Extracts (petroleum), residual oil solvent

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Extracts (petroleum), residual oil solvent

EC Number:

265-110-5

EC Name:

Extracts (petroleum), residual oil solvent

Cas Number:

64742-10-5

Molecular formula:

Not applicable

IUPAC Name:

Extracts (petroleum), residual oil solvent

Test material form:

liquid: viscous

Details on test material:

Concawe sample ref# S709

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation:
6-7 weeks at initiation of administration
- Weight at study initiation: Males 129-170g (target 100-300g) and Females 106-145g (target 100-200g)
- Fasting period before study:
No
- Housing:
Polycarbonate cages ( Makrolon type IV, height 18 cm or Makrolon type 2000P, height 21.5 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
Up to 5 animals of the same sex and same dosing group together.
- Diet (e.g. ad libitum):
Ad libitum, except during designated procedures
- Water (e.g. ad libitum):
Ad libitum, except during designated procedures
- Acclimation period:
12 days

DETAILS OF FOOD AND WATER QUALITY:
Results of analysis for nutritional components and environmental contaminants in feed are provided by the supplier and along with results of analysis of water are on file at the Test Facility. It is considered that there are no known contaminants in the feed or water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
Daily mean 20-22 deg C (Target 18-24 deg C)
- Humidity (%): Daily mean 45-75% (Target 40-70%)
Target value exceeded for 4 days only with a maximum of 75% and without a noticeable effect on the clinical condition of the animals or on the outcome of the study
- Air changes (per hr): Ten or more changes per hour
- Photoperiod (hrs dark / hrs light):
12 hours light/12 hours dark (Except during designated procedures)

IN-LIFE DATES: From: 17/03/2021 To: 29/06/2021

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The dietary route was selected as the test material is poorly soluble and difficult to administer by gavage on a repeated basis

Vehicle:

other: standard powdered diet

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared for use over a maximum of 3 weeks.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used to prepare pelleted diets. The test item was weighed on a layer of powder feed to prevent it sticking to the container. Acetone (in a ratio of 35,8 mL per kg diet) was added in a stepwise manner to facilitate mixing of the test item with the powder diet and to prevent test item from sticking to the walls of the container. Then the bulk of the diet was added in a stepwise manner, while mixing. Water (approximately 15% in total) was added to aid pelleting.
- Storage temperature of food: The pellets were kept for approximately 24 hours at 35°C before final storage,
to dry the pellets and to evaporate the acetone. The control animals received similarly prepared pellets but without the test item. Diets were kept at room temperature in the diet store room in the animal facility until use, if not used on the day of preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet samples were collected for analysis in weeks 1, 6 and 12 of dosing. Homogeneity samples taken for high and low dose groups only with homogeneity results averaged to provide concentration results for these groups. Concentration samples only were taken from the middle dose group.

Samples of approximately 60 g or 250 g were taken from the diets. For determination of accuracy, samples were taken at top, middle and bottom position (90%, 50% and 10% height) or at the random position. The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the diets.

In Week 6 and Week 12, separate samples were analyzed for males and females.

The concentrations analyzed in the diets of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).

The diets of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

A small response at the retention time of the test item was observed in the chromatograms of all Group 1 diets. The response is not considered to derive from the test item, as the same response was observed in blank QC samples and retention time of the observed response is shifted for ~0.2 min earlier from the test item.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Administered in diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Calculated mean intake 105 (males and females) mg/kg/day
Doses adjusted as follows: Weeks 1-4 1150ppm in diet (males and females), weeks 5-8 1450 ppm (males) 1250 ppm (females), weeks 9-13 1800 ppm (males) 1500 ppm (females)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Calculated mean intake 334 (males), 328 (females) mg/kg/day
Doses adjusted as follows: Weeks 1-4 3450ppm in diet (males and females), weeks 5-8 4300 ppm (males) 3800 ppm (females), weeks 9-13 5150 ppm (males) 4450 ppm (females)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Calculated mean intake 1102 (males), 1078 (females) mg/kg/day
Doses adjusted as follows: Weeks 1-4 11500 ppm in diet (males and females), weeks 5-8 14500 ppm (males) 12500 ppm (females), weeks 9-13 15600 ppm (males) 13800 ppm (females)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on the results of a range finding study
- Fasting period before blood sampling for clinical biochemistry: overnight with a maximum of 24 hours
- Other: The amount of test substance incorporated into the diet was adjusted every 4 weeks to approximate a constant intake in terms of mg/kg. Once a week, the group mean body weights and group mean food consumption (treating males and females separately) was used to predict expected food consumption and body weight and to calculate the amount of test item to be included in the diet to achieve the required intake.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
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)
Cageside observations: Once prior to first administration and at least once daily from start of administration onwards, up to the day prior to necropsy

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
weekly from week 1 and throughout the study and on the day of necropsy
- Arena observations: Once before the first administration of the test item and weekly during the Treatment Period.

