characterized by single or a few solitary tubules with basophilic tubular epithelium. The minimal degree (unilateral or bilateral) recorded in males of the control group and at 100 mg/kg/day was considered to be within background.
d) Inflammatory cell infiltrate, lymphocytic was recorded at an increased incidence and severity in all male rats at 1000 mg/kg/day (minimal or slight). The minimal inflammatory cell infiltrates recorded in the remaining dose groups including controls were regarded to be within the background pathology for male rats of this age and strain.
e) Immunohistochemistry for alpha 2μ-globulin of the male kidney showed hyaline droplets as well as the granular casts that stained positive for alpha 2μ-globulin.
- Adrenal gland (females only):
Cortical hypertrophy, zona fasciculata at minimal degree was observed in 2/10 females at 300 mg/kg/day and in 6/10 females at 1000 mg/kg/day.
- Vagina (females only):
Increased mucification of the vaginal epithelium was recorded in 3/10 females at 1000 mg/kg/day. The marked severity of increased vacuolation in one female rat at 300 mg/kg/day (Animal No. 61, found dead on Day 23 post coitum) was considered to be within normal limits for females at the end of the pregnancy period.
- Lung (males only):
A few male rats at 1000 mg/kg/day showed focal to multifocal areas with bronchiolo-alveolar inflammation, mainly confined to one lobe. These findings likely resulted from unintended aspiration of the test material formulation, were considered to be local findings and not regarded to represent a systemic treatment-related effect.
A summary of the findings are presented in Tables 22 23, and 24. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Reproductive function: oestrous cycle:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Length and regularity of the estrous cycle were unaffected by treatment. Most females had regular cycles of 4 to 5 days. One control female (No. 42) was acyclic but delivered a normal litter. Given that this concerned a control female, the finding was unrelated to treatment.
- Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- Stage dependent qualitative evaluation of spermatogenesis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
- Reproductive performance:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 1/10 couples of the control group (Male No. 3 and Female No. 43) and 1/10 couples in 100 mg/kg/day group (Male No. 13 and Female No. 53) were without offspring. For Female No. 53 the infertility was likely due to an imperforate vagina as evidenced by marked dilation with mucinous contents of the cervix. While this could be seen as a developmental abnormality, the single observation in one female at 300 mg/kg/day was regarded unrelated to treatment. No abnormalities were observed in the reproductive organs of the control couple, which could account for their lack of offspring. Data are summarized in Table 25.
Additionally, One female rat in the 300 mg/kg/day dose group (No. 61) was found dead on Day 23 post-coitum with pups in normal stage of development in the uterus and Male No. 21 was therefore regarded to be fertile. This death was regarded to be related to delivery difficulties.
There were no morphological findings in the reproductive organs of either sex which could be considered treatment-related. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Systemic Toxicity
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Reproductive Toxicity
- Key result
- Critical effects observed:
- no
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No clinical signs of toxicity were observed in the pups that were considered to be treatment-related.
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- Viability Index:
Viability index 1 (number of live offspring on PND 4 (before culling) as percentage of number of live offspring on PND 1) was unaffected by treatment with indices being 99, 99, 99, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.
Viability index 2 (number of live offspring on PND 7 after littering as percentage of number of live offspring on PND 4 (after culling)) was unaffected by treatment with indices being 100, 99, 100, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.
Viability index 3 (number of live offspring on PND 13 after littering as percentage of number of live offspring on PND 7) was unaffected by treatment with indices being 100% for all treatment groups.
One pup of the control, 100, and 300 mg/kg/day groups was found missing on PND 2 or 4 or was killed in extremis on PND 4 due to a wound on its back. No clinical signs were observed prior to the pups being noted as missing. These pups were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Lactation Index:
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment. The lactation indices were 100, 99, 100, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.
One pup of the 100 mg/kg/day group was found missing on PND 7. Pups missing were most likely cannibalized and no toxicological relevance was attributed to this since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Cumulative Survival Index:
The cumulative survival index ((number of live offspring on Day 13 after litter/number of live offspring on Day 4 (after culling)) × (number of live offspring on Day 4 before culling / total number of offspring born)) × 100% was considered unaffected by treatment. The indices were 97, 97, 97, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weights of pups were unaffected by treatment with the test material up to 300 mg/kg/day. However, at 1000 mg/kg/day, male, female, and combined pup body weights were lower compared to concurrent control values from PND 4 onwards (not statistically significant on PND 4 and 7). Mean body weights on PND 13 of male and female pups were 13 and 14% lower than controls, respectively.
- Food consumption and compound intake (if feeding study):
- not examined
- 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:
- no effects observed
- Description (incidence and severity):
- Serum T3, T4, and TSH levels in male and female PND 14-16 pups were considered unaffected by treatment.
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- no effects observed
- Description (incidence and severity):
- Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
- Nipple retention in male pups:
- no effects observed
- Description (incidence and severity):
- Treatment with the test material had no effect on areola/nipple retention. No nipples were observed on PND 13 in any of the examined male pups.
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Gross necropsy did not reveal any remarkable treatment-related findings in pups.
- Histopathological findings:
- not examined
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Sex Ratio:
Sex ratio was not affected by treatment with the test material.
Gestation Index and Duration:
Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were unaffected by treatment. Except for one female in the 300 mg/kg/day dose group, all pregnant females had live offspring. The gestation indices were 100, 100, 90, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.
Post-Implantation Survival Index:
The total number of offspring born compared to the total number of uterine implantations was considered unaffected by treatment with the test material. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 97, 91, 78, and 92% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.
Litter Size:
Litter size was not affected by treatment and live litter sizes were 10.6, 11.8, 10.9, and 11.4 living pups/litter for the control, 100, 300, and 1000 mg/kg/day groups, respectively.
Live Birth Index:
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment with the test material. The live birth indices were 98, 99, 98, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.
Two pups in the control and 300 mg/kg/day dose groups, and one pup in the 100 mg/kg/day dose group were found dead at first litter check. Absence of milk in the stomach was observed for the two control pups and the pup at 100 mg/kg/day. The two pups at 300 mg/kg/day were autolytic when found. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. - Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- ca. 300 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- other: Developmental toxicity
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- Based on the effects observed in this sub-chronic oral toxicity study, the systemic toxicity No Observed Adverse Effect Levels (NOAEL) of Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin was determined to be 100 mg/kg/day factoring in the alpha 2µ-globulin nephropathy observed in male rats and ≥1000 mg/kg/day excluding the alpha 2µ-globulin nephropathy since this is a male rat specific effect and of no toxicological relevance to human beings. In the absence of any adverse treatment-related reproductive toxicity observed at the highest dose tested, the NOAEL for reproductive toxicity was determined to be ≥1000 mg/kg/day.
- Executive summary:
A key OECD Guideline 408/422 combined 90-day repeated dose with the reproduction / developmental toxicity screening study was conducted to determine the potential toxic effects of the test material (Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin (EC 941-364-9)). The potential of the test material to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development was also evaluated. The test material was administered once daily to Han Wistar rats (10/sex/dose) via oral gavage in a corn oil vehicle at doses of 0, 100, 300, or 1000 mg/kg/day for a period of 90 days.
Mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, ophthalmology, estrous cycle, clinical pathology, measurement of thyroid hormones T3, T4, and TSH, gross necropsy findings, organ weights, and histopathologic evaluations were undertaken in the study. Additionally,reproductive toxicity parameters examined included mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, and maternal care while developmental parameters evaluated included sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, and measurement of thyroid hormones T3, T4 and TSH).
One female rat (No. 61) in the 300 mg/kg/day dose group was found dead on Day 23 post coitum (before scheduled necropsy). Based on the time of death and on the observed macroscopic findings, this death was considered most-likely related to labour difficulties and not treatment-related. There were no clinical signs of toxicity observed during the daily clinical or weekly arena observations. Slight salivation (dose-related) was observed post-dosing in treated animals through most of the treatment period. Taking into account the nature and minor severity of the effect and its time of occurrence (i.e., after dosing), this was considered to be a physiological response rather than a sign of treatment-related systemic toxicity.No treatment-related changes in body weights or body weight gain and food consumption were observed through the study period. Ophthalmology parameters in male and females rats were unaffected by treatment at doses up to 1000 mg/kg/day. Hearing ability, pupillary reflex, static righting reflex and grip strength were unaffected by treatment. Motor activity was considered unaffected by treatment in male rats up to 300 mg/kg/day and in females at all dose levels. At 1000 mg/kg/day, the number of total movements in male rats was decreased (0.72x of control). All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. In the absence of a dose-response trend, any changes in functional observations were considered unrelated to treatment or considered to derive from a relatively high mean control value (fore grip strength in males at 1000 mg/kg/day).
At 100 mg/kg/day, non-adverse changes such as decreased bile acid concentration in males; increased T4 levels in males; decreased T3 levels in females;and an increased incidence and/or severity of diffuse follicular cell hypertrophy and/or colloid alteration in the thyroid gland of both sexes was observed.
At 300 mg/kg/day, adverse alpha 2µ-globulin nephropathy correlating with increased urea and creatinine levels and enlargement, softening, and greenish discoloration of the kidneys was observed in the kidney of male rats. Correlating microscopic findings comprising hyaline droplet accumulation (confirmed by alpha 2µ-globulin immunostaining), granular casts, and tubular basophilia were also observed in male rats at this dose level. Additionally, at 300 mg/kg/day, non-adverse changes such as decreased bile acid concentration in males; decreased eosinophil count in females; increased T4 levels in males; decreased T3 levels in both sexes; enlargement of the liver in females; increased absolute and relative liver weights in males; increased absolute and relative adrenal gland weights in females; cortical hypertrophy of the zona fasciculata of the adrenal gland in females; increased incidence and/or severity of diffuse follicular cell hypertrophy and/or colloid alteration in the thyroid gland of both sexes were observed.
At 1000 mg/kg/day, adverse alpha 2µ-globulin nephropathy correlating with increased urea and creatinine levels; enlargement of the kidneys; and higher absolute and relative kidney weights was observed in the kidney of male rats. Correlating microscopic findings comprised of hyaline droplet accumulation (confirmed by alpha 2µ-globulin immunostaining), granular casts, tubular basophilia and inflammatory cell infiltrate. Additionally, at this dose levelnon-adverse observed included decreased total movements at motor activity assessment in males; increased white blood cell count; lymphocyte count and mean corpuscular volume in males; decreased red blood cell count; hemoglobin and hematocrit in both sexes; decreased eosinophil count in females; increased total bilirubin in both sexes; decreased bile acid concentration in males; increased potassium and inorganic phosphate levels in males; decreased T3 levels in both sexes; black-brown discoloration of the liver in both sexes; enlargement of the liver in females; increased absolute and relative liver weights in both sexes; centrilobular hepatocellular hypertrophy in both sexes; increased cytoplasmic rarefaction in females; enlargement and increased absolute and relative weight of the adrenal gland in females; cortical hypertrophy of the zona fasciculata of the adrenal gland in females; higher absolute and relative thyroid gland weights in males; increased incidence and/or severity of diffuse follicular cell hypertrophy and/or colloid alteration in the thyroid gland of both sexes; multifocal hypertrophy of adenohypophyseal cells in the pituitary gland in males; increased mucification of the vaginal epithelium in females; and bronchiolo-alveolar inflammation of the lung in males.
Length and regularity of the estrous cycle were unaffected by treatment with most females having a regular cycle of 4 to 5 days. One control female (No. 42) was acyclic but delivered a normal litter. Given that this concerned a control female, the finding was unrelated to treatment. 1/10 couples of the control group (Male No. 3 and Female No. 43) and 1/10 couples in 100 mg/kg/day group (Male No. 13 and Female No. 53) were without offspring. For Female No. 53 the infertility was likely due to an imperforate vagina as evidenced by marked dilation with mucinous contents of the cervix. While this could be seen as a developmental abnormality, the single observation in one female at 300 mg/kg/day was regarded unrelated to treatment. No abnormalities were observed in the reproductive organs of the control couple, which could account for their lack of offspring. Additionally, One female rat in the 300 mg/kg/day dose group (No. 61) was found dead on Day 23 post-coitum with pups in normal stage of development in the uterus and Male No. 21 was therefore regarded to be fertile. This death was regarded to be related to delivery difficulties. There were no morphological findings in the reproductive organs of either sex which could be considered treatment-related. Stage dependent qualitative evaluation of spermatogenesis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
The mating index and precoital time were unaffected by treatment with all females showing evidence of mating within 4 days, with the exception of one female at 1000 mg/kg/day, which showed evidence of mating after 14 days. This was considered to be a background finding commonly observed in rats of this age and strain and not related to treatment. The number of implantation sites was not affected by treatment. The mean number of implantation sites were 11.1, 13.0, 12.8, and 12.4 for the 0, 100, 300, and 1000 mg/kg/day dose groups, respectively. Fertility index was also not affected by treatment and fertility indices were 90, 90, 100, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively. One female in the 100 mg/kg/day group was not pregnant. The infertility was likely due to an imperforated vagina as evidenced by marked dilation with mucinous contents of the cervix. While this could be seen as a developmental abnormality, the single instance in one female at 300 mg/kg/day was regarded unrelated treatment. No signs of difficult or prolonged parturition were observed among the pregnant females, except for one female (No. 61) in the 300 mg/kg/day dose group. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.
Overall no signs of treatment-related reproductive toxicity were observed up to the highest dose level tested (1000 mg/kg/day) in this study.
Based on the effects observed in this sub-chronic oral toxicity study, the systemic toxicity No Observed Adverse Effect Levels (NOAEL) of Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin was determined to be 100 mg/kg/day factoring in the alpha 2µ-globulin nephropathy observed in male rats and ≥1000 mg/kg/day excluding the alpha 2µ-globulin nephropathy since this is a male rat specific effect and of no toxicological relevance to human beings. In the absence of any adverse treatment-related reproductive toxicity observed at the highest dose tested, the NOAEL for reproductive toxicity was determined to be ≥1000 mg/kg/day.
- Endpoint:
- extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)
- Type of information:
- experimental study planned (based on read-across)
- Study period:
- Subject to approval following ECHA/MSCA review of the testing proposal; projected start date for the EOGRTS in Q1 2019
- Justification for type of information:
- This study forms part of the overall Concawe testing strategy for petroleum substances and the VHGO category (see attached preliminary testing strategy document in Section 13).
TESTING PROPOSAL ON VERTEBRATE ANIMALS
[Please provide information for all of the points below. The information should be specific to the endpoint for which testing is proposed. Note that for testing proposals addressing testing on vertebrate animals under the REACH Regulation this document will be published on the ECHA website along with the third party consultation on the testing proposal(s).]
More detailed information on this testing proposal is found in the document "VHGO EOGRTs study proposal v9" attached below in the "attached justification" field.
NON-CONFIDENTIAL NAME OF SUBSTANCE:
This test is proposed to be conducted on one member of the VHGO category, with the results then read-across to other category members. Brief justification is given below, with additional support for the category given in the category justification document (attached to the category object and to Section 13 of the dossier) and in the document "VHGO EOGRTs study proposal v9" attached below in the "attached justification" field.
HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
VHGOs are UVCB substances grouped within established production boundaries based on phys-chem properties and hydrocarbon type (a full justification for this grouping is given in a separate document); substances within the category have qualitatively similar properties. The prediction for read-across in the category will be based on what is considered to be the worst-case substance.
It is hypothesised that the reproductive toxicity of VHGO will be related to the types and levels of aromatics present, and will generally follow a pattern of increasing severity with increasing ring number. Any trend for the developmental toxicity of gas oils would thus be hypothetically described in terms of increasing aromatic content and number of fused aromatic rings.
The predominant PACs in VHGOs are 2- and 3-ring aromatic compounds but some VHGOs may contain very low levels of PACs with four and more aromatic rings. It is hypothesised therefore that there is low potential for adverse effects in developmental reproductive toxicity tests from exposure to VHGOs.
In conclusion, there is a hypothetical case to suggest that any developmental reproductive effects observed in petroleum substances are associated with 4 – 7 ring (possible 3 – 7 ring) PACs, and there is in-vitro and in-vivo toxicity data to support this hypothesis. However, there is no comprehensive investigation of fertility and the implications of interaction with the AhR receptor are not yet fully evaluated (work in progress, as explained above). It is therefore proposed to complement the ongoing work through fully investigating the potential reproductive effects in this study by including cohort 1B.
CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
See detailed discussion of available in vitro and in vivo data on UVCB hydrocarbon classes as well as work currently in progress to help underpin the justifications for category read-across for toxicity to reproduction in "VHGO EOGRTs study proposal v9" document attached below.
• <3 ring polycyclic aromatics: no effects on reproductive organs and no selective developmental effects.
• ≥4 ring polycyclic aromatics (mainly 4-7 ring PACs) with specific structures (not necessarily present in gas oils) are associated with systemic toxicity (effects on liver, thymus and blood forming organs but not reproductive organs) and are potentially mutagenic and dermal carcinogens. In developmental studies they produce foetal death and resorption. Recent in vitro work suggests that ≥3 ring polycyclic aromatics can alter embryo development.
Based on recent analytical research work on representative samples of category members; the following substance has been chosen as a worst-case based on having the highest level of 4-7 ring PAH
- Name of the substance on which testing is proposed to be carried out
Gas oils (petroleum), light vacuum (CAS 64741-58-8, EC 265-059-9)
- Name of the substances for which the testing proposal will be used [if different from tested substance]
Condensates (petroleum), vacuum tower (CAS 64741-49-7, EC 265-049-4)
Gas oils (petroleum), light vacuum (CAS 64741-58-8, EC 265-059-9)
Distillates (petroleum), light hydrocracked (CAS 64741-77-1, EC 265-078-2)
Gas oils (petroleum), hydrodesulfurized light vacuum (CAS 64742-87-6, EC 265-190-1)
Fuels, diesel (CAS 68334-30-5, EC 269-822-7)
Fuel oil, no. 2 (CAS 68476-30-2, EC 270-671-4)
Fuel oil, no. 4 (CAS 68476-31-3, EC 270-673-5)
Fuels, diesel, no. 2 (CAS 68476-34-6, EC 270-676-1)
Gas oils (petroleum), hydrotreated light vacuum (CAS 92045-24-4, EC 295-407-5)
CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
· available GLP studies
There are no available GLP studies within the vacuum gas oils, hydrocracked gas oils, and distillate fuels (VHGO) group,that fully address the reproductive toxicity endpoints needed for classification.
· available non-GLP studies
There are no non-GLP studies available for the endpoint toxicity to reproduction.
· historical human data
No relevant human health data are available.
· (Q)SAR
There are no QSAR’s which are adequate to fully evaluate a UVCB substance (for more detailed explanation on [the complexity of] petroleum UVCBs see Section 2 of the document "VHGO EOGRTs study proposal v9" attached below).
· in vitro methods
There are no validated in vitro methods that fulfil this endpoint. An academic research programme applying an in vitro battery of developmental toxicity screening assays has been launched to underpin the reprotoxicity testing hypothesis that 4-7 ring Poly Aromatic Compounds (PAC) are the only constituents in petroleum substances that are associated with prenatal developmental toxicity (see section 2 of the document "VHGO EOGRTs study proposal v9" attached below for the testing hypothesis, a summary of this in-vitro project and some first published data). These data will be used in combination with in vivo data to further underpin grouping of these substances, in order to minimize animal testing as it supports the selection of the most representative worst case group member for testing and read-across of this test outcome to the other group members.
· weight of evidence
Not available according to ECHA guidance document (Chapter R.7a: Endpoint specific guidance Version 4.1 – October 2015). Concawe are undertaking a multi-year research project to support the grouping of petroleum substances for human health risk assessment (see below) and the data being generated from the various assays applied in this- and other ongoing projects may allow more substantial weight of evidence arguments to be developed in the future.
· grouping and read-across
This EOGRT will be conducted on one representative worst-case sample from the VHGO group (based on PAC content, see Section 2 of this testing proposal for the testing hypothesis and Section 4 for selection of the worst-case test sample). An ongoing research programme, in which high-content in-vitro screening assays and transcriptomic data in combination with phys/chem, analytical chemistry and already available in-vivo data are applied in an integrative analysis to further underpin grouping (www.concawe.eu/cat-app-project), will strengthen the read across assessment from the tested worst-case substance to the other group members. This is expected to significantly reduce the number of tests in vertebrate animals compared to conducting Individual substance evaluations.
