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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

June 2018

Deviations:

yes

Remarks:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

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.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

May 2008

Deviations:

yes

Remarks:

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

Test guideline 4

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.

Test guideline 5

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.

Test guideline 6

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

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.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

941-364-9

Molecular formula:

Not applicable to UVCB substance

IUPAC Name:

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

Test material form:

other: Low visocosity, liquid hydrocarbon

Details on test material:

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 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

Test animals

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)
- Age at study initiation: males: 6-7 weeks old, females: 6-7 weeks old
- Weight at study initiation: males: 161 and 216 g at onset of treatment, females: 121 and 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

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

corn oil

Remarks:

Sigma-Aldrich (Steinheim, Germany)

Details on oral exposure:

Test item 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 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 and test item. No correction was made for the purity/composition of the test item.

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

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control - corn oil)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 (Low dose)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3 (Intermedate dose)

Doses / concentrations 4

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 item.

- 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.

Examinations

Observations and examinations performed and frequency:

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).

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 animals, 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 5).
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

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.

Statistics:

Please see 'Any other information on materials and methods incl. tables' for information on statistics.

Results and discussion

Results of examinations

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.

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

Description (incidence and severity):

Statistically significant treatment-related organ weight changes were observed in the liver of both sexes, in the thyroid gland and kidney of male rats, and in the adrenal gland of female rats.

- Liver (males and females):
Statistically significant higher liver weights (absolute and relative to body weight) were recorded for male rats starting at 300 mg/kg/day and in female rats at 1000 mg/kg/day. The microscopic correlate at 1000 mg/kg/day was centrilobular hepatocellular hypertrophy. High absolute liver weight observed in female rats at 300 mg/kg/day was regarded to be related to the higher final body weight in this dose group.

- Thyroid gland (males only):
Statistically significant higher thyroid gland weights (absolute and relative to body weight) were recorded for male rats at 1000 mg/kg/day. The microscopic correlate was diffuse hypertrophy of follicular cells with colloid alteration. High absolute thyroid gland weight observed in female rats at 300 mg/kg/day was regarded to be related to the higher final body weight in this dose group.

- Kidney (males only):
Statistically significant higher kidney weights (absolute and relative to body weight) were recorded for male rats at 1000 mg/kg/day. This correlated to microscopic alpha 2µ-globulin nephropathy. High absolute kidney weight observed in female rats at 1000 mg/kg/day was regarded to be related to the slightly higher final body weight in this dose group.

- Adrenal gland (females only):
Statistically significant higher adrenal gland weights (absolute and relative to body weight) were recorded for female rats starting at 300 mg/kg/day. The microscopic correlate was cortical hypertrophy of the zona fasciculata. High absolute adrenal gland weight observed in male rats at 1000 mg/kg/day was regarded to be related to the slightly lower final body weight in this dose group and not regarded a direct treatment-related effect.

- Heart (males only):
Statistically significant organ weight changes observed in the heart of male rats included lower relative weight at 100 mg/kg/day; lower relative weight at 300 mg/kg/day; and lower absolute weight at 1000 mg/kg/day. These observations were considered to be of no toxicological significance as they occurred in the absence of a dose-related trend and without microscopic or macroscopic correlate.

A summary of the findings are presented in Tables 19, 20, and 21.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross necropsy revealed treatment-related macroscopic alterations in the liver of male and female rats; in the kidneys of male rats; and in the adrenal glands of female rats:

- Liver (males and females):
Black-brown discoloration observed in 6/10 males and 2/10 females at 1000 mg/kg/day. There was no microscopic correlate for this discoloration. Enlargement of the liver was observed in 1/10 females at 300 mg/kg/day and 3/10 females at 1000 mg/kg/day. Microscopic correlate for this enlargement was centrilobular hepatocellular hypertrophy and/or cytoplasmic rarefaction.

- Kidney (males only):
Enlargement of the kidney was observed in 1/10 males at 300 mg/kg/day and in 2/10 males at 1000 mg/kg/day. Soft kidneys were observed in 1/10 males at 300 mg/kg/day and greenish discoloration was observed in 1/10 males at 300 mg/kg/day. These necropsy findings correlated with the microscopic alpha 2µ-globulin nephropathy.

- Adrenal gland (females only):
Enlargement of the adrenal gland, associated with cortical hypertrophy of the zona fasciculata was observed in 2/10 females at 1000 mg/kg/day.

The findings are presented in Table 18.

Neuropathological findings:

not examined

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

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Systemic Toxicity

2

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

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

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 Over pre-mating	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 (10^9/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 (mmol/L) [G]	UREA (mmol/L) [G1]	CREAT (µmol/mL) [G1]	GLUC (mmol/L) [G1]	K (mmol/L) [G]	PHOS (mmol/L) [G]	T3 (ng/mL) [G]	T4 (ng/mL) [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 (ratio) [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 2 100 mg/kg/day	Group 3 300 mg/kg/day	Group 4 1000 mg/kg/day
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 Gland^a	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
Liver^a	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 Gland^a	10	10	10	10
Hypertrophy adenohypophyseal cells, multifocal
Minimal	-	-	-	3
Kidney^a	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 Gland^a	10	10	9	10
Hypertrophy cortical, zona fasciculata
Minimal	-	-	2	6
Vagina^a	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.

Applicant's summary and conclusion

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.

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 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.

 

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.

 

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.