BODY WEIGHT: Yes
- Time schedule for examinations:
At least weekly, from Day 1 and throughout the study (in order to monitor health status, animals may be weighed more often).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Weekly; from at least Day 1 and throughout the study. Quantitatively measured per cage.

WATER CONSUMPTION: regular basis throughout the study by visual inspection of water bottles

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
Pretreatment Period - All animals once (including spare animals). Dosing Period - during Week 13.
- Dose groups that were examined:
All control and top dose group animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Sampled between 7.00 and 10.30 from the retro-orbital sinus under anesthesia
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes overnight
- How many animals:
all animals
- Parameters below were examined.

White blood cell (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Large unstained cells (LUC) (absolute), Red blood cell (RBC), Reticulocytes (absolute), Red blood cell distribution width (RDW), Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets, Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Sampled between 7.00 and 10.30 from the retro-orbital sinus under anesthesia
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes overnight
- How many animals: all animals
- Parameters checked below were examined.
Alanine aminotransferase (ALT), Triglycerides, Aspartate aminotransferase (AST), HDL and LDL Cholesterol, Alkaline Phosphatase (ALP), Sodium, Total protein, Potassium, Albumin, Chloride, Total Bilirubin, Calcium, Urea, Inorganic Phosphate (Inorg. Phos), Creatinine, Triiodothyronine (T3), Glucose, Thyroxine (T4), Cholesterol, Thyroid-Stimulating Hormone (TSH)

URINALYSIS: Yes
- Time schedule for collection of urine:
Week 12 (all males)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: PAH analysis (non-GLP)


OTHER: Functional Tests
Once during the Dosing Period. The first 5 animals per sex per group during Week 12-13. These tests were performed after clinical observations (including arena observation, if applicable). Procedure: The following tests were performed:
• hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength were recorded as the mean of three measurements.
• locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported.

OTHER: Estrous Stage Determination
End of Treatment - on the day of necropsy, a vaginal smear will be taken to determine the stage of estrus from all females. Procedure:
Estrous stage was evaluated by examining the vaginal cytology of the samples obtained by vaginal smears procedures.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)

Statistics:

Constructed Variables
Body Weight Gains: Calculated between each scheduled interval.
Food Consumption: Calculated between each scheduled interval.
Test Item Intake: Calculated as concentration of test item in diet against food consumption.
Organ Weight Relative to Body Weight: Calculated against the terminal body weight.

Descriptive Statistical Analyses
Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences were reported as appropriate by dataset.

Inferential Statistical Analysis
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted.

Parametric/Non-parametric
Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis
test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Non-Parametric
The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.

ANCOVA
The data corresponding to a response variable of interest and to a related covariate were submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons were conducted using Dunnett’s test.

Incidence
A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test-item related clinical observations were noted.

Other findings (e.g. scabs) noted during the Dosing Period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

Animal No. 34 (male at 1000 mg/kg/day) was euthanized on Day 58 of the study because of severe body weight loss (20% between Days 36 and 58). Hunched posture was noted for a few of days prior to sacrifice.

The cause of the clinical observations leading to the pre-terminal euthanasia were not determined from the pathology evaluation. The most relevant observations in this animal were non-specific and included thin body condition and moderate decreased lymphoid cellularity in the thymus (correlating to small thymus macroscopically), which were consistent with a stress response. Moderate histiocytic infiltrates (multifocal macrophages aggregates) were also noted in the mandibular lymph node. The remainder of the findings were consistent with background findings in the rat. Considering that this animal was dosed at the high dose, a possible relationship to the test item cannot completely be excluded.

No other mortality occurred in this study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight and body weight gain of males at 100 mg/kg bw/day and females at 100 and 300 mg/kg bw/day remained in the same range as controls.

Body weight was decreased in males at 300 and 1000 mg/kg/day and in females at 1000 mg/kg/day from Day 9 onwards.

In males at 300 mg/kg bw/day, mean body weight was maximally 0.93x of control (statistically significant on Day 9 only) and differences at 1000 mg/kg bw/day were 0.88-0.93x of control (statistically significant on most occasions). In addition, body weight gain over the complete study period (Days 1-91) was lower in males at 1000 mg/kg bw/day (0.86x).