There are 9 CAS numbers in the VHGO group. Historically data for toxicity assessment has been generated via the dermal and inhalation route to replicate the route of human exposure to petroleum substances. However, to comply with regulatory requirements for hazard identification, oral exposure is required. Therefore, part of Concawe’s informed testing strategy is to supplement these historical data with targeted oral studies: the range-finding work to be conducted for this EOGRTS will be conducted as an OECD 422 guideline study (oral) which, together with OECD 422 data from other category members, will be used to inform repeated dose toxicity endpoints via the oral route (also underpinning the exposure route / dermal database) and further support grouping & read-across in the group.
· substance-tailored exposure driven testing [if applicable]
Not applicable
· [approaches in addition to above [if applicable]
Not applicable
· other reasons [if applicable]
Not applicable
CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Adaptation options as defined in Annexes VI to X were not applicable for this group and this endpoint. Regarding the column 2 rules for adaption, the data base was fully evaluated according to ‘Guidance on information requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance’.
FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
VHGO’s are widely used as fuels, but do not have significant nonfuel widespread use. Some VHGO’s contain 4 – 7 ring PACs which are believed to interact with the Aryl Hydrocarbon (Ah) receptor which may then lead to modifications of the estrogen receptor pathway; the rationale for this hypothesis and the data to support it are described in detail in section 2 of this testing proposal. As the available data base is limited and because the potential role of PAC’s in effects on fertility is not fully understood it is proposed that the study includes cohort 1B (second generation) to fully evaluate the effects on the developing offspring.
There are no pathological changes in the nervous system or CNS type clinical signs in the current database of dermal studies. However, in one inhalation study on a similar petroleum substance a minor effect on startle response was observed which may be indicative of neurotoxic effects. Due to uncertainties in the data base it is proposed that cohort 2 is included.
VHGOs are classified as H373: May cause damage to thymus, liver, and bone marrow through prolonged or repeated exposure, suggesting organs associated with the immune system are a target for toxicity. It is therefore proposed that cohort 3 for immunotoxicity is also included.
-Overall
With the aim to minimize -and avoid unnecessary- animal testing (on all 192 petroleum substances) while not underestimating the potential human health risks, a worst case testing approach and overall informed tiered testing strategy is proposed holistically across the entire portfolio of petroleum substances. Targeted CAS numbers will be subject to in-vivo testing covering all Concawe petroleum categories.
The sample selected for this test to cover the VHGO category is the CAS number with the highest % (w/w) of 4 – 7 ring PAC’s, as it is considered that this has the greatest potential for reproductive and developmental toxicity (see grouping of VHGO substances and testing hypothesis in section 2). The results of this test, supported with other targeted oral studies, historical data and mechanistic data from in-vitro assays, will be used as read-across for the remaining VHGO group members therefore avoiding additional high animal consuming in vivo studies.
The proposed EOGRTS on VHGOs is part of the overall informed testing strategy (see section 13 of the dossier). The results of this study is expected to help inform the decision making on potential further testing needs and design of similar studies which are required for other petroleum categories. The basic types of hydrocarbon molecules found in this substance and related petroleum category (ie straight and branched alkanes and alkenes, cycloalkanes and cycloalkenes, aromatic and aromatic cycloalkanes) are found in other petroleum categories and, although petroleum substances are UVCB substances of limited variability within product specifications, their similarities can help define a targeted approach to minimize animal use in further testing. - Reason / purpose for cross-reference:
- other: range finding study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
- Justification for study design:
- SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:
- Premating exposure duration for parental (P0) animals - 10 weeks
- Basis for dose level selection – an OECD 422 study by the oral (dietary) route is scheduled and results from this will be used as a basis for dose level selection.
- Inclusion/exclusion of extension of Cohort 1B – some of the 4 – 7 ring aromatic substances present in the VHGO may lead to interactions with the estrogen receptor and may be associated with foetal death and resorption. There are no indications of developmental effects in the absence of maternal toxicity nor robust evidence of endocrine disruptor activity. However, it is recognized that the data base is limited and the potential role of PACs is not fully understood and hence it is requested that the study includes a cohort to fully evaluate the effects on the developing offspring.
- Termination time for F2 – post natal day 4
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B – Based on a minor effect on startle response, cohort 2 is required
- Inclusion/exclusion of developmental immunotoxicity Cohort 3 – VHGO’s have a category 2 classification for repeated dose toxicity of the liver, thymus and bone marrow and therefore there is potentially risk to the developing immune system. Cohort 3 for the EOGRTS is requested
- Route of administration – It is considered that the most likely routes of exposure to VHGO are dermal and inhalation however, ECHA have previous indicated to Concawe that the route of administration for the study addressing this endpoint in Annex X would be oral. It should be noted that the oral route may result in a pattern and degree of exposure that is different to that observed following dermal application.
Concawe propose to conduct the range-finding and study and EOGRT’s via the dietary route. An OECD 422 will be conducted as the range-finding study.
This substance is an aspiration hazard and this is of concern, especially regarding the dose administration to neonates. In addition, VHGO’s are classified as irritating to the skin and petroleum substances are associated with the de-fatting of dermal tissue. Therefore the dietary route may ameliorate any possible gastrointestinal disturbances and irritancy.
Dietary administration is less invasive for the animals, therefore promoting animal welfare. In addition, the procedure of gavage administration may result in mild stress, which may influence some of the sensitive outcomes in this type of study.
The diet will be administered to achieve a constant mg/kg/day dose, the dietary concentration being adjusted to the weekly bodyweight. However, it is necessary to first ensure that homogeneous mixing with the diet and adequate concentration analysis and stability can be achieved and that the treated diet is palatable at sufficiently high concentrations; if this is not possible then the route of administration will revert to gavage. The following approach is suggested to the final design of the EOGRT’s.
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]- - Species:
- rat
- Sex:
- male/female
- Route of administration:
- oral: feed
- Details on exposure:
- This substance is an aspiration hazard and this is of concern, especially regarding the dose administration to neonates. In addition, VHGO’s are classified as irritating to the skin and petroleum substances are associated with the de-fatting of dermal tissue. Therefore the dietary route may ameliorate any possible gastrointestinal disturbances and irritancy.
Dietary administration is less invasive for the animals, therefore promoting animal welfare. In addition, the procedure of gavage administration may result in mild stress, which may influence some of the sensitive outcomes in this type of study.
The diet will be administered to achieve a constant mg/kg/day dose, the dietary concentration being adjusted to the weekly bodyweight. However, it is necessary to first ensure that homogeneous mixing with the diet and adequate concentration analysis and stability can be achieved and that the treated diet is palatable at sufficiently high concentrations; if this is not possible then the route of administration will revert to gavage. The following approach is suggested to the final design of the EOGRT’s. - Details on study design:
- an OECD 422 study by the oral (dietary) route is scheduled and results from this will be used as a basis for dose level selection.
Referenceopen allclose all
Mating index was unaffected by treatment with all females showed evidence of mating.
Precoital time:
Precoital time was considered unaffected by treatment. All females showed evidence of mating within 4 days, with the exception of one female at 1000 mg/kg/day, which showed evidence of mating after 14 days. This was considered to be a background finding commonly observed in rats of this age and strain and not related to treatment.
Number of Implantation Sites:
Number of implantation sites was not affected by treatment. The mean number of implantation sites were 11.1, 13.0, 12.8, and 12.4 for the 0, 100, 300, and 1000 mg/kg/day dose groups, respectively.
Fertility Index:
Fertility index was not affected by treatment. The fertility indices were 90, 90, 100, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.One female in the 100 mg/kg/day group was not pregnant. The infertility was likely due to an imperforated vagina as evidenced by marked dilation with mucinous contents of the cervix. While this could be seen as a developmental abnormality, the single instance in one female at 300 mg/kg/day was regarded unrelated treatment.
Parturition/ Maternal Care:
No signs of difficult or prolonged parturition were observed among the pregnant females, except for one female (No. 61) in the 300 mg/kg/day dose group. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.
No signs of treatment-related reproductive toxicity were observed up to the highest dose level tested (1000 mg/kg/day).
Accuracy
The concentrations analysed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (i.e., mean sample concentration results were within or equal to 90-110% of target concentration) and no test material was detected in the Group 1 formulations.
Homogeneity
The formulations of Groups 2, 3 and 4 were homogeneous (i.e., coefficient of variation ≤10%).
Table 7. Result of Formulation Analysis |
|||||
Date of analysis |
Concentration [mg/mL] |
Recovery [%] |
|||
Target |
Nominal |
Analyzed |
Individual |
Mean |
|
21 Apr 2021 |
25 |
25.2 |
24.3 |
96 |
96 |
25.2 |
24.0 |
95 |
|||
250 |
251 |
251 |
100 |
99 |
|
253 |
247 |
98 |
|||
26 May 2021 |
25 |
24.2 |
23.8 |
98 |
102 |
23.4 |
24.7 |
106 |
|||
250 |
250 |
245 |
98 |
97 |
|
248 |
237 |
96 |
|||
07 Jul 2021 |
25 |
24.9 |
24.9 |
100 |
105 |
26.5 |
29.0 |
110 |
|||
250 |
248 |
273 |
110 |
106 |
|
245 |
252 |
103 |
|||
Table 8. Body Weights (grams) Result Summary |
|||||
|
Group 1 (Control – 0 mg/kg/day) |
Group 2 (100 mg/kg/day) |
Group 3 (300 mg/kg/day) |
Group 4 (1000 mg/kg/day) |
|
Females |
|||||
Pre-mating |
|
||||
Day 1 Week 1 |
Mean |
141 |
147 |
144 |
141 |
St.dev |
11.3 |
6.4 |
7.4 |
11.9 |
|
N |
10 |
10 |
10 |
10 |
|
|
|||||
Day 8 Week 2 |
Mean |
160 |
166 |
163 |
163 |
St.dev |
12.8 |
7.5 |
8.9 |
12.4 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
||||
Day 15 Week 3 |
Mean |
174 |
180 |
177 |
181 |
St.dev |
12.1 |
9.6 |
14.5 |
14.4 |
|
N |
10 |
10 |
10 |
10 |
|
|
|||||
Day 8 Week 2 |
Mean |
160 |
166 |
163 |
163 |
St.dev |
12.8 |
7.5 |
8.9 |
12.4 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 15 Week 3 |
Mean |
174 |
180 |
177 |
181 |
St.dev |
12.1 |
9.6 |
14.5 |
14.4 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 22 Week 4 |
Mean |
189 |
194 |
191 |
196 |
St.dev |
16.0 |
9.6 |
13.9 |
17.4 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 29 Week 5 |
Mean |
198 |
201 |
203 |
204 |
St.dev |
16.1 |
9.4 |
13.2 |
18.2 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 36 Week 6 |
Mean |
203 |
209 |
207 |
211 |
St.dev |
15.1 |
9.3 |
12.2 |
16.6 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 43 Week 7 |
Mean |
210 |
213 |
212 |
215 |
St.dev |
14.2 |
10.0 |
14.4 |
17.8 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 50 Week 8 |
Mean |
213 |
218 |
218 |
217 |
St.dev |
14.8 |
11.0 |
13.2 |
18.3 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 57 Week 9 |
Mean |
216 |
221 |
222 |
216 |
St.dev |
18.1 |
11.4 |
11.3 |
21.0 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 64 Week 10 |
Mean |
219 |
223 |
223 |
219 |
St.dev |
17.3 |
10.8 |
11.9 |
18.2 |
|
N |
10 |
10 |
10 |
10 |
|
Mating Period |
|||||
Day 1 Week 10 |
Mean |
223 |
224 |
227 |
223 |
St.dev |
15.5 |
10.6 |
14.1 |
17.6 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 8 Week 2 |
Mean |
|
|
|
245 |
St.dev |
|
|
|
-- |
|
N |
|
|
|
1 |
|
|
|
|
|
|
|
Day 15 Week 3 |
Mean |
|
|
|
247 |
St.dev |
|
|
|
-- |
|
N |
|
|
|
1 |
|
|
|
|
|
|
|
Post Coitum |
|
|
|
|
|
F0-Generation |
|||||
Day 0 |
Mean |
221 |
230 |
232 |
226 |
St.dev |
14.3 |
12.2 |
13.4 |
19.4 |
|
N |
9 |
9 |
10 |
10 |
|
|
|||||
Day 4 |
Mean |
231 |
239 |
245 |
238 |
St.dev |
13.3 |
14.1 |
14.6 |
20.7 |
|
N |
9 |
9 |
10 |
10 |
|
|
|||||
Day 7 |
Mean |
237 |
249 |
253 |
245 |
St.dev |
15.1 |
16.1 |
14.6 |
19.4 |
|
N |
9 |
9 |
10 |
10 |
|
|
|||||
Day 11 |
Mean |
250 |
262 |
269* |
257 |
St.dev |
14.6 |
17.4 |
15.7 |
17.4 |
|
N |
9 |
9 |
10 |
10 |
|
|
|||||
Day 14 |
Mean |
258 |
274 |
278* |
265 |
St.dev |
16.5 |
15.9 |
18.4 |
19.3 |
|
N |
9 |
9 |
10 |
10 |
|
|
|||||
Day 17 |
Mean |
280 |
299 |
305* |
288 |
St.dev |
17.4 |
17.8 |
18.2 |
19.3 |
|
N |
9 |
9 |
10 |
10 |
|
|
|||||
Day 20 |
Mean |
315 |
343* |
346* |
325 |
St.dev |
19.1 |
22.9 |
23.0 |
22.8 |
|
N |
9 |
9 |
10 |
10 |
|
Lactation |
|
||||
Day 1 |
Mean |
249 |
251 |
262 |
252 |
St.dev |
19.9 |
16.6 |
15.6 |
20.7 |
|
N |
9 |
9 |
9 |
10 |
|
|
|||||
Day 4 |
Mean |
257 |
273 |
275 |
261 |
St.dev |
18.3 |
18.1 |
14.6 |
18.1 |
|
N |
9 |
9 |
9 |
10 |
|
|
|||||
Day 7 |
Mean |
270 |
280 |
282 |
269 |
St.dev |
19.5 |
18.4 |
13.9 |
18.7 |
|
N |
9 |
9 |
9 |
10 |
|
|
|||||
Day 13 |
Mean |
278 |
289 |
295 |
280 |
St.dev |
21.0 |
20.2 |
14.6 |
21.2 |
|
N |
9 |
9 |
9 |
10 |
|
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 9. Body Weight Gain (%) Result Summary |
|||||
|
|
Group 1 (Control – 0 mg/kg/day) |
Group 2 (100 mg/kg/day) |
Group 3 (300 mg/kg/day) |
Group 4 (1000 mg/kg/day) |
Females |
|||||
Pre-mating |
|
||||
Day 1 Week 1 |
Mean |
0 |
0 |
0 |
0 |
St.dev |
0.0 |
0.0 |
0.0 |
0.0 |
|
N |
10 |
10 |
10 |
10 |
|
|
|||||
Day 8 Week 2 |
Mean |
13 |
13 |
13 |
16* |
St.dev |
2.2 |
2.5 |
1.7 |
2.4 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 15 Week 3 |
Mean |
24 |
23 |
23 |
28* |
St.dev |
2.5 |
4.3 |
4.1 |
3.5 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 22 Week 4 |
Mean |
34 |
32 |
33 |
39* |
St.dev |
3.2 |
2.6 |
4.8 |
3.0 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 29 Week 5 |
Mean |
40 |
37 |
41 |
44* |
St.dev |
3.0 |
2.7 |
4.2 |
3.0 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 36 Week 6 |
Mean |
44 |
42 |
44 |
50** |
St.dev |
4.1 |
3.0 |
3.7 |
4.5 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 43 Week 7 |
Mean |
49 |
45 |
47 |
52 |
St.dev |
4.8 |
4.9 |
3.9 |
5.2 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 50 Week 8 |
Mean |
51 |
49 |
52 |
54 |
St.dev |
3.9 |
3.7 |
4.4 |
4.7 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 57 Week 9 |
Mean |
53 |
50 |
54 |
53 |
St.dev |
3.7 |
3.9 |
3.8 |
5.3 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Day 64 Week 10 |
Mean |
55 |
52 |
55 |
55 |
St.dev |
4.1 |
3.2 |
4.8 |
5.4 |
|
N |
10 |
10 |
10 |
10 |
|
Mating Period |
|||||
Day 1 Week 1 |
Mean |
58 |
53* |
58 |
58 |
St.dev |
5.4 |
4.6 |
4.7 |
4.3 |
|
N |
10 |
10 |
10 |
10 |
|
|
|||||
Day 8 Week 2 |
Mean |
|
|
|
70 |
St.dev |
|
|
|
-- |
|
N |
|
|
|
1 |
|
|
|||||
Day 15 Week 3 |
Mean |
|
|
|
72 |
St.dev |
|
|
|
-- |
|
N |
|
|
|
1 |
|
F0-Generation |
|||||
Post coitum |
|
||||
Day 0 |
Mean |
0 |
0 |
0 |
0 |
St.dev |
0.0 |
0.0 |
0.0 |
0.0 |
|
N |
9 |
9 |
10 |
10 |
|
|
|||||
Day 4 |
Mean |
5 |
4 |
6 |
5 |
St.dev |
1.7 |
1.6 |
1.8 |
2.0 |
|
N |
9 |
9 |
10 |
10 |
|
|
|
|
|
|
|
Day 7 |
Mean |
8 |
8 |
9 |
8 |
St.dev |
1.7 |
2.7 |
1.9 |
2.4 |
|
N |
9 |
9 |
10 |
10 |
|
|
|
|
|
|
|
Day 11 |
Mean |
13 |
14 |
16 |
14 |
St.dev |
2.3 |
3.1 |
2.4 |
3.8 |
|
N |
9 |
9 |
10 |
10 |
|
|
|
|
|
|
|
Day 14 |
Mean |
17 |
19 |
20 |
17 |
St.dev |
3.3 |
3.4 |
2.1 |
3.8 |
|
N |
9 |
9 |
10 |
10 |
|
|
|
|
|
|
|
Day 17 |
Mean |
27 |
30 |
31* |
28 |
St.dev |
3.9 |
4.3 |
2.7 |
3.6 |
|
|
N |
9 |
9 |
10 |
10 |
|
|
|
|
|
|
Day 20 |
Mean |
43 |
49* |
49* |
44 |
St.dev |
6.5 |
6.5 |
5.2 |
3.3 |
|
N |
9 |
9 |
9 |
10 |
|
Lactation |
|||||
Day 1
|
Mean |
0 |
0 |
0 |
0 |
St.dev |
0.0 |
0.0 |
0.0 |
0.0 |
|
N |
9 |
9 |
9 |
10 |
|
|
|||||
Day 4 |
Mean |
4 |
5 |
5 |
4 |
St.dev |
3.4 |
2.2 |
2.0 |
2.6 |
|
N |
9 |
9 |
9 |
10 |
|
|
|
||||
Day 7 |
Mean |
9 |
7 |
8 |
7 |
St.dev |
4.5 |
2.0 |
3.9 |
2.7 |
|
N |
9 |
9 |
9 |
10 |
|
|
|||||
Day 13 |
Mean |
12 |
11 |
13 |
11 |
St.dev |
5.2 |
3.2 |
4.0 |
4.8 |
|
N |
9 |
9 |
9 |
10 |
|
|
|||||
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 10. Food Consumption (g/animal/day) Result Summary |
|||||
|
Group 1 (Control – 0 mg/kg/day) |
Group 2 (100 mg/kg/day) |
Group 3 (300 mg/kg/day) |
Group 4 (1000 mg/kg/day) |
|
Females |
|||||
Pre-mating |
|
||||
Days 1-8 Weeks 1-2 |
Mean |
15 |
15 |
15 |
14 |
St.dev |
0.4 |
0.4 |
0.5 |
1.1 |
|
N(cage) |
2 |
2 |
2 |
2 |
|
|
|||||
Days 8-15 Weeks 2-3 |
Mean |
15 |
15 |
16 |
17 |
St.dev |
0.4 |
0.6 |
0.3 |
1.5 |
|
N(cage) |
2 |
2 |
2 |
2 |
|
|
|
|
|
|
|
Days 15-22 Weeks 3-4 |
Mean |
16 |
15 |
16 |
17 |
St.dev |
0.2 |
0.6 |
0.0 |
1.8 |
|
N(cage) |
2 |
2 |
2 |
2 |
|
|
|
|
|
|
|
Days 22-29 Weeks 4-5 |
Mean |
16 |
15 |
16 |
17 |
St.dev |
0.4 |
0.3 |
0.1 |
1.3 |
|
N(cage) |
2 |
2 |
2 |
2 |
|
|
|
|
|
|
|
Day 29-36 Weeks 5-6 |
Mean |
15 |
15 |
15 |
16 |
St.dev |
0.2 |
0.7 |
0.4 |
0.7 |
|
N(cage) |
2 |
2 |
2 |
2 |
|
|
|
|
|
|
|
Day 36-43 Week 6-7 |
Mean |
15 |
14 |
15 |
15 |
St.dev |
0.1 |
0.4 |
0.4 |
0.7 |
|
N(cage) |
2 |
2 |
2 |
2 |
|
|
|
|
|
|
|
Day 43-50 Weeks 7-8 |
Mean |
14 |
13 |
14 |
14 |
St.dev |
0.2 |
0.3 |
0.1 |
0.7 |
|
N(cage) |
2 |
2 |
2 |
2 |
|
|
|
|
|
|
|
Day 50-57 Weeks 8-9 |
Mean |
13 |
13 |
14 |
14 |
St.dev |
0.7 |
0.7 |
0.8 |
1.1 |
|
N(cage) |
2 |
2 |
2 |
2 |
|
|
|
|
|
|
|
Days 57-64 Weeks 9-10 |
Mean |
13 |
13 |
14 |
14 |
St.dev |
0.5 |
0.5 |
0.8 |
1.2 |
|
|
N(cage) |
2 |
2 |
2 |
2 |
|
|
|
|
|
|
Days 64-71 Weeks 10-11 |
Mean |
14 |
13 |
15 |
14 |
St.dev |
0.2 |
0.5 |
1.0 |
1.5 |
|
N(cage) |
2 |
2 |
2 |
2 |
|
|
|
|
|
|
|
Mean of Means |
Mean |
14 |
14 |
15 |
15 |
F0 -Generation |
|||||
Post Coitum |
|
||||
Days 0-4 |
Mean |
14 |
14 |
18* |
16 |
St.dev |
2.5 |
3.2 |
1.7 |
3.4 |
|
N |
9 |
9 |
10 |
10 |
|
|
|||||
Days 4-7 |
Mean |
17 |
19 |
19 |
19 |
St.dev |
1.8 |
3.0 |
2.6 |
2.5 |
|
N |
9 |
9 |
10 |
10 |
|
|
|||||
Days 7-11 |
Mean |
15 |
17 |
18* |
17 |
St.dev |
1.9 |
2.6 |
1.7 |
1.7 |
|
N |
9 |
9 |
10 |
10 |
|
|
|||||
Days 11-14 |
Mean |
19 |
21 |
21 |
22 |
St.dev |
3.8 |
3.0 |
2.2 |
3.3 |
|
N |
9 |
9 |
10 |
10 |
|
|
|||||
Days 14-17 |
Mean |
19 |
19 |
23* |
20 |
St.dev |
3.8 |
4.5 |
1.4 |
4.0 |
|
N |
9 |
9 |
10 |
10 |
|
|
|||||
Days 17-20 |
Mean |
23 |
25 |
26* |
24 |
St.dev |
2.0 |
2.5 |
2.7 |
1.7 |
|
N |
9 |
9 |
10 |
10 |
|
Mean of means |
|
18 |
19 |
21 |
20 |
Lactation |
|
||||
Days 1-4 |
Mean |
26 |
29 |
30 |
28 |
St.dev |
5.3 |
4.7 |
3.6 |
4.7 |
|
N |
9 |
9 |
9 |
9 |
|
|
|||||
Days 4-7 |
Mean |
41 |
42 |
40 |
39 |
St.dev |
6.4 |
4.9 |
3.5 |
3.5 |
|
N |
9 |
9 |
9 |
10 |
|
|
|||||
Days 7-13 |
Mean |
49 |
50 |
49 |
46 |
St.dev |
5.2 |
5.8 |
4.0 |
5.4 |
|
N |
9 |
9 |
9 |
10 |
|
Mean of means |
|
39 |
41 |
40 |
38 |
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 11. Select Hematological Results |
|||
Dose (mg/kg/day): |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
|
Sex: |
Female |
Male |
Female |
Number of animals: |
9 |
4 |
10 |
White Blood Cells (WBC) |
- |
1.17x |
- |
Lymphocytes (LYMPH) |
- |
1.16x |
- |
Eosinophils (EOS) |
0.54x* |
- |
0.41x** |
Red blood cells (RBC) |
- |
0.92x |
0.94x |
Hemoglobin (HGB) |
- |
0.95x |
0.93x** |
Hematocrit (HCT) |
- |
0.95x |
0.92x** |
Mean Corpuscular Volume (MCV) |
- |
1.04x* |
- |
- indicates absence of change.
Numerical values indicate fold change of the treated group mean value relative to the control group value.
Mean values were statistically different from control at * P < 0.05, ** P <0.01
Table 12. Summary of Select Hematology Values: F0 Generation |
|||||
Day: 93 Relative to Start Date (Males)
Day: 94 Relative to Start Date (Females) |
|
Reporting Hematology |
|||
Males |
Females |
||||
MCV (fL) [G] |
EOS (109/L) [G1] |
HGB (g/L) [G] |
HCT (L/L) [G1] |
||
Group 1 (Control – 0 mg/kg/day) |
Mean |
51.94 |
0.076 |
141.2 |
0.4333 |
SD |
1.24 |
0.024 |
5.4 |
0.0149 |
|
N |
5 |
9 |
9 |
9 |
|
|
|||||
Group 2 (100 mg/kg/day) |
Mean |
51.74 |
0.061 |
136.4 |
0.4188 |
SD |
0.77 |
0.029 |
6.5 |
0.0253 |
|
N |
5 |
9 |
9 |
9 |
|
tCtrl |
1.00 |
0.81 |
0.97 |
0.97 |
|
|
|||||
Group 3 (300 mg/kg/day) |
Mean |
51.72 |
0.041* |
136.8 |
0.4170 |
SD |
0.56 |
0.013 |
2.7 |
0.0085 |
|
N |
5 |
9 |
9 |
9 |
|
tCtrl |
1.00 |
0.54 |
0.97 |
0.96 |
|
|
|||||
Group 4 (1000 mg/kg/day) |
Mean |
53.78* |
0.031** |
131.9** |
0.4005** |
SD |
0.74 |
0.014 |
6.8 |
0.0239 |
|
N |
4 |
10 |
10 |
10 |
|
tCtrl |
1.04 |
0.41 |
0.93 |
0.92 |
|
[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01
[G1] - Kruskal-Wallis & Dunn: * = p ≤ 0.05; ** = p ≤ 0.01
Table 13. Select Clinical Chemistry Results |
||||||
Dose (mg/kg/day): |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
|||
Sex: |
Male |
Female |
Male |
Female |
Male |
Female |
Number of animals: |
10 |
9 |
10 |
9 |
10 |
10 |
Total bilirubin (TBIL) |
- |
- |
- |
- |
1.12x* |
1.17x* |
Bile acids (BILEAC) |
0.73x |
- |
0.32x** |
- |
0.31x** |
- |
Urea (UREA) |
- |
- |
1.20x** |
- |
1.28x** |
- |
Creatinine (CREAT) |
- |
- |
1.14x** |
- |
1.25x** |
- |
Potassium (K) |
- |
- |
- |
- |
1.10x** |
- |
Inorganic Phosphate (PHOS) |
- |
- |
- |
- |
1.13x* |
- |
(-) indicates absence of change.
Numerical values indicate fold change of the treated group mean value relative to the control group value.
Mean values were statistically different from control at * P < 0.05, ** P <0.01.
Table 14. Summary of Select Clinical Chemistry Values: F0 Generation |
||||||||||||
Day: 93 Relative to Start Date (Males) |
Reporting Clinical Chemistry |
|||||||||||
Males |
||||||||||||
ALT (U/L) [G] |
AST (U/L) [G1] |
TBIL (µmol/L) [G1] |
BILEAC [G] |
UREA [G1] |
CREAT (µmol/mL) [G1] |
GLUC (mmol/L) [G1] |
K (mmol/L) [G] |
PHOS (mmol/L) [G] |
T3 (ng/mL) |
T4 [G] |
||
Group 1 (Control – 0 mg/kg/day) |
Mean |
39.8 |
84.2 |
2.97 |
54.33 |
5.21 |
40.5 |
8.989 |
4.82 |
2.080 |
0.353 |
42.50 |
SD |
5.7 |
9.9 |
0.22 |
33.54 |
0.53 |
2.0 |
1.278 |
0.23 |
0.256 |
0.047 |
7.52 |
|
N |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
|||||||
Group 2 (100 mg/kg/day) |
Mean |
37.9 |
102.3* |
2.79 |
39.67 |
5.55 |
42.2 |
7.369* |
4.92 |
2.199 |
0.326 |
55.24** |
SD |
8.3 |
9.9 |
0.32 |
29.24 |
0.60 |
2.6 |
1.089 |
0.26 |
0.143 |
0.050 |
9.91 |
|
N |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
|
tCtrl |
0.95 |
1.21 |
0.94 |
0.73 |
1.07 |
1.04 |
0.82 |
1.02 |
1.06 |
0.92 |
1.30 |
|
|
|
|
|
|
|
|||||||
Group 3 (300 mg/kg/day) |
Mean |
30.0* |
82.6 |
2.75 |
17.13** |
6.24** |
46.2** |
8.755 |
5.02 |
2.124
|
0.266** |
51.15 |
SD |
3.8 |
10.7 |
0.23 |
6.61 |
0.83 |
2.1 |
0.952 |
0.26 |
0.248 |
0.041 |
10.65 |
|
N |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
|
tCtrl |
0.75 |
0.98 |
0.93 |
0.32 |
1.20 |
1.14 |
0.97 |
1.04 |
1.02 |
0.75 |
1.20 |
|
|
|
|
|
|
|
|||||||
Group 4 (1000 mg/kg/day) |
Mean |
39.6 |
100.4* |
3.32* |
16.92** |
6.65** |
50.7** |
8. 020 |
5.29** |
2.353* |
0.267* |
40.39 |
SD |
10.2 |
22.9 |
0.40 |
6.68 |
0.90 |
3.6 |
1.423 |
0.44 |
0.167 |
0.096 |
7.00 |
|
N |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
|
tCtrl |
0.99 |
1.19 |
1.12 |
0.31 |
1.28 |
1.25 |
0.89 |
1.10 |
1.13 |
0.76 |
0.95 |
|
[G] - Kruskal-Wallis & Dunn: * = p ≤ 0.05; ** = p ≤ 0.01
[G1] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01
Table 15. Summary of Select Clinical Chemistry Values: F0 Generation |
|||||
Day: 94 Relative to Start Date (Females) |
Reporting Clinical Chemistry |
||||
Females |
|||||
A/G [G] |
LDL (mmol/L) [G] |
TBIL (µmol/L) [G] |
T3 (ng/mL) [G] |
||
Group 1 (Control – 0 mg/kg/day) |
Mean |
1.27 |
0.204 |
2.72 |
0.341 |
SD |
0.07 |
0.035 |
0.32 |
0.074 |
|
N |
9 |
9 |
9 |
9 |
|
|
|||||
Group 2 (100 mg/kg/day) |
Mean |
1.26 |
0.158** |
2.67 |
0.248* |
SD |
0.05 |
0.034 |
0.40 |
0.044 |
|
N |
9 |
9 |
9 |
9 |
|
tCtrl |
0.99 |
0.77 |
0.98 |
0.73 |
|
|
|||||
Group 3 (300 mg/kg/day) |
Mean |
1.28 |
0.167* |
2.81 |
0.284 |
SD |
0.04 |
0.022 |
0.25 |
0.080 |
|
N |
9 |
9 |
9 |
9 |
|
tCtrl |
1.01 |
0.82 |
1.03 |
0.83 |
|
|
|||||
Group 4 (1000 mg/kg/day) |
Mean |
1.35** |
0.143** |
3.19* |
0.247* |
SD |
0.05 |
0.032 |
0.47 |
0.062 |
|
N |
10 |
10 |
10 |
10 |
|
tCtrl |
1.07 |
0.70 |
1.17 |
0.72 |
|
[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01
Table 16. Functional Observations Summary |
|||||
|
Group 1 (Control – 0 mg/kg/day) |
Group 2 (100 mg/kg/day) |
Group 3 (300 mg/kg/day) |
Group 4 (1000 mg/kg/day) |
|
Males |
|||||
End of Treatment |
|
||||
Hearing Score 0/1 |
Median |
0 |
0 |
0 |
0 |
N |
10 |
10 |
10 |
10 |
|
|
|||||
Pupil L Score 0/1 |
Median |
0 |
0 |
0 |
0 |
N |
10 |
10 |
10 |
10 |
|
|
|||||
Pupil R Score 0/1 |
Median |
0 |
0 |
0 |
0 |
N |
10 |
10 |
10 |
10 |
|
|
|||||
Static R Score 0/1 |
Median |
0 |
0 |
0 |
0 |
N |
10 |
10 |
10 |
10 |
|
|
|||||
Grip Fore gram |
Mean |
1421 |
1402 |
1294 |
1114* |
SD |
59 |
105 |
196 |
253 |
|
N |
10 |
10 |
10 |
10 |
|
|
|||||
Grip Hind gram |
Mean |
759 |
902* |
754 |
737 |
SD |
155 |
73 |
96 |
91 |
|
N |
10 |
10 |
10 |
10 |
|
Females |
|||||
At Lactation |
|||||
Hearing Score 0/1 |
Median |
0 |
0 |
0 |
0 |
N |
9 |
9 |
9 |
10 |
|
|
|||||
Pupil L Score 0/1 |
Median |
0 |
0 |
0 |
0 |
N |
9 |
9 |
9 |
10 |
|
|
|||||
Pupil R Score 0/1 |
Median |
0 |
0 |
0 |
0 |
N |
9 |
9 |
9 |
10 |
|
|
|||||
Static R Score 0/1 |
Median |
0 |
0 |
0 |
0 |
N |
9 |
9 |
9 |
10 |
|
|
|||||
Grip Fore gram |
Mean |
1235 |
1303 |
1220 |
1068 |
SD |
202 |
121 |
165 |
130 |
|
N |
9 |
9 |
9 |
10 |
|
|
|||||
Grip Hind gram |
Mean |
696 |
764 |
662 |
651 |
SD |
76 |
108 |
97 |
80 |
|
N |
9 |
9 |
9 |
10 |
|
|
|
|
|
|
|
+/++ Steel-test significant at 5% (+) or 1% (++) level
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 17. Motor Activity test summary |
||||
Males |
||||
Total Movements |
Group 1 (Control) 0 mg/kg/day |
Group 2 mg/kg/day |
Group 3 |
Group 4 |
Mean |
3097 |
3114 |
2649 |
2241** |
N |
10 |
10 |
10 |
10 |
St. Dev |
740 |
844 |
404 |
465 |
* indicates a p-value <0.05, ** indicates a p-value <0.01
MEAN and STDEV values are calculated per group, from each animal's total Total Movements over all intervals
Table 18. Summary of Macroscopic Findings |
||||
|
Group 1 (Control – 0 mg/kg/day) |
Group 2 (100 mg/kg/day) |
Group 3 (300 mg/kg/day) |
Group 4 (1000 mg/kg/day) |
Males |
||||
End of Treatment |
|
|||
Animals examined |
10 |
10 |
10 |
10 |
Animals without findings |
7 |
9 |
6 |
4 |
Animals affected |
3 |
1 |
4 |
6 |
|
||||
Liver |
|
|||
Reduced in size |
0 |
0 |
1 |
0 |
Discolouration |
1 |
0 |
0 |
6 |
|
|
|
|
|
Kidneys |
|
|
|
|
Pelvic dilation |
0 |
1 |
1 |
2 |
Enlarged |
0 |
0 |
1 |
2 |
Soft |
0 |
0 |
1 |
0 |
Discolouration |
0 |
0 |
1 |
0 |
|
|
|
|
|
Epididymides |
|
|
|
|
Nodule(s) |
1 |
0 |
1 |
0 |
|
|
|
|
|
Seminal vesicles |
|
|
|
|
Reduced in size |
1 |
0 |
0 |
0 |
Thyroid gland |
|
|
|
|
Enlarged |
0 |
0 |
0 |
1 |
Agenesis |
0 |
0 |
0 |
1 |
Skin |
|
|
|
|
Alopecia |
0 |
0 |
0 |
1 |
Body cavities |
|
|
|
|
Nodule(s) |
0 |
0 |
1 |
0 |
|
|
|
|
|
Females |
||||
Intercurrent death |
|
|
|
|
Animals examined |
|
|
1 |
|
Animals affected |
|
|
1 |
|
|
|
|||
Stomach |
|
|
|
|
Contents: |
|
|
1 |
|
Uterus |
|
|
|
|
Contents: |
|
|
1 |
|
|
|
|||
End of Treatment |
|
|||
Animals examined |
10 |
10 |
9 |
10 |
Animals without findings |
9 |
7 |
7 |
3 |
|
|
|
|
|
Animals affected |
1 |
3 |
2 |
7 |
|
|
|
|
|
Stomach |
|
|||
Discolouration |
0 |
0 |
1 |
0 |
Liver |
|
|||
Enlarged |
0 |
0 |
1 |
3 |
Reduced in size |
0 |
1 |
0 |
0 |
Discolouration |
0 |
0 |
0 |
2 |
Kidneys |
|
|||
Cyst(s) |
0 |
0 |
0 |
1 |
Cervix |
|
|||
Enlarged |
0 |
1 |
0 |
0 |
Contains fluid |
0 |
1 |
0 |
0 |
Clitoral glands |
|
|||
Focus/foci |
0 |
1 |
0 |
3 |
Grown together with: |
1 |
0 |
0 |
0 |
Discolouration |
0 |
0 |
0 |
1 |
Agenesis |
0 |
0 |
0 |
1 |
Adrenal glands |
|
|
|
|
Enlarged |
0 |
0 |
0 |
2 |
Mandibular Lymph n |
|
|
|
|
Discolouration |
0 |
0 |
0 |
1 |
Skin |
|
|
|
|
Alopecia |
1 |
0 |
1 |
1 |
# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level
Table 19. Select Organ Weights: Mean Percent Weight Differences from Control Groups |
||||||
Sex: |
Males |
Females |
||||
Dose (mg/kg/day): |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Liver |
|
|||||
Absolute |
1 |
18* |
29** |
3 |
16** |
23** |
Relative to body weight |
3 |
14** |
35** |
-3 |
8 |
21** |
|
||||||
Thyroid Gland |
|
|||||
Absolute |
3 |
20 |
44** |
21 |
27* |
18 |
Relative to body weight |
5 |
16 |
50** |
13 |
19 |
15 |
|
||||||
Kidney |
|
|||||
Absolute |
4 |
11 |
24** |
5 |
9 |
9* |
Relative to body weight |
5 |
8 |
29** |
0 |
3 |
7 |
|
||||||
Adrenal Gland |
|
|||||
Absolute |
11 |
15 |
9 |
8 |
26** |
24** |
Relative to body weight |
17 |
17 |
17* |
4 |
21* |
21** |
*: P<0.05, **: P<0.01
Table 20. Select Organ Weights (grams) Summary |
|||||
End of Treatment |
Group 1 (Control – 0 mg/kg/day) |
Group 2 (100 mg/kg/day) |
Group 3 (300 mg/kg/day) |
Group 4 (1000 mg/kg/day) |
|
Males |
|||||
Liver (Gram) |
Mean |
9.17 |
9.30 |
10.79* |
11.80** |
SD |
0.91 |
1.34 |
1.13 |
1.49 |
|
N |
10 |
10 |
10 |
10 |
|
|
|||||
Thyroids (Gram) |
Mean |
0.0171 |
0.0176 |
0.0205 |
0.0246** |
SD |
0.0033 |
0.0017 |
0.0021 |
0.0053 |
|
N |
10 |
10 |
10 |
9 |
|
|
|||||
Kidneys (Gram) |
Mean |
2.51 |
2.60 |
2.79 |
3.12** |
SD |
0.20 |
0.19 |
0.34 |
0.59 |
|
N |
10 |
10 |
10 |
10 |
|
|
|||||
Heart (Gram) |
Mean |
1.108 |
1.011* |
1.014 |
0.998* |
SD |
0.091 |
0.065 |
0.090 |
0.098 |
|
N |
10 |
10 |
10 |
10 |
|
Females |
|||||
Liver (Gram) |
Mean |
11.83 |
12.13 |
13.71** |
14.60** |
SD |
1.06 |
1.63 |
0.95 |
1.43 |
|
N |
10 |
10 |
9 |
10 |
|
|
|||||
Thyroid (Gram) |
Mean |
0.0146 |
0.0176 |
0.0185* |
0.0172 |
SD |
0.0032 |
0.0030 |
0.0022 |
0.0036 |
|
N |
10 |
10 |
9 |
10 |
|
|
|||||
Adrenals (Gram) |
Mean |
0.066 |
0.071 |
0.083** |
0.082** |
SD |
0.013 |
0.008 |
0.009 |
0.010 |
|
N |
10 |
10 |
9 |
10 |
|
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 21. Organ/Body Weight Ratios (%) Summary |
|||||
End of Treatment |
Group 1 (Control – 0 mg/kg/day) |
Group 2 (100 mg/kg/day) |
Group 3 (300 mg/kg/day) |
Group 4 (1000 mg/kg/day) |
|
Males |
|||||
Liver (%) |
Mean |
2.37 |
2.43 |
2.71** |
3.19** |
SD |
0.14 |
0.26 |
0.10 |
0.16 |
|
N |
10 |
10 |
10 |
10 |
|
|
|||||
Thyroids (%) |
Mean |
0.0044 |
0.0046 |
0.0051 |
0.00066** |
SD |
0.0010 |
0.0005 |
0.0004 |
0.0013 |
|
N |
10 |
10 |
10 |
10 |
|
|
|||||
Kidneys (%) |
Mean |
0.65 |
0.68 |
0.70 |
0.84** |
SD |
0.05 |
0.03 |
0.05 |
0.11 |
|
N |
10 |
10 |
10 |
10 |
|
|
|||||
Heart (%) |
Mean |
0.287 |
0.265* |
0.255** |
0.272 |
SD |
0.025 |
0.014 |
0.012 |
0.021 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Adrenals (%) |
Mean |
0.012 |
0.014 |
0.014 |
0.014* |
SD |
0.001 |
0.001 |
0.001 |
0.002 |
|
N |
10 |
10 |
10 |
10 |
|
|
|
|
|
|
|
Females |
|||||
Liver (%) |
Mean |
4.35 |
4.20 |
4.71 |
5.26** |
SD |
0.42 |
0.38 |
0.31 |
0.34 |
|
N |
10 |
10 |
9 |
10 |
|
|
|||||
Adrenals (%) |
Mean |
0.024 |
0.025 |
0.029* |
0.029** |
SD |
0.005 |
0.002 |
0.004 |
0.002 |
|
N |
10 |
10 |
9 |
10 |
|
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level
Table 22. Select Microscopic Findings: Thyroid Gland and Liver (Males and Females) |
||||||||
Sex: |
Males |
Females |
||||||
Dose (mg/kg/day): |
Group 1 (Control) 0 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Group 1 (Control) 0 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Thyroid Glanda |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
Follicular cell hypertrophy, diffuse |
|
|||||||
Minimal |
2 |
5 |
8 |
6 |
1 |
6 |
7 |
5 |
Slight |
- |
1 |
- |
2 |
- |
- |
1 |
1 |
Colloid alteration |
|
|||||||
Minimal |
1 |
6 |
8 |
3 |
- |
1 |
3 |
3 |
Slight |
- |
- |
- |
6 |
- |
- |
1 |
3 |
|
||||||||
Livera |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
Centrilobular hepatocellular hypertrophy |
|
|||||||
Minimal |
- |
- |
- |
3 |
- |
- |
- |
3 |
Cytoplasmic rarefaction |
|
|||||||
Minimal |
- |
- |
- |
- |
1 |
1 |
2 |
9 |
a= Number of tissues examined from each group.