In females at 1000 mg/kg bw/day, lower body weights were noted between Days 9 and 91 (0.90-0.92x; statistically significant on most occasions). Body weight gain over Days 1-91 was 0.79x in these females.

Statistically significant decreased body weight gain in males at 100, 300 and 1000 mg/kg bw/day, and any other statistically significant changes in body weight gain were considered to be unrelated to administration of the test item since no trend was apparent regarding dose and/or duration of the administration with the test item.

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 recorded.

Food consumption in males at 100, 300 and 1000 mg/kg bw/day and in females at 300 mg/kg bw/day was slightly higher compared to controls. As this involves an increased in food consumption and no trend was apparent regarding dose, this was considered not toxicologically relevant.

The apparent lower food consumption over Days 78-85 in males of all groups was due to the urine collection on Day 78 and was considered unrelated to administration of the test item.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological parameters were considered unaffected by the administration of the test item in males and females at 100 mg/kg bw/day.

In males, hemoglobin (HGB), hematocrit (HCT) and eosiniophil count (EOS) were decreased and reticulocyte (RETIC) increased at 300 and 1000 mg/kg/day with red blood cell count (RBC) and platelet count (PLT) also decreased and red blood cell distribution width (RDWG) increased at 1000 mg/kg/day only.

In females, eosiniophil count (EOS) was decreased and reticulocyte count (RETIC) was increased at 300 and 1000 mg/kg/day (EOS count not statistically significant at 300 mg/kg/day), with platelet count (PLT), mean corpuscular volume (MCV), hemoglobin (HGB) and hematocrit (HCT) also decreased and large unstained cells count (LUC) increased at 1000 mg/kg/day (HGB and HCT not statistically significant).

Platelet clumps were observed in two males at 100 mg/kg bw/day and one male and one female at 1000 mg/kg bw/day which, at the incidence observed and in the absence of a clear dose-related response, were not considered to be test item related.

Coagulation parameters were considered unaffected by the administration of the test item in males and females up to 1000 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, aspartate aminotransferase (AST) activity and potassium concentration was increased at 300 and 1000 mg/kg bw/day, with urea concentration, colesterol, HDL cholesterol, LDL cholesterol and thyroid stimulating hormone (TSH) increased at 1000 mg/kg/day only.

In females, urea concentration was increased and sodium concentration was decreased at 300 and 1000 mg/kg bw/day, with aspartate aminotransferase (AST) activity, albumin (ALB), cholesterol, HDL cholesterol, LDL cholesterol, potassium concentration and thyroid stimulating hormone (TSH) increased and thyroxine (T4) level was decreased at 1000 mg/kg/day only.

The high level of TSH at 1000 mg/kg bw/day was mainly caused by two males and three females. Individual high TSH levels were also seen in a few females at 100 and 300 mg/kg bw/day.

Remaining differences in clinical chemistry parameters (i.e. total bilirubin, total protein, creatinine, glucose, chloride and calcium), regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Description (incidence and severity):

See "Any other information on results incl. tables" below for details of urine PAH analysis (this was not conducted to GLP and was performed at Biochemical Institute for Environmental Carcinogens, Prof. Dr. Gernot Grimmer-Foundation, Lurup 4, D-22927 Grosshansdorf, Germany).

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The following changes were observed and considered test item related:
• Absolute thymus weight was decreased in males at 1000 mg/kg/day.
• Increased absolute and relative liver weight in males at 1000 mg/kg/day.
• Increased absolute and relative spleen weight in males at 1000 mg/kg/day
• Increased relative liver weight in males at 1000 mg/kg/day
• Increased relative spleen weight in males at 1000 mg/kg/day

There were no macroscopic or microscopic correlates to the liver weight changes. Differences in liver weights noted in males at 100 and 300 mg/kg bw/day were interpreted as not likely test item-related as these were of a small magnitude and not statistically significant as absolute values.

There was no microscopic correlate to the spleen weights; macroscopic enlargement noted macroscopically in one female (Animal No. 80) but was also without histologic correlate.

There was no macroscopic or microscopic correlate to the thymus changes.