Shaded dose levels regarded to represent treatment-related changes.
Table 23. Select Microscopic Findings: Pituitary gland and Kidney (Males) |
||||
Dose (mg/kg/day): |
Group 1 (Control) 0 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Pituitary Glanda |
10 |
10 |
10 |
10 |
Hypertrophy adenohypophyseal cells, multifocal |
|
|||
Minimal |
- |
- |
- |
3 |
|
||||
Kidneya |
10 |
10 |
10 |
10 |
Hyaline droplet accumulation |
|
|||
Minimal |
4 |
6 |
1 |
- |
Slight |
2 |
3 |
6 |
2 |
Moderate |
- |
- |
3 |
7 |
Marked |
- |
- |
- |
1 |
Granular cast(s) |
|
|||
Minimal |
- |
- |
7 |
4 |
Slight |
- |
- |
- |
2 |
Moderate |
- |
- |
- |
2 |
Marked |
- |
- |
- |
2 |
Basophilia tubule |
|
|||
Minimal |
5 |
8 |
4 |
- |
Slight |
- |
- |
5 |
5 |
Moderate |
- |
- |
- |
4 |
Marked |
- |
- |
- |
1 |
Infiltrate inflammatory cell, lymphocytic |
|
|||
Minimal |
3 |
6 |
7 |
8 |
Slight |
- |
- |
- |
2 |
a= Number of tissues examined from each group.
Shaded dose levels regarded to represent treatment-related changes.
Table 24. Select Microscopic Findings: Adrenal gland and Vagina (Females) |
||||
Dose (mg/kg/day): |
Group 1 (Control) 0 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Adrenal Glanda |
10 |
10 |
9 |
10 |
Hypertrophy cortical, zona fasciculata |
|
|||
Minimal |
- |
- |
2 |
6 |
|
||||
Vaginaa |
10 |
10 |
9 |
10 |
Increased mucification |
|
|||
Minimal |
- |
- |
- |
3 |
Marked |
- |
- |
1 |
- |
a= Number of tissues examined from each group.
Shaded dose levels regarded to represent treatment-related changes.
Table 25. Reproduction Data Summary |
||||
|
Group 1 (Control – 0 mg/kg/day) |
Group 2 (100 mg/kg/day) |
Group 3 (300 mg/kg/day) |
Group 4 (1000 mg/kg/day) |
Females paired |
10 |
10 |
10 |
10 |
Females mated |
10 |
10 |
10 |
10 |
Pregnant females |
9 |
9 |
10 |
10 |
Females dead on post coitum Day 23 |
0 |
0 |
1 |
0 |
Females with living pups on Day 1 |
9 |
9 |
9 |
10 |
|
||||
Mating index (%): (Females mated / Females paired) * 100 |
100 |
100 |
100 |
100 |
Fertility index (%): (Pregnant females / Females mated) * 100 |
90 |
90 |
100 |
100 |
Gestation index (%): (Females with living pups on Day 1 / Pregnant females) * 100 |
100 |
100 |
90 |
100 |
Table 26. Precoital Time (F0 – Generation: Post Coitum) |
||||
Day of the Pairing Period |
Group 1 (Control – 0 mg/kg/day) |
Group 2 (100 mg/kg/day) |
Group 3 (300 mg/kg/day) |
Group 4 (1000 mg/kg/day) |
Number of Females Mated |
||||
Day 1 |
3 |
3 |
2 |
2 |
Day 2 |
2 |
3 |
- |
2 |
Day 3 |
2 |
1 |
2 |
1 |
Day 4 |
3 |
3 |
6 |
4 |
Day 14 |
- |
- |
- |
1 |
|
||||
Median Precoital Time |
3 |
2 |
4 |
4 |
Mean Precoital Time |
2.5 |
2.4 |
3.2 |
3.9 |
N |
10 |
10 |
10 |
10 |
+/++ Steel-test significant at 5% (+) or 1% (++) level
Table 27. Summary of Implantation Sites (Females) |
|||||
|
Group 1 (Control – 0 mg/kg/day) |
Group 2 (100 mg/kg/day) |
Group 3 (300 mg/kg/day) |
Group 4 (1000 mg/kg/day) |
|
At necropsy |
|
||||
Implantations |
Mean |
11.1 |
13.0 |
12.8 |
12.4 |
Std Dev |
2.4 |
1.4 |
1.5 |
1.3 |
|
N |
9 |
9 |
10 |
10 |
|
Interpretation of results will be in accord with that described in OECD Testing Guideline 443.
Effect on fertility: 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:
- One key substance specific Guideline study in rats available for assessment.
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Oral
A key OECD Guideline 408/422 combined 90-day repeated dose with the reproduction / developmental toxicity screening study was conducted to determine the potential toxic effects of the test material (Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin (EC 941-364-9)). The potential of the test material to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development was also evaluated. The test material was administered once daily to Han Wistar rats (10/sex/dose) via oral gavage in a corn oil vehicle at doses of 0, 100, 300, or 1000 mg/kg/day for a period of 90 days (Charles River Laboratories Den Bosch B.V., 2022).
Mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, ophthalmology, estrous cycle, clinical pathology, measurement of thyroid hormones T3, T4, and TSH, gross necropsy findings, organ weights, and histopathologic evaluations were undertaken in the study. Additionally,reproductive toxicity parameters examined included mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, and maternal care while developmental parameters evaluated included sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, and measurement of thyroid hormones T3, T4 and TSH).
One female rat (No. 61) in the 300 mg/kg/day dose group was found dead on Day 23 post coitum (before scheduled necropsy). Based on the time of death and on the observed macroscopic findings, this death was considered most-likely related to labour difficulties and not treatment-related. There were no clinical signs of toxicity observed during the daily clinical or weekly arena observations. Slight salivation (dose-related) was observed post-dosing in treated animals through most of the treatment period. Taking into account the nature and minor severity of the effect and its time of occurrence (i.e., after dosing), this was considered to be a physiological response rather than a sign of treatment-related systemic toxicity.No treatment-related changes in body weights or body weight gain and food consumption were observed through the study period. Ophthalmology parameters in male and females rats were unaffected by treatment at doses up to 1000 mg/kg/day. Hearing ability, pupillary reflex, static righting reflex and grip strength were unaffected by treatment. Motor activity was considered unaffected by treatment in male rats up to 300 mg/kg/day and in females at all dose levels. At 1000 mg/kg/day, the number of total movements in male rats was decreased (0.72x of control). All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. In the absence of a dose-response trend, any changes in functional observations were considered unrelated to treatment or considered to derive from a relatively high mean control value (fore grip strength in males at 1000 mg/kg/day).
At 100 mg/kg/day, non-adverse changes such as decreased bile acid concentration in males; increased T4 levels in males; decreased T3 levels in females;and an increased incidence and/or severity of diffuse follicular cell hypertrophy and/or colloid alteration in the thyroid gland of both sexes was observed.
At 300 mg/kg/day, adverse alpha 2µ-globulin nephropathy correlating with increased urea and creatinine levels and enlargement, softening, and greenish discoloration of the kidneys was observed in the kidney of male rats. Correlating microscopic findings comprising hyaline droplet accumulation (confirmed by alpha 2µ-globulin immunostaining), granular casts, and tubular basophilia were also observed in male rats at this dose level. Additionally, at 300 mg/kg/day, non-adverse changes such as decreased bile acid concentration in males; decreased eosinophil count in females; increased T4 levels in males; decreased T3 levels in both sexes; enlargement of the liver in females; increased absolute and relative liver weights in males; increased absolute and relative adrenal gland weights in females; cortical hypertrophy of the zona fasciculata of the adrenal gland in females; increased incidence and/or severity of diffuse follicular cell hypertrophy and/or colloid alteration in the thyroid gland of both sexes were observed.
At 1000 mg/kg/day, adverse alpha 2µ-globulin nephropathy correlating with increased urea and creatinine levels; enlargement of the kidneys; and higher absolute and relative kidney weights was observed in the kidney of male rats. Correlating microscopic findings comprised of hyaline droplet accumulation (confirmed by alpha 2µ-globulin immunostaining), granular casts, tubular basophilia and inflammatory cell infiltrate. Additionally, at this dose levelnon-adverse observed included decreased total movements at motor activity assessment in males; increased white blood cell count; lymphocyte count and mean corpuscular volume in males; decreased red blood cell count; hemoglobin and hematocrit in both sexes; decreased eosinophil count in females; increased total bilirubin in both sexes; decreased bile acid concentration in males; increased potassium and inorganic phosphate levels in males; decreased T3 levels in both sexes; black-brown discoloration of the liver in both sexes; enlargement of the liver in females; increased absolute and relative liver weights in both sexes; centrilobular hepatocellular hypertrophy in both sexes; increased cytoplasmic rarefaction in females; enlargement and increased absolute and relative weight of the adrenal gland in females; cortical hypertrophy of the zona fasciculata of the adrenal gland in females; higher absolute and relative thyroid gland weights in males; increased incidence and/or severity of diffuse follicular cell hypertrophy and/or colloid alteration in the thyroid gland of both sexes; multifocal hypertrophy of adenohypophyseal cells in the pituitary gland in males; increased mucification of the vaginal epithelium in females; and bronchiolo-alveolar inflammation of the lung in males.
Length and regularity of the estrous cycle were unaffected by treatment with most females having a regular cycle of 4 to 5 days. One control female (No. 42) was acyclic but delivered a normal litter. Given that this concerned a control female, the finding was unrelated to treatment. 1/10 couples of the control group (Male No. 3 and Female No. 43) and 1/10 couples in 100 mg/kg/day group (Male No. 13 and Female No. 53) were without offspring. For Female No. 53 the infertility was likely due to an imperforate vagina as evidenced by marked dilation with mucinous contents of the cervix. While this could be seen as a developmental abnormality, the single observation in one female at 300 mg/kg/day was regarded unrelated to treatment. No abnormalities were observed in the reproductive organs of the control couple, which could account for their lack of offspring. Additionally, One female rat in the 300 mg/kg/day dose group (No. 61) was found dead on Day 23 post-coitum with pups in normal stage of development in the uterus and Male No. 21 was therefore regarded to be fertile. This death was regarded to be related to delivery difficulties. There were no morphological findings in the reproductive organs of either sex which could be considered treatment-related. Stage dependent qualitative evaluation of spermatogenesis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
The mating index and precoital time were unaffected by treatment with all females showing evidence of mating within 4 days, with the exception of one female at 1000 mg/kg/day, which showed evidence of mating after 14 days. This was considered to be a background finding commonly observed in rats of this age and strain and not related to treatment. The number of implantation sites was not affected by treatment. The mean number of implantation sites were 11.1, 13.0, 12.8, and 12.4 for the 0, 100, 300, and 1000 mg/kg/day dose groups, respectively. Fertility index was also not affected by treatment and fertility indices were 90, 90, 100, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively. One female in the 100 mg/kg/day group was not pregnant. The infertility was likely due to an imperforated vagina as evidenced by marked dilation with mucinous contents of the cervix. While this could be seen as a developmental abnormality, the single instance in one female at 300 mg/kg/day was regarded unrelated treatment. No signs of difficult or prolonged parturition were observed among the pregnant females, except for one female (No. 61) in the 300 mg/kg/day dose group. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.
Overall no signs of treatment-related reproductive toxicity were observed up to the highest dose level tested (1000 mg/kg/day) in this study.
Based on the effects observed in this sub-chronic oral toxicity study, the systemic toxicity No Observed Adverse Effect Levels (NOAEL) of Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin was determined to be 100 mg/kg/day factoring in the alpha 2µ-globulin nephropathy observed in male rats and ≥1000 mg/kg/day excluding the alpha 2µ-globulin nephropathy since this is a male rat specific effect and of no toxicological relevance to human beings. In the absence of any adverse treatment-related reproductive toxicity observed at the highest dose tested, the NOAEL for reproductive toxicity was determined to be ≥1000 mg/kg/day.
Some indication of the likely effect of a test substance on reproductive organs can be gained from the results of repeated-dose toxicity studies with closely related materials, as summarized in Table 1. Robust Study summaries for these studies can be found in the respective Concawe dossiers.
Table 1. Summaries of data on reproductive organs from subchronic studies with related substances. (Robust study summaries are provided in Concawe Section 7.5 Repeated Dose Toxicity) |
||||
Test Material |
Route, Species, Doses, Exposure Regimen |
Endpoints |
Results |
Reference |
Diesel fuel |
Inhalation. Rat. 350, 880, 1710 mg/m3 4 hr/day, twice/wk, 13 wk |
Weight and histopathology of testes |
No treatment-related effect noted |
Lock et al, 1984 |
Cherry point diesel fuel No. 2 CAS 68476-34-6 |
Dermal. Rat. 0.5, 2.0, 5.0 ml/kg 5 d/wk, 4 wk |
Weight and histopathology of testes and ovaries |
No treatment-related effect noted |
ARCO, 1986a |
Naval distillate Watson CAS 68334-30-5 |
Dermal. Rat. 0.25, 2.0, 5.0 ml/kg 5 d/wk, 4 wk |
Weight and histopathology of testes and ovaries |
No effect noted. Testicular degeneration in 2/10 males considered spontaneous. |
ARCO, 1986b |
Watson diesel fuel #2 68476-34-6 |
Dermal. Rat. 0.5, 2.0, 5.0 ml/kg 5 d/wk, 4 wk |
Weight and histopathology of testes and ovaries |
No treatment-related effect noted |
ARCO, 1986c |
Diesel fuel CAS 68476-34-6 |
Dermal. Rat. 0.5, 1.0, 2.5, 5.0, 10.0 ml/kg 5 d/wk, 4 wk |
Weight and histopathology of testes and ovaries |
No treatment-related effect noted |
ARCO, 1988 |
Naval distillate CAS 68334-30-5 |
Dermal. Rat. 0.0001, 0.005, 0.5 ml/kg 5 d/wk, 4 wk |
Weight and histopathology of testes and ovaries |
No treatment-related effect noted |
ARCO, 1992a |
Naval distillate CAS 68334-30-5 |
Dermal. Rat. 0.50 ml/kg 5 d/wk, 4 wk |
Weight of testes and ovaries. No histopathology was done. |
No treatment-related effect noted |
ARCO, 1994a |
Diesel fuel (F-237) 68334-30-5 |
Dermal. Rat. 0.01, 0.10, 1.00 ml/kg, 5 d/wk, 13 wk |
Weight of testes and ovaries. Histopathology of testis, ovary, prostate, uterus |
No treatment-related effect noted |
ARCO, 1994b |
Based on test results from the above studies, it is considered unlikely that exposure will affect reproductive performance. However, an extended one-generation reproductive toxicity test (OECD 443) has been proposed by Concawe to fulfil the data requirement for the VHGC Category. As this Category contains substances that are structurally related to the registered material it is proposed that the reproductive toxicity endpoint will be fulfilled by read-across to the Concawe study once complete.
Effects on developmental toxicity
Description of key information
Developmental Toxicity NOAEL (Rat): 1000 mg/kg/day (OECD 414)
Developmental Toxicity NOAEL (Rabbit): 1000 mg/kg/day (OECD 414)
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2021-SEP-16 to 2022-SEP-21
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- June 2018
- Deviations:
- yes
- Remarks:
- Deviations did not impact the overall integrity of the study or the interpretation of the study results and conclusions.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.31 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- May 2008
- Deviations:
- yes
- Remarks:
- Deviations did not impact the overall integrity of the study or the interpretation of the study results and conclusions.
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- August 1998
- Deviations:
- yes
- Remarks:
- Deviations did not impact the overall integrity of the study or the interpretation of the study results and conclusions.
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- yes
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Shell Global Solutions (Spain); Batch number: 204377961
- Purity, including information on contaminants, isomers, etc.: 100% (UVCB)
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable for at least 24 hours at room temperature under normal laboratory light conditions and for at least 8 days in the refrigerator over the concentration range 2 to 800 mg/mL (solutions).
FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid
OTHER SPECIFICS
- Expiration Date: 2022-Aug-20
- Specific gravity: 0.8319 - Species:
- rat
- Strain:
- Wistar
- Remarks:
- CRL:WI(Han)
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories Deutschland (Sulzfeld, Germany)
- Age at study initiation: Young adult females, 11-15 weeks old at mating
- Weight at study initiation: 201 - 269 g at onset of treatment
- Fasting period before study: Not specified
- Housing: Individually housed in Makrolon type III polycarbonate cages
- Diet (e.g. ad libitum): Pelleted SM R/M-Z (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) ad libitum
- Water (e.g. ad libitum): Municipal tap water (in water bottles) ad libitum
- Acclimation period:5- 6 days before the commencement of dosing
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20°C (target: 18 to 24°C)
- Humidity (%): 45-69% (target: 40-70%)
- Air changes (per hr): 10 or more air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
IN-LIFE DATES: From: 2021-SEP-22/24 To: 2021-OCT-15 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Remarks:
- Sigma-Aldrich (Steinheim, Germany)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test material was formulated in the vehicle (corn oil). The formulations were homogenised to visibly acceptable levels at appropriate concentrations to meet dose level requirements. Formulations were prepared at least weekly, filled out in daily portions and stored in a refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle (0.92) and test item (0.8319). No correction was made for the purity/composition of the test item as it is a UVCB.
A dose volume of 4 mL/kg body weight was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weight of the animals.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on results of the trial formulation, corn oil was selected as vehicle for this study
- Concentration in vehicle: 0, 25, 75, or 250 mg/mL for the 0, 100, 300, and 1000 mg/kg bw/day dose levels, respectively.
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no. (if required): Source: Sigma-Aldrich; Batch Number: MKCN9742
- Purity: Not specified - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Dose formulation samples (duplicate) were collected for analysis on Week 1 of treatment. Samples for concentration analysis were taken from the middle for Groups 1, 2, 3, and 4 while samples for homogeneity analysis were taken from approximately the top, middle, and bottom for Groups 2, 3, and 4 only.
Analyses were performed using a validated analytical procedure (Test Facility Study No. 20276345).
Concentration and Homogeneity Analysis: at 21°C
For concentration: mean sample concentration results within or equal to ± 10% solutions of theoretical concentration were considered acceptable.
For homogeneity: relative standard deviation (RSD) of concentrations of ≤10% for each group was considered acceptable.
Stability Analysis:
Stability analyses was performed previously in conjunction with the method development and validation study (Test Facility Study No. 20276345). This demonstrated that the test material was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. - Details on mating procedure:
- Impregnation procedure: purchased timed pregnant (females arrived on Day 0 or Day 1 post-coitum)
- Duration of treatment / exposure:
- Daily from day 6 to 20 post coitum
- Frequency of treatment:
- Once daily
- Duration of test:
- From day 0 to 21 post coitum (Caesarean section and necropsy)
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- Group 1 (Control)
- Dose / conc.:
- 100 mg/kg bw/day
- Remarks:
- Group 2 (Low dose)
- Dose / conc.:
- 300 mg/kg bw/day
- Remarks:
- Group 3 ( (Intermediate dose)
- Dose / conc.:
- 1 000 mg/kg bw/day
- Remarks:
- Group 4 (High dose)
- No. of animals per sex per dose:
- 22 females/dose group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
Dose levels were selected based on the results of a 14-day dose range finder and a combined 90-day repeated dose toxicity study with the reproduction / developmental toxicity screening test with oral gavage administration of Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin (EC 941-364-9) in rats (Test Facility StudyNos. 20276348 and 20276349, respectively), and in an attempt to produce graded responses to the test item. For both studies, no toxicity was observed at 300 and 1000 mg/kg/day. Therefore, a maximum dose level of 1000 mg/kg/day was selected for the current study. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- Rationale for animal assignment: The animals were assigned to groups at random
- Fasting period before blood sampling for (rat) dam thyroid hormones: Not fasted
- Time of day for (rat) dam blood sampling: Sampled between 07.00 and 09.00 from the jugular vein - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Except on days of receipt and necropsy (where frequency was at least once daily), all F0 animals were observed at least twice daily (beginning upon arrival through termination) for mortality. Cage side observations were conducted 0 to 1 hours post-dose at least once daily starting on Day 6 post-coitum up to the day prior to necropsy.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observtions were conducted for all animals on Days 2, 6, 15, and 21 post-coitum.
BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of all animals were recorded on Days 2, 6, 9, 12, 15, 18, and 21 post-coitum.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; over Days 2-6, 6-9, 9-12, 12-15, 15-18, and 18-21 post coitum.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes; Regular basis throughout the study. Water consumption was monitored by visual inspection of the water bottles. Data were used for health monitoring of the animals only and not reported.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 21 post coitum
- Organs examined: Animals surviving until scheduled euthanasia were anaesthetized by a mixture of carbon dioxide and dioxide (graduate fill to a ratio of 60%/40%) and euthanized by a graduate fill of carbon dioxide on Day 21 post-coitum. The thyroid gland was weighed at necropsy for all animals. Paired organs were weighed together. Organ to body weight ratio (using the body weight on Day 21 post-coitum) was calculated. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- OTHER:
- The number and distribution of live and dead fetuses
- The sex of each fetus based on the anogenital distance - Blood sampling:
- - Plasma: No
- Serum: Yes
- Volume collected : 1.0 mL
Blood of F0-animals was collected on the day of scheduled necropsy (Day 21 post-coitum). Animals were not fasted overnight. Samples were collected between 7:00 and 9:00 a.m. from the jugular vein of all F0 animals. Blood samples were processed for serum, and serum was analyzed for Triiodothyronine (T3), Thyroxine (T4), and Thyroid-Stimulating Hormone (TSH). - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes, the anogenital distance of each foetus was measured
Live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity using a small metal feeding tube.
Litters of females surviving to scheduled necropsy were subjected to detailed external, visceral and skeletal examinations. External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).
External Examinations:
Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and its weight was determined. The anogenital distance (AGD) was measured for all viable fetuses and normalized to the cube root of the fetal body weight.
Visceral Examinations:
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected and this examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development .
Tissues with variations or malformations were stored in 10% formalin.
Head Examinations:
The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique.
Skeletal Examinations:
All fetuses were eviscerated, followed by fixation in 96% aqueous ethanol, and maceration in potassium hydroxide. Thereafter, they were processed for double staining with Alcian Blue 8GX and Alizarin Red S. Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). - Statistics:
- For information on statistics, please see 'Any other information on materials and methods incl. tables'.
- Indices:
- Caesarean Section and Necropsy Data:
1) Pre-implantation loss (%, group mean): (Number of corpora lutea - Number of implantations / Number of corpora lutea) x100
2) Post-implantation loss (%, group mean): (Number of implantations - Number of live foetuses / Number of implantations) x100
Foetal Data:
1) Sex distribution (%, group mean): (Number of male (female) foetuses / Number of foetuses) x 100
2) External abnormalities/litter (%, group mean): (Number of foetuses with abnormality / Number of foetuses) x 100
3) Visceral abnormalities/litter (%, group mean): (Number of foetuses with abnormality / Number of examined foetuses) x 100
4) Skeletal abnormalities/litter (%, group mean): (Number of foetuses with abnormality / Number of examined foetuses) x 100 - Historical control data:
- Historical control data is provided below under 'Attachments' in this Robust Study Summary
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No clinical signs of toxicity were observed through the study period. Salivation was observed in 1/22, 2/22, 14/22, and 19/22 females in the control, 100, 300, and 1000 mg/kg/day dose groups, respectively. This was considered to be a physiological response (rather than a sign of systemic toxicity) and not toxicologically relevant taking into account the nature and minor severity of the effect and time of occurrence (i.e., after dosing).
Other clinical signs observed (incidental abnormal breathing sounds, fur loss, thin fur cover, and skin scabs) were considered to be of no toxicological relevance as they occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study or were observed during a single day in only one female. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- No mortality was observed through the study period.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- At 100 and 300 mg/kg/day, mean body weight, body weight gain and body weight gain corrected for gravid uterus weight were observed to be comparable to the corresponding controls. Mean gravid uterus weight was considered to be comparable to the control animals for all treatment groups. Mean gravid uterus weight at 100 mg/kg/day was slightly lower than control (13%; not statistically significant). This was mainly based on two females (Nos. 24 and 39) with small litters (3 fetuses each). In the absence of a dose-related effect, this was not considered to be toxicologically relevant.
At 1000 mg/kg/day, mean body weight gain was statistically significantly reduced (p<0.01) between Days 6-9 post-coitum (mean gain 0.9 g vs 9.5 g in the control group). During this period, 7/22 females were observed to have slight body weight loss (up to 3% on Day 9 compared to Day 6 post-coitum). From Day 9 post coitum onwards body weight gain was comparable to control. Mean body weight gain corrected for gravid uterus at 1000 mg/kg/day was statistically significantly lower (p<0.05) than control (18.86 g vs 25.90 g, respectively).
As the body weight gain recovered from Day 12 post-coitum onwards to levels comparable to control, and in the absence of other signs of maternal toxicity, these findings were considered not to represent an adverse effect of the test material. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Mean food consumption at 100 and 300 mg/kg/day was considered to be comparable to the corresponding controls. At 1000 mg/kg/day, mean food consumption was statistically significantly decreased over (p<0.01) Days 6-12 post-coitum (up to 23% lower than control) but recovered to values comparable to control from Day 12 post coitum onwards. As a result of the initial decrease, overall food consumption (Days 6 21 post-coitum) was statistically significantly (p<0.05) lower (8%) than control.
As food consumption recovered from Day 12 post-coitum onwards to levels comparable to control, and in the absence of other signs of maternal toxicity, these findings were considered not to represent an adverse effect of the test material. - Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Description (incidence and severity):
- Water consumption was monitored by visual inspection of the water bottles. Data were used for health monitoring of the animals only and not reported.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Endocrine findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Mean serum levels of T3 at 100 mg/kg/day were in the same range as the corresponding control animals. Mean serum levels of Triiodothyronine (T3) were statistically significantly reduced (p<0.01) at 300 and 1000 mg/kg/day (0.75 and 0.63x of control, respectively), and were considered treatment-related. Nonetheless, at 300 mg/kg/day, the mean T3 level remained within the available historical control range. However, at 1000 mg/kg/day, the mean T3 level (and individual serum levels of 16/22 females) were below the lower limit of the available historical control range. The downstream biological consequences of thyroid hormone changes were not assessed within this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed. This finding was not taken into account when determining the maternal NOAEL.
Mean serum levels of total T4 at 100 mg/kg/day were slightly increased (1.20x of control). Mean serum levels of total Thyroxine (T4) were considered unaffected by treatment at 300 and 1000 mg/kg/day and mean T4 serum levels at all dose levels remained within the available historical control range.
.
Mean serum levels of Thyroid Stimulating Hormone (TSH) showed an apparent dose-related trend towards an increase across the dose groups, reaching statistical significance (p<0.01) at 1000 mg/kg/day only (2.38x of control). This effect was considered treatment-related. However, mean TSH levels for all groups remained within the available historical control range. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- No treatment-related effects were observed on thyroid gland weight/s.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Gross necropsy did not reveal any treatment-related effects in the thyroid glands.
Other findings observed amongst the control and/or treated animals were considered to be of no toxicological significance since they remained within the range of biological variation for rats of this age and strain. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment-related microscopic observations were seen in the thyroid glands. The recorded microscopic findings in the thyroid gland were within the range of background pathology encountered in rats of this age and strain.
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Number of abortions:
- no effects observed
- Description (incidence and severity):
- All females were gravid with viable fetuses, except for one female (No. 38) in the low dose group (100 mg/kg/day), which was non gravid.
- Pre- and post-implantation loss:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The percentage of pre-implantation loss was higher than control in females treated at 100 and 300 mg/kg/day. However, as treatment was only initiated after implantation was completed and in the absence of a dose response, this was considered a chance finding.
The percentage of post-implantation loss in the control and test groups was similar and in the range of normal biological variation. - Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- The mean number of corpora lutea and implantation sites in the control and test groups were similar and in the range of normal biological variation. All females were gravid with viable fetuses, except for one female (No. 38) in the low dose group (100 mg/kg/day), which was non gravid.
- Other effects:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: Systemic Toxicity
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Mean fetal weights (male, female and combined) in the control and test groups were considered similar and unaffected by treatment. Higher fetal weights observed at 100 mg/kg/day (males, females and combined; only statistically significant for females) were considered a chance finding as this occurred in the absence of a dose response.
- Reduction in number of live offspring:
- no effects observed
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- The male:female ratio was unaffected by treatment up to 1000 mg/kg/day.
- Changes in litter size and weights:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment-related effects on litter size were observed in any group. The mean number of fetuses per litter was slightly lower at 100 mg/kg/day (10.0 vs 11.7 in the control group). In the absence of a dose-related effect or statistically significant difference, this was considered unrelated to treatment.
- Anogenital distance of all rodent fetuses:
- no effects observed
- Description (incidence and severity):
- Anogenital distance (absolute and normalized for body weight) in male and female pups was unaffected by treatment up to 1000 mg/kg/day.
- Changes in postnatal survival:
- no effects observed
- External malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- One fetus (No. 28 L1) in the low dose group (100 mg/kg/day) presented with an external malformation (polydactyly of a hindpaw) which was confirmed at skeletal examination. This single occurrence of a malformation in a low-dose fetus did not suggest any treatment-related effect and was therefore considered a chance finding. No external variations were observed through the study period.
- Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Skeletal malformations occurred in one fetus each in the low- and high-dose groups. The fetus (No. 28-L1) in the low dose group (100 mg/kg/day) presented extra hindpaw phalanges, consistent with polydactyly observed in this fetus at external examination while the fetus (No. 71-L1) in the high dose group (1000 mg/kg/day) had an extra lumbar vertebra. Due to the single occurrences of these malformations, they were deemed chance findings. All skeletal variations observed occurred in the absence of a dose-related incidence trend, infrequently and/or in control fetuses only. Therefore, they were not considered treatment-related.
- Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Visceral malformations were observed in two fetuses, one in the control group and the other in the high dose group. The fetus in the control group (Fetus No. 21-L2) had fused lung lobes while the fetus in the high dose group (1000 mg/kg/day) presented situs inversus and fused lung lobes. Given the single occurrences of these malformations and/or occurrence in the control group, they were deemed chance findings.
Visceral variations were limited to supernumerary liver lobes, absent renal papilla, and convoluted or dilated ureters. The low incidences, group distribution, and occurrence in control fetuses ruled out any relationship to treatment with the test material. - Other effects:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Developmental Toxicity
- Key result
- Abnormalities:
- effects observed, non-treatment-related
- Key result
- Developmental effects observed:
- no
- Conclusions:
- Based on the effects observed, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Petroleum Gas Oil Fraction, Co-Processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) was determined to be ≥1000 mg/kg/day in the rat.
- Executive summary:
A key OECD Guideline 414 pre-natal developmental toxicity study was conducted to evaluate the potential of the test material (Petroleum Gas Oil Fraction, Co Processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9)) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested.
The test material was administered to time-mated female Wistar Han rats (22/dose) once daily via oral gavage in a corn oil vehicle at doses of 0, 100, 300, or 1000 mg/kg/day from Days 6 to 20 post-coitum, inclusive. The parameters evaluated for the maternal animals (F0 generation) included mortality / moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), macroscopic examination, organ weights (thyroid gland), uterine contents, microscopic examination (thyroid gland), corpora lutea, implantation sites and pre- and post-implantation loss. Additionally, the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations were evaluated for the pups (F1 generation).
No mortality or clinical signs of toxicity were observed through the study period. At 1000 mg/kg/day, mean food consumption was decreased over Days 6-12 post-coitum and mean body weight gain was reduced between Day 6-9 post-coitum. As a result, body weight gain corrected for gravid uterus weight was also slightly reduced. Food consumption and body weight gain recovered from Day 12 post-coitum onwards to levels comparable to control and in the absence of other signs of maternal toxicity, these findings were not considered to represent an adverse treatment-related effect.
Mean serum levels of T3 at 100 mg/kg/day were in the same range as the corresponding control animals. Mean serum levels of Triiodothyronine (T3) were statistically significantly reduced (p<0.01) at 300 and 1000 mg/kg/day (0.75 and 0.63x of control, respectively), and were considered treatment-related. Nonetheless, at 300 mg/kg/day, the mean T3 level remained within the available historical control range. However, at 1000 mg/kg/day, the mean T3 level (and individual serum levels of 16/22 females) were below the lower limit of the available historical control range. The downstream biological consequences of thyroid hormone changes are not assessed in this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed and this finding was not taken into account when determining the maternal NOAEL.
Mean serum levels of total T4 at 100 mg/kg/day were slightly increased (1.20x of control). Mean serum levels of total Thyroxine (T4) were considered unaffected by treatment at 300 and 1000 mg/kg/day and mean T4 serum levels at all dose levels remained within the available historical control range.
Mean serum levels of Thyroid Stimulating Hormone (TSH) showed an apparent dose-related trend towards an increase across the dose groups, reaching statistical significance (p<0.01) at 1000 mg/kg/day only (2.38x of control). This effect was considered treatment-related. However, mean TSH levels for all groups remained within the available historical control range. No treatment-related effects were observed on thyroid gland weight/s and gross necropsy as well as histopathology did not reveal any treatment-related effects in the thyroid glands.
All females were gravid with viable fetuses, except for one female (No. 38) in the low dose group (100 mg/kg/day), which was non gravid. The percentage of pre-implantation loss was higher than control in females treated at 100 and 300 mg/kg/day. However, as treatment was only initiated after implantation was completed and in the absence of a dose response, this was considered a chance finding. The percentage of post-implantation loss in the control and test groups was similar and in the range of normal biological variation. The mean number of corpora lutea and implantation sites in the control and test groups were similar and in the range of normal biological variation.
Mean fetal weights (male, female and combined) in the control and test groups were considered similar and unaffected by treatment. The sex ratio was also unaffected by treatment up to 1000 mg/kg/day and no treatment-related effects on litter size were observed in any group. Anogenital distance (absolute and normalized for body weight) in male and female pups was unaffected by treatment up to 1000 mg/kg/day.
One fetus (No. 28 L1) in the low dose group (100 mg/kg/day) presented with an external malformation (polydactyly of a hindpaw) which was confirmed at skeletal examination. This single occurrence of a malformation in a low-dose fetus did not suggest a treatment-related effect and was therefore considered a chance finding. No external variations were observed through the study period.
Visceral malformations were observed in two fetuses, one in the control group and the other in the high dose group. The fetus in the control group (Fetus No. 21-L2) had fused lung lobes while the fetus in the high dose group (1000 mg/kg/day) presented situs inversus and fused lung lobes. Given the single occurrences of these malformations and/or occurrence in the control group, they were deemed chance findings.
Visceral variations were limited to supernumerary liver lobes, absent renal papilla, and convoluted or dilated ureters. The low incidences, group distribution, and occurrence in control fetuses ruled out any relationship to treatment with the test material.
Skeletal malformations occurred in one fetus each in the low- and high-dose groups. The fetus (No. 28-L1) in the low dose group (100 mg/kg/day) presented extra hindpaw phalanges, consistent with polydactyly observed in this fetus at external examination while the fetus (No. 71-L1) in the high dose group (1000 mg/kg/day) had an extra lumbar vertebra. Due to the single occurrences of these malformations, they were deemed chance findings. All skeletal variations observed occurred in the absence of a dose-related incidence trend, infrequently and/or in control fetuses only. Therefore, they were not considered treatment-related.
Based on the effects observed, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Petroleum Gas Oil Fraction, Co-Processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) was determined to be≥1000 mg/kg/day in the rat.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2021-Jun-21 to 2021-Nov-26
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 2018
- Deviations:
- no
- Remarks:
- There were no deviations from the above regulations that affected the overall integrity of the study or the interpretation of the study results and conclusions.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.31 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 2008
- Deviations:
- no
- Remarks:
- There were no deviations from the above regulations that affected the overall integrity of the study or the interpretation of the study results and conclusions.
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 1998
- Deviations:
- no
- Remarks:
- There were no deviations from the above regulations that affected the overall integrity of the study or the interpretation of the study results and conclusions.
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Shell Global Solutions (Spain); Batch number: 204377961
- Purity, including information on contaminants, isomers, etc.: UVCB
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable for at least 24 hours at room temperature under normal laboratory light conditions and for at least 8 days in the refrigerator over the concentration range 2 to 800 mg/mL (solutions).
FORM AS APPLIED IN THE TEST (if different from that of starting material) : Liquid
OTHER SPECIFICS
- Expiration Date: 2022-Aug-20
- Storage: At room temperature
- Specific gravity: 0.8319 - Species:
- rabbit
- Strain:
- New Zealand White
- Remarks:
- Females (time-mated)
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 18-20 weeks
- Weight at study initiation: 3042 - 4187 grams
- Fasting period before study: Not specified
- Housing: individually housed in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles
- Diet (e.g. ad libitum): KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding pellets (Granovit AG, Kaiseraugst, Switzerland); On arrival animals received approximately 25 grams pelleted diet and 140-160 grams per day subsequently. Additionally, pressed hay, fresh fruits and/or vegetables were provided during the study period
- Water (e.g. ad libitum): Municipal tap water via water bottles ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 19°C
- Humidity (%): 52 to 92%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light (except during designated procedures)
IN-LIFE DATES: From: 2021-Sep-08/10 To: 2021-Oct-08 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Remarks:
- (Sigma-Aldrich, Steinheim, Germany)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Trial preparations were performed to select a suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Dose formulations were divided into aliquots where required to allow dispensing on each dosing occasion.
Test material formulations (w/w) were prepared at least weekly in daily portions and homogenized by magnetic stirring to visually acceptable levels. For dosing with 1000 mg/kg/day, aliquots of pure test material were prepared. Storage of formulations was at 4°C. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing and adjustments were made for specific gravity of the test material.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil (selected based on trials undertaken at the Test Facility)
- Concentration in vehicle: 0, 100, 300, or 831.9 mg/mL for Groups 1 (control), 2 (100 mg/kg/day), 3 (300 mg/kg/day), & 4 (1000 mg/kg/day), respectively.
- Amount of vehicle (if gavage): 1 mL/kg for the 0, 100, and 300 mg/kg/day dose groups; 1.20 mL/kg for the 1000 mg/kg/day dose group
- Lot/batch no. (if required): Not specified
- Purity: Not specified - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analyses were performed using a validated analytical procedure (Test Facility Study No. 2027634533).
Concentration and Homogeneity Analysis: at 4°C
For concentration: mean sample concentration results within or equal to ± 10% solutions of theoretical concentration were considered acceptable.
For homogeneity: relative standard deviation (RSD) of concentrations of ≤10% for each group was considered acceptable.
Stability Analysis:
Stability analyses was performed previously in conjunction with the method development and validation study (Test Facility Study No. 20276345). This demonstrated that the test material was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. - Details on mating procedure:
- - Impregnation procedure: purchased timed pregnant (females arrived on Day 0 or Day 2 post-coitum)
- Duration of treatment / exposure:
- Day 7 to Day 28 post-coitum
- Frequency of treatment:
- Once daily
- Duration of test:
- Day 7 to Day 28 post-coitum
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Group 1 - Control
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- Group 2
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Remarks:
- Group 3
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- Group 4
- No. of animals per sex per dose:
- 22 females/dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
The dose levels were selected based on the results of the Dose Range Finder (Test Facility Study No. 20276356)
- Rationale for animal assignment (if not random): At arrival, animals were assigned to groups at random
- Fasting period before blood sampling for dam thyroid hormones: Not specified
- Time of day for dam blood sampling: Not specified
- Other: The New Zealand White rabbit was chosen as the animal model for this study as it is an accepted non-rodent species for developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test material.
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test material. - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All F0 animals were observed for mortality at least twice daily beginning upon arrival through termination/release (except on days of receipt and necropsy where frequency was at least once daily).
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were conducted for all F0 animals at least once daily post-dose; starting on Day 7 post-coitum up to the day prior to necropsy. Animals were observed during the dosing procedure and cage debris was examined to detect premature birth.
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of all F0 animals were recorded on Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum. Data for body weight Day 0 post-coitum were provided by the Supplier (non GLP).
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; Food consumption of all F0 animals was quantitatively measured daily from Day 3 post-coitum onwards.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption of all F0 animals was monitored on a regular basis throughout the study. Water consumption was monitored by visual inspection of the water bottles. Data was used for health monitoring of the animals only and not reported.
POST-MORTEM EXAMINATIONS: Yes
Unscheduled Deaths - F0-Generation
One female (No. 39; in the 100 mg/kg/day dose group) was sacrificed in extremis on Day 12 post-coitum. The animal presented with labored breathing, decreased activity and pale skin after dosing. One female rabbit dosed at 100 mg/kg/day (Female No. 42) was euthanized on Day 27 post-coitum, because it started to deliver its offspring that day. These animals were euthanized by an intravenous injection of sodium pentobarbital, underwent necropsy, and specified tissues were retained.
Scheduled Euthanasia - F0-Generation
Animals surviving until scheduled euthanasia were euthanized by an intravenous injection of sodium pentobarbital on Day 29 post-coitum.
- Sacrifice on Day 29 post-coitum
- Organs examined: All animals including one animal that was sacrificed before planned necropsy (Female No. 39, 100 mg/kg/day) and one female that delivered before the day of scheduled necropsy (Female No. 42, 100 mg/kg/day) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The number and distribution of live and dead fetuses - Blood sampling:
- - Plasma: No
- Serum: No - Fetal examinations:
- Method of Euthanasia - F1-Generation:
Live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity using a small metal feeding tube.
Fetal Examinations (Unscheduled) - F1-Generation:
For late resorptions, recognizable fetuses or normal implantations in development of females sacrificed before planned necropsy (Female No 39, 100 mg/kg/day) or that delivered before the day of scheduled necropsy (Female No. 42, 100 mg/kg/day), a gross external examination was performed.
Fetal Examinations (Scheduled) - F1-Generation
Litters of females surviving to scheduled necropsy were subjected to detailed external, visceral and skeletal examinations. External, visceral, and skeletal findings were recorded as developmental variations or malformations.
as detailed below:
- External examinations: Yes: [all per litter]
Each viable fetus was examined in detail to detect macroscopic visible abnormalities, and their weight was determined. Nonviable fetuses were examined and weighed. For late resorptions, a gross external examination was performed. Late resorptions with malformations were fixed in 10% buffered formalin.
- Soft tissue examinations: Yes: [all per litter]
The sex of all fetuses was confirmed by internal examination and all fetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (1972). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar (1984).
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique (1965). After examination, the tissues without variation or malformations were discarded and tissues with variations or malformations were stored in 10% formalin.
- Skeletal examinations: Yes: all per litter
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and processed for double staining with Alcian Blue 8GX and Alizarin Red S by a method similar to that described by Dawson (1926) and Inouye (1976). The skeletal examination was done on all fetuses from all groups.
- Head examinations: Yes: half per litter
The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.
- Anogenital distance of all live pups: Not evaluated - Statistics:
- Please see 'Any other information on materials and methods incl. tables' for information on statistics.
- Indices:
- Maternal Indices:
1) Pregnancy Rate (%): (No. of pregnant females / No. of mated females) x 100
Litter Indices:
1) Male Fetuses (%): (No. of male fetuses / No. fetuses) x 100
2) Female Fetuses (%): (No. of female fetuses / No. fetuses) x 100
3) Pre-Implantation Loss (%): (No. of corpora lutea – No. of implantations / No. of corpora lutea) x 100
4) Post-Implantation Loss (%): (No. of implantations – No. of live fetuses / No. of implantations) x 100
5) Litter % of Fetuses with Abnormalities: (No. of fetuses in litter with a given finding / No. of fetuses in litter examined) x 100 - Historical control data:
- Historical control data (2016 - 2020) included in Appendix 16 of the final study report.
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment-related clinical signs were observed.
All clinical signs noted during the dosing period occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent relation to dose level and/or duration of dosing. At the incidence observed, these were considered to be unrelated to treatment with the test material.
One animal (No. 45) in Group 3 (300 mg/kg/day) presented with red liquid on the cage tray on Day 28 post coitum. This animal was pregnant with a normal litter, therefore, this finding was considered not toxicologically relevant. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- No treatment-related mortality was observed through the study period.
One female (No. 39) in Group 2 (100 mg/kg/day) was sacrificed in extremis on Day 12 post-coitum. This animal presented with labored breathing, decreased activity, and pale skin after dosing. At necropsy, red content was found in the thoracic body cavity and trachea, as well as dark red foci on the lungs which confirmed that the poor condition of this animal was related to the gavage procedure and as such not test treatment-related.
Another female dosed at 100 mg/kg/day (No. 42) was euthanized on Day 27 post coitum, because it started to deliver its offspring that day. No further abnormalities were noted for this animal. This was considered incidental and not test treatment-related. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- No toxicologically relevant changes in body weight and body weight gain were observed following treatment with the test material.
At 1000 mg/kg/day (Group 4), females were observed with a slight mean body weight loss during the first two days of dosing (i.e. Days 7-9 post-coitum; not statistically significant). Subsequently, mean body weight gain remained in the same range as control, except for the period from Days 15-18 post-coitum, in which a slightly lower mean body weight gain was noted (statistically significant: p<0.05). No toxicological relevance was attached to these findings as changes compared to control were only slight and transient.
Mean body weight gain corrected for gravid uterus weight and mean uterus weight were unaffected by treatment with the test material. The lower mean uterus weight observed in the 100 mg/kg/day group (20% below control) was considered unrelated to treatment as no dose-related response was evident and as this was most likely due to the lower mean number of fetuses per female in this group. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- No toxicologically relevant changes in food consumption were observed following treatment with the test material.
At 1000 mg/kg/day (Group 4) , food consumption was increased compared to controls from Day 21 post coitum onwards. This was due to low (individual) control values compared to the daily provided ration and was therefore considered not treatment-related. - Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Description (incidence and severity):
- Water consumption data was used for health monitoring of the animals only and not reported.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Endocrine findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Lower mean uterus weight was observed in the 100 mg/kg/day group (20% below control) and considered unrelated to treatment with the test material, as no dose-related response was evident and as this was most likely due to the lower mean number of fetuses per female in this group.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Macroscopic observations of animals surviving until scheduled necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test material.
Incidental findings among control and treated animals included abnormalities of the spleen (ectopic splenic tissue), oviduct (watery clear cysts), kidney (brown foci), liver (prominent lobular architecture), lung (foci; irregular surface), placenta (dark, red clot), skin (scab; thin hair). These findings are occasionally seen among rabbits used in these types of study and in the absence of a dose-related trend they were considered changes of no toxicological significance. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Number of abortions:
- no effects observed
- Description (incidence and severity):
- Seven females were not gravid at the time of scheduled necropsy: three control females (Nos. 11, 13, 14) and two females in the 300 and 1000 mg/kg/day groups each (Nos. 64, 65, and 79, 84, respectively). Excluding non-pregnant females and the two females in the 100 mg/kg/day group that did not survive until scheduled necropsy, there were 19, 20, 20 and 20 females with viable litters in the control, 100, 300, and 1000 mg/kg/day groups, respectively.
- Pre- and post-implantation loss:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Mean numbers of implantation sites, and post implantation loss in the control and test groups were in the range of normal biological variation. Pre-implantation loss in treated groups was higher compared to controls without a dose response. This was considered not relevant because treatment with the test material started after completion of implantation.
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- Mean numbers of pregnant females in the control and test groups were in the range of normal biological variation.
- Other effects:
- no effects observed
- Description (incidence and severity):
- Mean numbers of corpora lutea in the control and test groups were in the range of normal biological variation.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: Systemic Toxicity
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related effects observed on fetal body weights (both sexes). All mean fetal weights (males, females and combined sexes) remained within the normal range of biological variation.
- Reduction in number of live offspring:
- effects observed, non-treatment-related
- Description (incidence and severity):
- One litter (No. 23) in the 100 mg/kg/day group (Group 2) had only two fetuses, one live male and one dead female fetus (post-implantation loss of 50% which contributed to the lower mean no. of live foetuses in this group). As this concerned the low dose group, a possible relation to treatment with the test material could be excluded.
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- The male:female ratio was considered to be unaffected by treatment.
- Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related effects on litter size of any group. Mean live litter sizes were 9.7, 8.0, 10.0 and 9.8 fetuses/litter for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
- Anogenital distance of all rodent fetuses:
- not examined
- Changes in postnatal survival:
- no effects observed
- External malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- External malformations only occurred in two late resorptions. Both these resorptions (Nos. 45-L1 and 68-R8 from the 300 and 1000 mg/kg/day groups, respectively) had hyperflexed forepaws and due to single occurrences, these were deemed chance findings and not treatment-related.
- Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were 1 (1), 4 (2), 0 (0) and 2 (2) fetuses (litters) with skeletal malformations in the control, 100, 300 and 1000 mg/kg/day groups, respectively. More than half of the cases concerned supernumerary lumbar vertebrae, that occurred in the affected control fetus (No. 21-R9), two fetuses at 100 mg/kg/day (Nos. 40-L2 and 43-L7) and one fetus at 1000 mg/kg/day (No. 83-L3). Remaining malformations were absent and/or fused ribs, vertebrae, centra or sternebrae (low dose fetuses No. 43-R9 and -R10) and sternoschisis (high-dose fetus No. 88-R4). Due to the group distribution, low incidence and different nature of all above malformations any relation with the test material was ruled out.
Skeletal variations occurred across a variety of structures and the low incidences and group distribution of these findings did not indicate any relation to treatment with the test material. - Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Visceral malformations only occurred at 300 mg/kg/day (Group 3). One fetus (No. 56-R8) had a retroesophageal right subclavian artery and another fetus (No. 57-L5) was observed with misshapen eye lenses during fixed head examination. The single occurrences at 300 mg/kg/day does not indicate a test material relationship and consequently both malformations were considered to have occurred by chance.
Among visceral variations more fetuses with retrocaval ureter were observed at the highest dose of 1000 mg/kg/day (Group 4). Comparison to the historical control data revealed that the mean litter incidence of fetuses did not exceed the upper limit and was therefore considered not related to treatment.
Remaining visceral variations occurred across a variety of structures and the low incidences and group distribution of these findings did not indicate any effects to treatment with the test material. - Other effects:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Developmental Toxicity
- Key result
- Abnormalities:
- effects observed, non-treatment-related
- Key result
- Developmental effects observed:
- no
- Conclusions:
- Based on the results of this prenatal developmental toxicity study in rabbits, the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Petroleum Gas Oil Fraction, Co-Processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) were determined to be at least 1000 mg/kg/day.
- Executive summary:
A key OECD Guideline 414 developmental toxicity study was conducted to determine the potential of the test material (Petroleum Gas Oil Fraction, Co-Processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9)) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Days 7 to 28 post-coitum, inclusive.
The test material (in corn oil vehicle) was administered once daily via oral gavage to groups of 22 time-mated female New Zealand White rabbits at dose levels of 0, 100, 300, or 1000 mg/kg/day from Days 7 to 28 post-coitum, inclusive. Control group animals received the vehicle, corn oil, alone. The parameters and end points evaluated for the maternal animals (F0-generation) included mortality/moribundity, clinical signs, body weights, food consumption, macroscopic examination, uterine contents, corpora lutea, implantation sites, and pre- and post-implantation loss. Additionally, parameters determined for the F1-generation included the number of live and dead fetuses, fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.
No mortality or treatment-related signs of clinical toxicity were observed through the study period. In Group 2 (100 mg/kg/day), one female was sacrificed in extremis due to poor condition as a result of the gavage procedure which was not considered to be treatment-related. No toxicologically relevant changes in body weight, body weight gain, or food consumption were observed following treatment with the test material. Gross necropsy did not reveal any remarkable treatment-related findings and while a lower mean uterus weight was observed in the 100 mg/kg/day dose group (20% below control), this was considered unrelated to treatment as no dose-related response was evident and it was most likely due to the lower mean number of fetuses per female in this group.
Seven females (F0) were not gravid at the time of scheduled necropsy: three control females (Nos. 11, 13, 14) and two females in the 300 and 1000 mg/kg/day dose groups each (Nos. 64, 65, and 79, 84, respectively). Excluding non-pregnant females and the two females in the 100 mg/kg/day group that did not survive until scheduled necropsy, there were 19, 20, 20 and 20 females with viable litters in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Pre-implantation loss in treated groups was higher compared to controls but without a dose-response. This was considered not relevant because treatment with the test material started after completion of implantation. Mean numbers of pregnant females, corpora lutea and implantation sites, and post-implantation in the control and test groups were in the range of normal biological variation.
Overall, no maternal toxicity was observed in the 100, 300, and 1000 mg/kg/day groups.
There were no test treatment-related effects on litter size and sex ratio and fetal body weights (both sexes) remained unaffected. External malformations only occurred in two late resorptions. Both these resorptions (Nos. 45-L1 and 68-R8 from the 300 and 1000 mg/kg/day groups, respectively) had hyperflexed forepaws and due to single occurrences, deemed chance findings and not treatment-related. There were 1 (1), 4 (2), 0 (0) and 2 (2) fetuses (litters) with skeletal malformations in the control, 100, 300 and 1000 mg/kg/day groups, respectively. More than half of the cases concerned supernumerary lumbar vertebrae, that occurred in the affected control fetus (No. 21-R9), two fetuses at 100 mg/kg/day (Nos. 40-L2 and 43-L7) and one fetus at 1000 mg/kg/day (No. 83-L3). Remaining malformations were absent and/or fused ribs, vertebrae, centra or sternebrae (low dose fetuses No. 43-R9 and -R10) and sternoschisis (high-dose fetus No. 88-R4). Due to the group distribution, low incidence and different nature of all above malformations, any relation with the test material was ruled out. Skeletal variations occurred across a variety of structures and the low incidences and group distribution of these findings did not indicate any relation to treatment with the test material.
Visceral malformations only occurred at 300 mg/kg/day (Group 3). One fetus (No. 56-R8) had a retroesophageal right subclavian artery and another fetus (No. 57-L5) was observed with misshapen eye lenses during fixed head examination. The single occurrences at 300 mg/kg/day does not indicate a test material relationship and consequently both malformations were considered to have occurred by chance. Among visceral variations more fetuses with retrocaval ureter were observed at the highest dose 1000 mg/kg/day (Group 4). Comparison to the historical control data revealed that the mean litter incidence of fetuses did not exceed the upper limit and this observation was therefore considered not related to treatment. Remaining visceral variations occurred across a variety of structures and the low incidences and group distribution of these findings did not indicate any effects of treatment with the test material.
Overall, no developmental toxicity was observed in the 100, 300, and 1000 mg/kg/day groups.
Based on the results of this prenatal developmental toxicity study in rabbits, the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Petroleum Gas Oil Fraction, Co-Processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) were determined to be at least 1000 mg/kg/day.
Referenceopen allclose all
Formulation Analysis:
Concentration analysis:
The concentrations analyzed in the formulation of Groups 2, 3, and 4 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 90-110% of target concentration) and no test material was detected in the Group 1 formulation.
Homogeneity:
The formulation of Groups 2, 3, and 4 were homogeneous (i.e., coefficient of variation ≤ 10%).