Some organ weight differences were statistically significant when compared to the control group but were considered to be the result of a test item-related effect on final body weight (i.e., heart, kidney, spleen, testes of males at 1000 mg/kg bw/day). Lower brain weight in females was in line with the lower body weight of the animals in this group (reflecting slower overall growth). Any other differences, including those that reached statistical significance were considered not to be test item-related due to the direction of the change, lack of doserelated pattern, and/or general overlap and variability in individual values.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Enlargement of the spleen was noted in one female at 1000 mg/kg bw/day (Animal No. 80; no microscopic correlate; high individual weight) and may be test item-related. This correlated with higher individual spleen weight and mean spleen weight in this group was higher than the control females (statistically significant relative to body weight, 30%).

Spleen enlargement in one male at 300 mg/kg bw/day was interpreted as likely not test item related considering the single incidence, lack of spleen weight differences in this group, and absence of this observation at higher dose levels in males.

In one male at 1000 mg/kg bw/day (Animal No. 37) the papillary process of the liver was enlarged, dark red, and hard. These observations were consistent with a partial lobe torsion, which is a common spontaneous lesion in the rat, particularly at this location. The weight of the liver from this animal was high as a result of this spontaneous lesion and not representative of the group, therefore, it was excluded from the calculation and analysis of the group mean.

In the preputial gland of males, discoloration (green or red) and tan foci were observed only in test item-treated animals however these are common background findings in rats and were without any dose relationship and were not considered test item-related.

The few remaining recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain and/or were of low incidence and without a dose relationship.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the thyroid gland starting at 100 mg/kg bw/day in males and 300 mg/kg bw/day in females.

In the thyroid gland an increased incidence and severity of diffuse follicular cell hypertrophy recorded in males and females at 1000 mg/kg bw/day, with colloid alteration recorded in males starting at 100 mg/kg bw/day (increased incidence/severity compared to the controls) and in females starting at 300 mg/kg bw/day. The colloid alteration was visible as basophilic granular or clumped deposits in the colloid-filled follicles of the thyroid gland. The incidence and severity of diffuse follicular cell hypertrophy in a single female at 300 mg/kg bw/day was comparable to the control group and was regarded to be within background.

Possible test item-related findings were noted in the brain and spinal cord of a few females at 300 mg/kg bw/day and 1000 mg/kg bw/day.

Axonal dystrophy was noted in the brain of four females: 1/10 at 300 mg/kg bw/day (Animal No. 61, moderate) and 3/10 at 1000 mg/kg bw/day (Animal Nos. 72 and 80, moderate; Animal No. 78, minimal). This finding was localized to the gracile and cuneate nucleus in the caudal medulla and characterized by the presence of swollen axons (spheroids) bilaterally. Axonal dystrophy was also noted in the associated tracts of the spinal cord (except for Animal No. 78), specifically in the fasiculus gracilus and cuneatus. Axonal dystrophy at this location is a well described background finding in aged rats (Fujisawa and Shiraki 1978; Kaufmann W, 2012), and has also been reported in rats from sub-chronic (90 day) studies (Eisenbrandt et al., 1990). Although the observation is described in the literature, it is infrequently observed at the test facility. Therefore, the occurrence in 1/10 females at 300 mg/kg bw/day and 3/10 females at 1000 mg/kg bw/day is noteworthy and a potential relationship to the test item could not completely be excluded.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

mortality

2

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Results of urine PAH analysis (not conducted to GLP)

Urine PAH Analysis (ug/l)
	Males
mg/kg/day	0	100	300	1000
no examined	5	5	5	5
1+2 Naphthol 1+2 (sum)	1.262	2.425	3.304	4.49
1-Hydroxyphenanthrene	0.281	0.264	0.188	0.144
2-Hydroxyphenanthrene	0.153	0.098	0.081	0.065
3-Hydroxyphenathrene	0.527	0.293	0.216	0.143
4-Hydroxyphenanthrene	0.904	0.509	0.339	0.253
9-Hydroxyphenanthrene	0.253	0.135	0.118	0.092
1-Hydroxypyrene	0.298	0.291	0.206	0.197
1,6+1,8 Dihydroxypyrene (sum)	0.557	0.408	0.323	0.287

Applicant's summary and conclusion

Conclusions:

In conclusion, administration of Residual Aromatic Extract (CAS number 64742-10-5) via diet for at least 90 days resulted in unscheduled death of one male at 1000 mg/kg bw/day and in adverse microscopic findings (axonal dystrophy in the brain and spinal cord) in females at 300 and 1000 mg/kg bw/day. A possible relationship with the test item could not be excluded for both the unscheduled death and the microscopic findings in the brain.

Based on the results of the study, a No Observed Adverse Effect Level (NOAEL) of 300 mg/kg bw/day for males and 100 mg/kg bw/day for females was established.