Table 2. Results of Formulation Analysis |
|||||
Date of analysis |
Concentration (mg/mL) |
Recovery (%) |
|||
Target |
Nominal |
Analyzed |
Individual |
Mean |
|
2021-SEP-27 |
25 |
22.6 |
24.3 |
108 |
104 |
24.8 |
24.8 |
100 |
|||
250 |
247 |
254 |
103 |
102 |
|
255 |
255 |
100 |
|||
Table 3. Summary of Body Weights: Gestation (F0: Female) |
||||||||
Group |
|
Day(s) Relative to Mating (Litter: A) |
||||||
2 |
6 |
9 |
12 |
15 |
18 |
21 |
||
Group 1 Control 0 mg/kg/day |
Mean |
220.0 |
232.7 |
242.1 |
256.5 |
270.5 |
303.6 |
340.3 |
SD |
13.5 |
14.6 |
15.4 |
15.3 |
17.5 |
21.8 |
27.3 |
|
N |
22 |
22 |
22 |
22 |
22 |
22 |
20 |
|
|
||||||||
Group 2 100 mg/kg/day |
Mean |
215.4 |
228.8 |
237.7 |
251.8 |
264.9 |
293.8 |
331.3 |
SD |
11.3 |
12.0 |
12.4 |
12.3 |
13.8 |
17.3 |
22.3 |
|
N |
21 |
21 |
21 |
21 |
21 |
21 |
20 |
|
% Diff |
-2.1 |
-1.7 |
-1.8 |
-1.9 |
-2.1 |
-3.3 |
-2.6 |
|
|
||||||||
Group 3 300 mg/kg/day |
Mean |
220.7 |
234.1 |
242.0 |
257.3 |
270.6 |
303.2 |
340.9 |
SD |
12.1 |
13.0 |
12.5 |
15.6 |
16.8 |
18.9 |
23.6 |
|
N |
22 |
22 |
22 |
22 |
22 |
22 |
21 |
|
% Diff |
0.3 |
0.6 |
-0.1 |
0.3 |
0.0 |
-0.1 |
0.2 |
|
|
||||||||
Group 4 1000 mg/kg/day |
Mean |
218.6 |
231.8 |
232.7 |
246.5 |
261.2 |
294.2 |
326.7 |
SD |
13.2 |
14.7 |
13.8 |
15.5 |
17.8 |
20.9 |
28.8 |
|
N |
22 |
22 |
22 |
22 |
22 |
22 |
20 |
|
% Diff |
-0.7 |
-0.4 |
-3.9 |
-3.9 |
-3.4 |
-3.1 |
-4.0 |
|
Anova & Dunnett
Table 4. Summary of Body Weight Gains (g): Gestation (F0: Females) |
|||||||
Group |
|
Day(s) Relative to Mating (Litter: A) |
|||||
6 → 9 [G] |
9 → 12 [G1] |
12 → 15 [G1] |
15 → 18 [G1] |
18 → 21 [G1] |
6 → 21 [G1] |
||
Group 1 Control 0 mg/kg/day |
Mean |
9.5 |
14.5 |
13.9 |
33.1 |
37.7 |
108.2 |
SD |
3.5 |
3.2 |
4.8 |
6.6 |
6.5 |
15.7 |
|
N |
22 |
22 |
22 |
22 |
20 |
20 |
|
|
|||||||
Group 2 100 mg/kg/day |
Mean |
8.9 |
14.1 |
13.1 |
28.9 |
36.1 |
101.6 |
SD |
2.4 |
4.5 |
4.2 |
6.3 |
9.2 |
19.2 |
|
N |
21 |
21 |
21 |
21 |
20 |
20 |
|
|
|||||||
Group 3 300 mg/kg/day |
Mean |
7.9 |
15.3 |
13.3 |
32.6 |
36.3 |
106.2 |
SD |
4.4 |
5.7 |
4.7 |
5.2 |
6.9 |
14.7 |
|
N |
22 |
22 |
22 |
22 |
21 |
21 |
|
|
|||||||
Group 4 1000 mg/kg/day |
Mean |
0.9** |
13.8 |
14.7 |
33.0 |
33.7 |
95.8 |
SD |
5.2 |
4.5 |
5.3 |
5.8 |
10.0 |
18.2 |
|
N |
22 |
22 |
22 |
22 |
20 |
20 |
|
[G] - Kruskal-Wallis & Dunn: ** = p ≤ 0.01
[G1] - Anova & Dunnett
Table 5. Summary of Gravid Uterine Weights and Gravid Uterus Adjusted Body Weights: Gestation (F0: Female) |
|||||
Day(s) Relative to Mating (Litter: A) |
|
Group 1 Control 0 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Bodyweight on Day 6 (g) [G] |
Mean |
232.7 |
228.8 |
234.1 |
231.8 |
SD |
14.6 |
12.0 |
13.0 |
14.7 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-1.7 |
0.6 |
-0.4 |
|
|
|||||
Terminal Body Weight (g) [G] |
Mean |
340.3 |
331.3 |
340.9 |
326.7 |
SD |
27.3 |
22.3 |
23.6 |
28.8 |
|
N |
20 |
20 |
21 |
20 |
|
% Diff |
- |
-2.6 |
0.2 |
-4.0 |
|
|
|||||
Gravid Uterus Weight (g) [G] |
Mean |
82.10 |
71.70 |
76.12 |
78.13 |
SD |
15.9 |
19.10 |
13.09 |
16.33 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-12.67 |
-7.29 |
-4.84 |
|
|
|||||
Adjusted BWG (6-abw) (g) [G] |
Mean |
25.90 |
29.01 |
28.80 |
18.86* |
SD |
9.15 |
8.48 |
8.25 |
10.74 |
|
N |
20 |
20 |
21 |
20 |
|
% Diff |
- |
12.03 |
11.22 |
-27.19 |
|
[G] - Anova & Dunnett: * = p ≤ 0.05
Table 6. Summary of Food Consumption: Gestation (F0: Females) |
|||||||
Group |
|
Day(s) Relative to Mating (Litter: A) |
|||||
6 → 9 |
9 → 12 |
12 → 15 |
15 → 18 |
18 → 21 |
6 → 21 |
||
Group 1 Control 0 mg/kg/day |
Mean |
20.50 |
19.94 |
20.59 |
21.83 |
21.18 |
20.81 |
SD |
2.11 |
1.61 |
1.81 |
1.82 |
1.76 |
1.51 |
|
N |
22 |
22 |
22 |
22 |
22 |
22 |
|
|
|||||||
Group 2 100 mg/kg/day |
Mean |
21.35 |
19.83 |
20.92 |
21.97 |
21.57 |
21.13 |
SD |
2.46 |
2.73 |
2.11 |
2.37 |
2.23 |
1.89 |
|
N |
21 |
21 |
21 |
21 |
21 |
21 |
|
% Diff |
4.14 |
-0.57 |
1.60 |
0.62 |
1.84 |
1.53 |
|
|
|||||||
Group 3 300 mg/kg/day |
Mean |
20.24 |
19.09 |
20.50 |
22.41 |
21.71 |
20.79 |
SD |
3.98 |
2.61 |
3.05 |
2.81 |
2.74 |
2.40 |
|
N |
22 |
22 |
22 |
22 |
22 |
22 |
|
% Diff |
-1.26 |
-4.26 |
-0.44 |
2.64 |
2.50 |
-0.09 |
|
|
|||||||
Group 4 1000 mg/kg/day |
Mean |
15.88** |
16.97** |
19.03 |
22.59 |
20.95 |
19.08* |
SD |
2.94 |
2.60 |
2.33 |
1.85 |
2.88 |
1.91 |
|
N |
22 |
22 |
22 |
22 |
22 |
22 |
|
% Diff |
-22.54 |
-14.89 |
-7.58 |
3.47 |
-1.07 |
-8.29 |
|
Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01
Table 7. Summary of Thyroid Hormone Values (F0 Female) |
||||
Day: 21 Relative to Mating (Litter: A) |
Reporting Special Chemistry |
|||
Group |
|
T3 (ng/mL) [G] |
T4 (ng/mL) [G] |
TSH (ng/mL) [G] |
Group 1 Control 0 mg/kg/day |
Mean |
0.386 |
20.01 |
0.2425 |
SD |
0.051 |
3.11 |
0.1698 |
|
N |
22 |
22 |
22 |
|
|
||||
Group 2 100 mg/kg/day |
Mean |
0.382 |
24.03** |
0.3191 |
SD |
0.079 |
4.91 |
0.1531 |
|
N |
21 |
21 |
21 |
|
tCtrl |
0.99 |
1.20 |
1.32 |
|
|
||||
Group 3 300 mg/kg/day |
Mean |
0.291** |
21.23 |
0.3443 |
SD |
0.057 |
3.85 |
0.2992 |
|
N |
22 |
22 |
22 |
|
tCtrl |
0.75 |
1.06 |
1.42 |
|
|
||||
Group 4 1000 mg/kg/day |
Mean |
0.244** |
18.16 |
0.5770** |
SD |
0.074 |
3.63 |
0.3300 |
|
N |
22 |
22 |
22 |
|
tCtrl |
0.63 |
0.91 |
2.38 |
|
[G] - Anova & Dunnett: ** = p ≤ 0.01
Table 8. Summary of Organ Weights (F0: Female) |
|||||
Day(s) Relative to Mating (Litter: A) |
|
Group 1 Control 0 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Terminal Body Weight (g) [G] |
Mean |
340.3 |
331.3 |
340.9 |
326.7 |
SD |
27.3 |
22.3 |
23.6 |
28.8 |
|
N |
20 |
20 |
21 |
20 |
|
% Diff |
- |
-2.6 |
0.2 |
-4.0 |
|
|
|||||
Gland, Thyroid Weight (g) - [G] |
Mean |
0.01362 |
0.01404 |
0.01423 |
0.01473 |
SD |
0.00268 |
0.00272 |
0.00264 |
0.00236 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
3.04908 |
4.47114 |
8.14147 |
|
|
|||||
Gland, Thyroid (%bw) - [G] |
Mean |
0.00394 |
0.00427 |
0.00421 |
0.00449 |
SD |
0.00079 |
0.00077 |
0.00071 |
0.00076 |
|
N |
20 |
20 |
21 |
20 |
|
[G] - Anova & Dunnett
Table 9. Summary of Macroscopic Pathology: Incidence (F0: Female) |
||||
Incidence |
Group 1 Control 0 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Number of Animals |
22 |
22 |
22 |
22 |
GLAND, SALIVARY, MANDIBULAR |
|
|||
Submitted |
1 |
0 |
0 |
0 |
Focus, dark; red |
1 |
- |
- |
- |
GLAND, THYROID |
|
|||
Submitted |
22 |
22 |
22 |
22 |
No Visible Lesions |
21 |
22 |
22 |
21 |
Focus, dark; red |
1 |
0 |
0 |
0 |
Enlargement |
0 |
0 |
0 |
1 |
Small |
0 |
0 |
0 |
1 |
SKIN |
|
|||
Submitted |
1 |
1 |
0 |
0 |
Scab; cervical, dorsal |
1 |
0 |
- |
- |
Thin hair coat |
1 |
0 |
- |
- |
Thin hair coat; forelimb,left |
0 |
1 |
- |
- |
Table 10. Summary of Microscopic Pathology: Incidence (F0: Female) |
||||
Incidence |
Group 1 Control 0 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Number of Animals |
22 |
22 |
22 |
22 |
GLAND, THYROID |
|
|||
Examined |
22 |
22 |
22 |
22 |
No Visible Lesions |
22 |
21 |
21 |
19 |
Hypertrophy; follicular cell |
0 |
1 |
1 |
2 |
.... minimal |
0 |
1 |
1 |
2 |
Ectopia; thymic corpuscle |
0 |
0 |
0 |
1 |
Table 11. Summary of Maternal Performance and Mortality (F0: Female) |
|||||
Day(s) Relative to Mating (Litter: A) |
|
Group 1 Control 0 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Group Size - Females |
|
22 |
22 |
22 |
22 |
Number of Females Pregnant [f] |
N+ve |
22 |
21 |
22 |
22 |
% |
100.0 |
95.5 |
100.0 |
100.0 |
|
|
|||||
Female with Live Fetuses [f] |
N+ve |
22 |
21 |
22 |
22 |
% |
100.0 |
100.0 |
100.0 |
100.0 |
|
|
|||||
Total Resorptions [f] |
N+ve |
0 |
0 |
0 |
0 |
% |
0.0 |
0.0 |
0.0 |
0.0 |
|
|
|||||
Female with all Nonviable [f] |
N+ve |
0 |
0 |
0 |
0 |
% |
0.0 |
0.0 |
0.0 |
0.0 |
|
|
|||||
Terminal Euthanasia [f] |
N+ve |
22 |
22 |
22 |
22 |
% |
100.0 |
100.0 |
100.0 |
100.0 |
|
|
|||||
Unscheduled Death/Euthanasia [f] |
N+ve |
0 |
0 |
0 |
0 |
% |
0.0 |
0.0 |
0.0 |
0.0 |
|
[f] - Fisher's Exact
Table 12. Summary of Ovarian and Uterine Examinations and Litter Observations (F0: Female) |
|||||
Day(s) Relative to Mating (Litter: A) |
|
Group 1 Control 0 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Female with Live Fetuses |
N+ve |
22 |
21 |
22 |
22 |
% |
100.0 |
100.0 |
100.0 |
100.0 |
|
|
|||||
Number of Corpora Lutea [k] |
Mean |
13.6 |
12.7 |
13.6 |
13.0 |
SD |
1.5 |
1.3 |
1.3 |
2.4 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-6.8 |
0.0 |
-4.7 |
|
|
|||||
Number of Implantations [k] |
Mean |
12.6 |
10.8 |
11.8 |
12.0 |
SD |
1.5 |
3.0 |
1.8 |
2.9 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-14.5 |
-6.1 |
-4.3 |
|
|
|||||
Pre-implantation Loss (%) [k] |
Mean |
7.50 |
15.97 |
12.84 |
7.69 |
SD |
7.52 |
21.14 |
13.52 |
12.32 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
112.98 |
71.19 |
2.48 |
|
|
|||||
Total Number of Fetuses [k] |
Mean |
11.7 |
10.0 |
11.2 |
11.6 |
SD |
2.2 |
3.0 |
2.0 |
2.8 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-14.3 |
-4.7 |
-0.8 |
|
|
|||||
Number of Live Fetuses [k] |
Mean |
11.7 |
10.0 |
11.2 |
11.6 |
SD |
2.2 |
3.0 |
2.0 |
2.8 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-14.3 |
-4.7 |
-0.8 |
|
|
|||||
Number of Dead Fetuses [k] |
Mean |
0.0 |
0.0 |
0.0 |
0.0 |
SD |
0.0 |
0.0 |
0.0 |
0.0 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
- |
- |
- |
|
|
|||||
Number of Early Resorptions [k] |
Mean |
0.9 |
0.7 |
0.6 |
0.4 |
SD |
1.2 |
1.1 |
0.9 |
0.6 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-17.3 |
-26.3 |
-52.6 |
|
|
|||||
Number of Late Resorptions [k] |
Mean |
0.0 |
0.0 |
0.0 |
0.0 |
SD |
0.0 |
0.0 |
0.0 |
0.0 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
- |
- |
- |
|
|
|||||
Total Number of Resorptions [k] |
Mean |
0.9 |
0.7 |
0.6 |
0.4 |
SD |
1.2 |
1.1 |
0.9 |
0.6 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-17.3 |
-26.3 |
-52.6 |
|
|
|||||
Post-implantation Loss (%) [k] |
Mean |
7.40 |
6.89 |
5.61 |
3.52 |
SD |
11.16 |
11.13 |
7.66 |
5.52 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-7.00 |
-24.27 |
-52.44 |
|
|
|||||
Number of Live Male Fetuses [k] |
Mean |
6.0 |
5.2 |
4.9 |
5.3 |
SD |
2.3 |
2.1 |
2.4 |
1.7 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-12.0 |
-17.6 |
-11.5 |
|
|
|||||
Number of Live Female Fetuses [k] |
Mean |
5.8 |
4.8 |
6.3 |
6.4 |
SD |
2.1 |
2.5 |
2.2 |
2.1 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-16.7 |
8.7 |
10.2 |
|
|
|||||
Live Male Fetus/Litter (%) [k] |
Mean |
50.37 |
53.71 |
43.02 |
45.30 |
SD |
15.55 |
16.94 |
19.10 |
11.51 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
6.64 |
-14.58 |
-10.06 |
|
|
|||||
Live Female Fetuses/Litter (%) [k] |
Mean |
49.63 |
46.29 |
56.98 |
54.70 |
SD |
15.55 |
16.94 |
19.10 |
11.51 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-6.73 |
14.79 |
10.21 |
|
|
|||||
Mean Fetal Weight males (g) [G] |
Mean |
5.328 |
5.434 |
5.253 |
5.211 |
SD |
0.181 |
0.261 |
0.296 |
0.246 |
|
N |
22 |
21 |
21 |
22 |
|
% Diff |
- |
1.985 |
-1.398 |
-2.195 |
|
|
|||||
Mean Fetal Weight females (g) [G] |
Mean |
5.052 |
5.212* |
5.004 |
4.973 |
SD |
0.217 |
0.265 |
0.206 |
0.157 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
3.172 |
-0.941 |
-1.558 |
|
|
|||||
Mean Fetal Weight all (g) [G] |
Mean |
5.193 |
5.333 |
5.105 |
5.086 |
SD |
0.176 |
0.242 |
0.216 |
0.192 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
2.696 |
-1.696 |
-2.072 |
|
|
|||||
Mean Fetal AGD males (mm) [G1] |
Mean |
2.92 |
2.93 |
2.79 |
2.74 |
SD |
0.27 |
0.23 |
0.25 |
0.32 |
|
N |
22 |
21 |
21 |
22 |
|
% Diff |
- |
0.25 |
-4.51 |
-6.24 |
|
|
|||||
Mean Fetal AGD females (mm) [G1] |
Mean |
1.39 |
1.33 |
1.32 |
1.39 |
SD |
0.29 |
0.32 |
0.26 |
0.34 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-3.90 |
-5.19 |
-0.10 |
|
|
|||||
Mean Normalized Fetal AGD m [G] |
Mean |
1.672 |
1.667 |
1.604 |
1.579 |
SD |
0.146 |
0.137 |
0.130 |
0.176 |
|
N |
22 |
21 |
21 |
22 |
|
% Diff |
- |
-0.301 |
-4.042 |
-5.573 |
|
|
|||||
Mean Normalized Fetal AGD f [G]
|
Mean |
0.810 |
0.771 |
0.770 |
0.813 |
SD |
0.168 |
0.186 |
0.152 |
0.202 |
|
N |
22 |
21 |
22 |
22 |
|
% Diff |
- |
-4.850 |
-4.938 |
0.423 |
|
[G] - Anova & Dunnett: * = p ≤ 0.05
[G1] - Anova & Dunnett
[k] - Kruskal-Wallis & Dunn
Table 13. Summary of Fetal Abnormalities by Finding (F1: External) |
|||||
|
|
Group 1 Control 0 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Number of Fetuses Examined: |
|
258 |
211 |
246 |
256 |
Number of Fetuses Evaluated: |
|
258 |
211 |
246 |
256 |
Number of Litters Examined: |
|
22 |
21 |
22 |
22 |
Number of Litters Evaluated: |
|
22 |
21 |
22 |
22 |
|
|||||
Paw/Digit |
|
||||
Hindpaw, Left, Polydactyly - Malformation |
Fetuses N(%) |
0 (0.00) |
1 (0.37) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
1 (4.8) |
0 (0.0) |
0 (0.0) |
|
Table 14. Summary of Fetal Abnormalities by Finding (F1: FreshVisBody) |
|||||
|
|
Group 1 Control 0 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Number of Fetuses Examined: |
|
127 |
104 |
121 |
127 |
Number of Fetuses Evaluated: |
|
258 |
211 |
246 |
256 |
Number of Litters Examined: |
|
22 |
21 |
22 |
22 |
Number of Litters Evaluated: |
|
22 |
21 |
22 |
22 |
|
|||||
General |
|
||||
General, Situs inversus - Malformation |
Fetuses N(%) |
0 (0.00) |
0 (0.00) |
0 (0.00) |
1 (0.65) |
Litters N(%) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (4.5) |
|
Kidney |
|
||||
Renal papilla, Right, Absent - Variation |
Fetuses N(%) |
0 (0.00) |
1 (0.68) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
1 (4.8) |
0 (0.0) |
0 (0.0) |
|
Liver |
|
||||
Lobe, Left medial, Supernumerary - Variation |
Fetuses N(%) |
3 (2.06) |
1 (0.79) |
1 (0.76) |
2 (1.41) |
Litters N(%) |
3 (13.6) |
1 (4.8) |
1 (4.5) |
2 (9.1) |
|
Lobe, Right medial, Supernumerary - Variation |
Fetuses N(%) |
0 (0.00) |
2 (1.90) |
2 (1.41) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
1 (4.8) |
2 (9.1) |
0 (0.0) |
|
Lung |
|
||||
Lobe, Left cranial, Fused - Malformation |
Fetuses N(%) |
0 (0.00) |
0 (0.00) |
0 (0.00) |
1 (0.65) |
Litters N(%) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (4.5) |
|
Lobe, Right medial, Fused - Malformation |
Fetuses N(%) |
1 (0.65) |
0 (0.00) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
1 (4.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|
Ureter |
|
||||
Ureter, Left, Convoluted - Variation |
Fetuses N(%) |
1 (0.76) |
1 (0.79) |
1 (0.76) |
0 (0.00) |
Litters N(%) |
1 (4.5) |
1 (4.8) |
1 (4.5) |
0 (0.0) |
|
Ureter, Right, Convoluted - Variation |
Fetuses N(%) |
1 (0.65) |
2 (1.63) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
1 (4.5) |
2 (9.5) |
0 (0.0) |
0 (0.0) |
|
Ureter, Right, Dilatation, Minimal - Variation |
Fetuses N(%) |
0 (0.00) |
1 (0.68) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
1 (4.8) |
0 (0.0) |
0 (0.0) |
|
[Fetuses %] - Kruskal-Wallis & Dunn
FetusesN(%) N=Group Fetal Incidence;(%)=Mean Litter % of Fetuses with the Abnormality
Table 15. Summary of Fetal Abnormalities by Finding (F1: Skeletal) |
|||||
|
|
Group 1 Control 0 mg/kg/day |
Group 2 100 mg/kg/day |
Group 3 300 mg/kg/day |
Group 4 1000 mg/kg/day |
Number of Fetuses Examined: |
|
131 |
107 |
125 |
129 |
Number of Fetuses Evaluated: |
|
258 |
211 |
246 |
256 |
Number of Litters Examined: |
|
22 |
21 |
22 |
22 |
Number of Litters Evaluated: |
|
22 |
21 |
22 |
22 |
|
|||||
Forelimb |
|
||||
Forepaw phalanges, 1 or more, Unossified - Variation |
Fetuses N(%) |
0 (0.00) |
0 (0.00) |
1 (0.65) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
0 (0.0) |
1 (4.5) |
0 (0.0) |
|
Hindlimb |
|
||||
Hindpaw phalanges, 1 or more, Supernumerary - Malformation |
Fetuses N(%) |
0 (0.00) |
1 (0.68) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
1 (4.8) |
0 (0.0) |
0 (0.0) |
|
Metatarsal, 1 or more, Malpositioned - Variation |
Fetuses N(%) |
0 (0.00) |
1 (0.79) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
1 (4.8) |
0 (0.0) |
0 (0.0) |
|
Pelvic Girdle |
|
||||
Ilium, Both, Misaligned - Variation |
Fetuses N(%) |
10 (7.03) |
4 (4.05) |
3 (1.95) |
11 (9.22) |
Litters N(%) |
6 (27.3) |
4 (19.0) |
2 (9.1) |
9 (40.9) |
|
Rib |
|
||||
Costal cartilage, 1 or more, Fused - Variation |
Fetuses N(%) |
2 (1.41) |
1 (0.79) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
2 (9.1) |
1 (4.8) |
0 (0.0) |
0 (0.0) |
|
Rib, 1 or more, Wavy rib - Variation |
Fetuses N(%) |
12 (8.87) |
10 (8.95) |
2 (1.41) |
3 (2.32) |
Litters N(%) |
7 (31.8) |
6 (28.6) |
2 (9.1) |
2 (13.6) |
|
Skull |
|
||||
Frontal, Left, Incomplete ossification - Variation |
Fetuses N(%) |
0 (0.00) |
1 (1.19) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
1 (4.8) |
0 (0.0) |
0 (0.0) |
|
Interparietal, Incomplete ossification - Variation |
Fetuses N(%) |
3 (2.16) |
5 (4.97) |
1 (0.76) |
0 (0.00) |
Litters N(%) |
3 (13.6) |
4 (19.0) |
1 (4.5) |
0 (0.0) |
|
Parietal, Both, Incomplete ossification - Variation |
Fetuses N(%) |
2 (1.52) |
1 (1.19) |
1 (0.76) |
0 (0.00) |
Litters N(%) |
2 (9.1) |
1 (4.8) |
1 (4.5) |
0 (0.0) |
|
Parietal, Right, Incomplete ossification - Variation |
Fetuses N(%) |
0 (0.00) |
1 (1.19) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
1 (4.8) |
0 (0.0) |
0 (0.0) |
|
Squamosal, Both, Incomplete ossification - Variation |
Fetuses N(%) |
1 (0.76) |
1 (1.19) |
2 (1.52) |
0 (0.00) |
Litters N(%) |
1 (4.5) |
1 (4.8) |
1 (4.5) |
0 (0.0) |
|
Supraoccipital, Incomplete ossification - Variation |
Fetuses N(%) |
1 (0.76) |