Executive summary:

Wistar Han rats were treated with Residual Aromatic Extract (CAS number 64742-10-5) for 13 weeks by dietary administration at dose levels of 100, 300 and 1000 mg/kg bw/day. The animals of the control group received the standard rodent diet, alone. Dietary concentrations were adjusted every 4 weeks to maintain a stable mg/kg/day dose.

There was one unscheduled death: Male No. 34 (1000 mg/kg bw/day) was sacrificed unscheduled on Day 58 of the study, based on signs of discomfort and severe body weight loss. The cause of the clinical observations could not be determined from the anatomic pathology evaluation, however, a possible association with the test item cannot be completely excluded.

The lower body weight and body weight gain in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day observed from Day 9 onwards was considered not adverse at the slight severity observed or the absence of corroborative findings.

The slightly higher food consumption in males at 100, 300 and 1000 mg/kg bw/day and females at 300 mg/kg bw/day, might be indicative for a lower food efficiency. At this severity observed, this is considered not adverse.

Changes in hematology parameters in males comprised a decrease in hemoglobin (HGB),  hematocrit (HCT) and eosiniophil count (EOS) with reticulocyte (RETIC) increased at 300 and 1000 mg/kg/day. Red blood cell count (RBC) and platelet count (PLT) also decreased and red blood cell distribution width (RDWG) increased at 1000 mg/kg/day only.

In females, eosiniophil count (EOS) was decreased and reticulocyte count (RETIC) was increased at 300 and 1000 mg/kg/day (EOS count not statistically significant at 300 mg/kg/day), with platelet count (PLT), mean corpuscular volume (MCV), hemoglobin (HGB) and hematocrit (HCT) also decreased and large unstained cells count (LUC) increased at 1000 mg/kg/day (HGB and HCT not statistically significant. In absence of corroborative findings or histopathological correlation, these findings are considered to be not adverse.

Clinical chemistry

In males, aspartate aminotransferase (AST) activity and potassium concentration was increased at 300 and 1000 mg/kg bw/day, with urea concentration, colesterol, HDL cholesterol and LDL cholesterol increased at 1000 mg/kg/day only.

In females, urea concentration was increased and sodium concentration was decreased at 300 and 1000 mg/kg bw/day, with aspartate aminotransferase (AST) activity, albumin (ALB), cholesterol, HDL cholesterol, LDL cholesterol and potassium concentration increased at 1000 mg/kg/day only. In the absence of any corroborative findings or a histopathological correlation, these findings were considered to be not adverse.

Thyroid stimulating hormone (TSH) concentration was increased in males and females at 1000 mg/kg bw/day (2.74x of control in males, 14.57x of control in females). The high level of TSH at 1000 mg/kg bw/day was mainly caused by two males and three females. Individual high TSH levels were also seen in a few females at 100 and 300 mg/kg bw/day. As the change was only noted in a few animals of each group and the majority of the animals had comparable values as the control group and also no clear histopathological correlation was seen, this finding was considered not toxicologically relevant. Thyroxine (T4) level was decreased in females at 1000 mg/kg bw/day (0.62x). At the severity observed, this finding was considered to be not adverse.

Organ weight differences were of low magnitude and mostly without correlates and were considered non-adverse. These included: lower thymus weight in males at 1000 mg/kg bw/day, higher liver weight in males and females at 1000 mg/kg bw/day, and higher mean spleen weight in males and females at 1000 mg/kg bw/day, which correlated with macroscopic with enlarged spleen in one female.

Microscopic changes observed in the thyroid gland starting at 100 mg/kg bw/day in males and 300 mg/kg bw/day in females (colloid alteration) and at 1000 mg/kg bw/day (follicular cell hypertrophy) were without correlative changes in thyroid weight or macroscopic appearance.

Axonal degeneration noted in the brain (cuneate and gracile nuclei) and associated spinal cord tracts (funiculus cuneatus and gracilus) of a few females at 300 mg/kg bw/day (1/10) and 1000 mg/kg bw/day (3/10), was morphologically consistent with a known background finding in rats (Fujisawa and Shiraki 1978; Eisenbrandt et al., 1990, McMartin et al., 1997, Kaufmann W, 2012;). This finding, though well described in the literature, has been observed only rarely

at this facility. The total incidence in this study and the distribution to the mid and high dose levels only are reason for cautious interpretation. These findings may represent a background change with an unfortunate distribution, but a relationship of this adverse finding to the test item cannot be excluded.

No test item-related or toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e., clinical observations, functional observations, ophthalmoscopy, and coagulation parameters).