1 (0.95) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
1 (4.5) |
1 (4.8) |
0 (0.0) |
0 (0.0) |
|
Zygomatic arch, Both, Incomplete ossification - Variation |
Fetuses N(%) |
1 (0.76) |
0 (0.00) |
3 (2.27) |
0 (0.00) |
Litters N(%) |
1 (4.5) |
0 (0.0) |
1 (4.5) |
0 (0.0) |
|
Zygomatic arch, Left, Incomplete ossification - Variation |
Fetuses N(%) |
0 (0.00) |
1 (1.19) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
1 (4.8) |
0 (0.0) |
0 (0.0) |
|
Zygomatic arch, Right, Incomplete ossification - Variation |
Fetuses N(%) |
0 (0.00) |
2 (2.38) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
1 (4.8) |
0 (0.0) |
0 (0.0) |
|
Sternebra |
|
||||
Sternebra, 1 or more, Branched - Variation |
Fetuses N(%) |
1 (0.76) |
0 (0.00) |
1 (0.65) |
0 (0.00) |
Litters N(%) |
1 (4.5) |
0 (0.0) |
1 (4.5) |
0 (0.0) |
|
Sternebra, 1 or more, Misaligned - Variation |
Fetuses N(%) |
3 (2.32) |
1 (4.76) |
2 (1.56) |
1 (0.76) |
Litters N(%) |
3 (13.6) |
1 (4.8) |
2 (9.1) |
1 (4.5) |
|
Sternebra, 1 or more, Unossified - Variation |
Fetuses N(%) |
0 (0.00) |
1 (4.76) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
1 (4.8) |
0 (0.0) |
0 (0.0) |
|
Sternebra, 1 or more, Incomplete ossification - Variation |
Fetuses N(%) |
0 (0.00) |
1 (4.76) |
2 (1.41) |
2 (1.41) |
Litters N(%) |
0 (0.0) |
1 (4.8) |
2 (9.1) |
2 (9.1) |
|
Supernumerary rib |
|
||||
Cervical, 1 or more, Full - Variation |
Fetuses N(%) |
2 (1.41) |
0 (0.00) |
0 (0.00) |
0 (0.00) |
Litters N(%) |
2 (9.1) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|
Cervical, 1 or more, Short - Variation |
Fetuses N(%) |
5 (4.39) |
4 (7.30) |
5 (4.13) |
6 (4.11) |
Litters N(%) |
4 (18.2) |
3 (14.3) |
5 (22.7) |
5 (22.7) |
|
Thoracolumbar, 1 or more, Full - Variation |
Fetuses N(%) |
15 (10.46) |
5 (4.29) |
9 (7.49) |
17 (11.96) |
Litters N(%) |
10 (45.5) |
3 (14.3) |
6 (27.3) |
9 (40.9) |
|
Thoracolumbar, 1 or more, Short - Variation |
Fetuses N(%) |
83 (64.09) |
53 (49.56) |
81 (63.82) |
80 (63.40) |
Litters N(%) |
20 (90.9) |
20 (95.2) |
22 (100.0) |
21 (95.5) |
|
Vertebra |
|
||||
Cervical arch, 1 or more, Small - Variation |
Fetuses N(%) |
0 (0.00) |
0 (0.00) |
1 (0.91) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
0 (0.0) |
1 (4.5) |
0 (0.0) |
|
Lumbar vertebra, 1 or more, Supernumerary - Malformation |
Fetuses N(%) |
0 (0.00) |
0 (0.00) |
0 (0.00) |
1 (0.76) |
Litters N(%) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (4.5) |
|
Thoracic centrum, 1 or more, Incomplete ossification - Variation |
Fetuses N(%) |
0 (0.00) |
0 (0.00) |
1 (0.91) |
0 (0.00) |
Litters N(%) |
0 (0.0) |
0 (0.0) |
1 (4.5) |
0 (0.0) |
|
[Fetuses %] - Kruskal-Wallis & Dunn
FetusesN(%) N=Group Fetal Incidence;(%)=Mean Litter % of Fetuses with the Abnormality
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 300 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rabbit
- Quality of whole database:
- Two key substance specific OECD 414 Guideline study in rodents and non-rodents available for assessment.
Effect on developmental toxicity: via dermal route
- Species:
- rat
Additional information
Oral Route:
A key OECD Guideline 414 pre-natal developmental toxicity study was conducted to evaluate the potential of the test material (Petroleum Gas Oil Fraction, Co Processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9)) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested (Charles River Laboratories Den Bosch B.V, 2022)
The test material was administered to time-mated female Wistar Han rats (22/dose) once daily via oral gavage in a corn oil vehicle at doses of 0, 100, 300, or 1000 mg/kg/day from Days 6 to 20 post-coitum, inclusive. The parameters evaluated for the maternal animals (F0 generation) included mortality / moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), macroscopic examination, organ weights (thyroid gland), uterine contents, microscopic examination (thyroid gland), corpora lutea, implantation sites and pre- and post-implantation loss. Additionally, the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations were evaluated for the pups (F1 generation).
No mortality or clinical signs of toxicity were observed through the study period. At 1000 mg/kg/day, mean food consumption was decreased over Days 6-12 post-coitum and mean body weight gain was reduced between Day 6-9 post-coitum. As a result, body weight gain corrected for gravid uterus weight was also slightly reduced. Food consumption and body weight gain recovered from Day 12 post-coitum onwards to levels comparable to control and in the absence of other signs of maternal toxicity, these findings were not considered to represent an adverse treatment-related effect.
Mean serum levels of T3 at 100 mg/kg/day were in the same range as the corresponding control animals. Mean serum levels of Triiodothyronine (T3) were statistically significantly reduced (p<0.01) at 300 and 1000 mg/kg/day (0.75 and 0.63x of control, respectively), and were considered treatment-related. Nonetheless, at 300 mg/kg/day, the mean T3 level remained within the available historical control range. However, at 1000 mg/kg/day, the mean T3 level (and individual serum levels of 16/22 females) were below the lower limit of the available historical control range. The downstream biological consequences of thyroid hormone changes are not assessed in this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed and this finding was not taken into account when determining the maternal NOAEL.
Mean serum levels of total T4 at 100 mg/kg/day were slightly increased (1.20x of control). Mean serum levels of total Thyroxine (T4) were considered unaffected by treatment at 300 and 1000 mg/kg/day and mean T4 serum levels at all dose levels remained within the available historical control range.
Mean serum levels of Thyroid Stimulating Hormone (TSH) showed an apparent dose-related trend towards an increase across the dose groups, reaching statistical significance (p<0.01) at 1000 mg/kg/day only (2.38x of control). This effect was considered treatment-related. However, mean TSH levels for all groups remained within the available historical control range. No treatment-related effects were observed on thyroid gland weight/s and gross necropsy as well as histopathology did not reveal any treatment-related effects in the thyroid glands.
All females were gravid with viable fetuses, except for one female (No. 38) in the low dose group (100 mg/kg/day), which was non gravid. The percentage of pre-implantation loss was higher than control in females treated at 100 and 300 mg/kg/day. However, as treatment was only initiated after implantation was completed and in the absence of a dose response, this was considered a chance finding. The percentage of post-implantation loss in the control and test groups was similar and in the range of normal biological variation. The mean number of corpora lutea and implantation sites in the control and test groups were similar and in the range of normal biological variation.
Mean fetal weights (male, female and combined) in the control and test groups were considered similar and unaffected by treatment. The sex ratio was also unaffected by treatment up to 1000 mg/kg/day and no treatment-related effects on litter size were observed in any group. Anogenital distance (absolute and normalized for body weight) in male and female pups was unaffected by treatment up to 1000 mg/kg/day.
One fetus (No. 28 L1) in the low dose group (100 mg/kg/day) presented with an external malformation (polydactyly of a hindpaw) which was confirmed at skeletal examination. This single occurrence of a malformation in a low-dose fetus did not suggest a treatment-related effect and was therefore considered a chance finding. No external variations were observed through the study period.
Visceral malformations were observed in two fetuses, one in the control group and the other in the high dose group. The fetus in the control group (Fetus No. 21-L2) had fused lung lobes while the fetus in the high dose group (1000 mg/kg/day) presented situs inversus and fused lung lobes. Given the single occurrences of these malformations and/or occurrence in the control group, they were deemed chance findings.
Visceral variations were limited to supernumerary liver lobes, absent renal papilla, and convoluted or dilated ureters. The low incidences, group distribution, and occurrence in control fetuses ruled out any relationship to treatment with the test material.
Skeletal malformations occurred in one fetus each in the low- and high-dose groups. The fetus (No. 28-L1) in the low dose group (100 mg/kg/day) presented extra hindpaw phalanges, consistent with polydactyly observed in this fetus at external examination while the fetus (No. 71-L1) in the high dose group (1000 mg/kg/day) had an extra lumbar vertebra. Due to the single occurrences of these malformations, they were deemed chance findings. All skeletal variations observed occurred in the absence of a dose-related incidence trend, infrequently and/or in control fetuses only. Therefore, they were not considered treatment-related.
Based on the effects observed, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Petroleum Gas Oil Fraction, Co-Processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) was determined to be ≥1000 mg/kg/day in the rat.
Another key OECD Guideline 414 developmental toxicity study was conducted to determine the potential of the test material (Petroleum Gas Oil Fraction, Co-Processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9)) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Days 7 to 28 post-coitum, inclusive (Charles River Laboratories Den Bosch B.V., 2022).
The test material (in corn oil vehicle) was administered once daily via oral gavage to groups of 22 time-mated female New Zealand White rabbits at dose levels of 0, 100, 300, or 1000 mg/kg/day from Days 7 to 28 post-coitum, inclusive. Control group animals received the vehicle, corn oil, alone. The parameters and end points evaluated for the maternal animals (F0-generation) included mortality/moribundity, clinical signs, body weights, food consumption, macroscopic examination, uterine contents, corpora lutea, implantation sites, and pre- and post-implantation loss. Additionally, parameters determined for the F1-generation included the number of live and dead fetuses, fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.
No mortality or treatment-related signs of clinical toxicity were observed through the study period. In Group 2 (100 mg/kg/day), one female was sacrificed in extremis due to poor condition as a result of the gavage procedure which was not considered to be treatment-related. No toxicologically relevant changes in body weight, body weight gain, or food consumption were observed following treatment with the test material. Gross necropsy did not reveal any remarkable treatment-related findings and while a lower mean uterus weight was observed in the 100 mg/kg/day dose group (20% below control), this was considered unrelated to treatment as no dose-related response was evident and it was most likely due to the lower mean number of fetuses per female in this group.
Seven females (F0) were not gravid at the time of scheduled necropsy: three control females (Nos. 11, 13, 14) and two females in the 300 and 1000 mg/kg/day dose groups each (Nos. 64, 65, and 79, 84, respectively). Excluding non-pregnant females and the two females in the 100 mg/kg/day group that did not survive until scheduled necropsy, there were 19, 20, 20 and 20 females with viable litters in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Pre-implantation loss in treated groups was higher compared to controls but without a dose-response. This was considered not relevant because treatment with the test material started after completion of implantation. Mean numbers of pregnant females, corpora lutea and implantation sites, and post-implantation in the control and test groups were in the range of normal biological variation.
Overall, no maternal toxicity was observed in the 100, 300, and 1000 mg/kg/day groups.
There were no test treatment-related effects on litter size and sex ratio and fetal body weights (both sexes) remained unaffected. External malformations only occurred in two late resorptions. Both these resorptions (Nos. 45-L1 and 68-R8 from the 300 and 1000 mg/kg/day groups, respectively) had hyperflexed forepaws and due to single occurrences, deemed chance findings and not treatment-related. There were 1 (1), 4 (2), 0 (0) and 2 (2) fetuses (litters) with skeletal malformations in the control, 100, 300 and 1000 mg/kg/day groups, respectively. More than half of the cases concerned supernumerary lumbar vertebrae, that occurred in the affected control fetus (No. 21-R9), two fetuses at 100 mg/kg/day (Nos. 40-L2 and 43-L7) and one fetus at 1000 mg/kg/day (No. 83-L3). Remaining malformations were absent and/or fused ribs, vertebrae, centra or sternebrae (low dose fetuses No. 43-R9 and -R10) and sternoschisis (high-dose fetus No. 88-R4). Due to the group distribution, low incidence and different nature of all above malformations, any relation with the test material was ruled out. Skeletal variations occurred across a variety of structures and the low incidences and group distribution of these findings did not indicate any relation to treatment with the test material.
Visceral malformations only occurred at 300 mg/kg/day (Group 3). One fetus (No. 56-R8) had a retroesophageal right subclavian artery and another fetus (No. 57-L5) was observed with misshapen eye lenses during fixed head examination. The single occurrences at 300 mg/kg/day does not indicate a test material relationship and consequently both malformations were considered to have occurred by chance. Among visceral variations more fetuses with retrocaval ureter were observed at the highest dose 1000 mg/kg/day (Group 4). Comparison to the historical control data revealed that the mean litter incidence of fetuses did not exceed the upper limit and this observation was therefore considered not related to treatment. Remaining visceral variations occurred across a variety of structures and the low incidences and group distribution of these findings did not indicate any effects of treatment with the test material.
Overall, no developmental toxicity was observed in the 100, 300, and 1000 mg/kg/day groups.
Based on the results of this prenatal developmental toxicity study in rabbits, the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Petroleum Gas Oil Fraction, Co-Processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) were determined to be ≥1000 mg/kg/day in the rabbit.
A supporting OECD Guideline 408/422 combined 90-day repeated dose with the reproduction / developmental toxicity screening study (Charles River Laboratories Den Bosch B.V., 2022) was conducted to determine the potential toxic effects of the test material (Petroleum gas oil fraction, co-processed with renewable hydrocarbons of plant and/or animal origin (EC 941-364-9)). The potential of the test material to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development was also evaluated. The test material was administered once daily to Han Wistar rats (10/sex/dose) via oral gavage in a corn oil vehicle at doses of 0, 100, 300, or 1000 mg/kg/day for a period of 90 days (Charles River Laboratories Den Bosch BV, 2022).
Mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, ophthalmology, estrous cycle, clinical pathology, measurement of thyroid hormones T3, T4, and TSH, gross necropsy findings, organ weights, and histopathologic evaluations were undertaken in the study. Additionally,reproductive toxicity parameters examined included mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, and maternal care while developmental parameters evaluated included sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, and measurement of thyroid hormones T3, T4 and TSH).
Based on lower pup body weights (PND 4 onwards) observed at 1000 mg/kg/day, the developmental toxicity NOAEL was determined to be 300 mg/kg/day.
Read-across
It is noted that any supporting developmental toxicity robust study summaries for read-across substances are all available in the Concawe VHGO dossiers for those substances. For clarity reasons, where data are available on “Petroleum gas oil fraction, co-processed with organic substances of plant and/or animal origin” (EC 941-364-9), the dossier is focused on data generated on the substance itself and there is reference to those robust study summaries in the Concawe dossiers for the remaining read across data which are part of the WoE
Justification for classification or non-classification
The information available currently on reproduction toxicity parameters is insufficient to determine the impact on human fertility. No classification is considered appropriate at this time but a testing proposal based on Read Across substance EC 265 -059 -9 is included for an extended one generation fertility study to meet data requirements for this endpoint.
Classification for developmental toxicity is not considered appropriate based on the data from key OECD 414 developmental studies conducted on substance EC 941-364-9
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
