[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August 2019 - 15 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

According to ECHA Decision No CCH-D-2114453248-46-01/F "modified to include urinalysis and a full histopathological examination which is to include immunohistochemical investigation of renal pathology to determine if the pathology is mediated by alpha-2u globulin nephropathy."

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one

EC Number:

251-020-3

EC Name:

[3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one

Cas Number:

32388-55-9

Molecular formula:

C17H26O

IUPAC Name:

1-((3R,3aR,7R,8aS)-3,6,8,8-tetramethyl-2,3,4,7,8,8a-hexahydro-1H-3a,7-methanoazulen-5-yl)ethanone

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Methyl Cedryl Ketone
- Physical state: Liquid
- Purity test date: 31-07-2012
- Lot/batch No.: 0100172226
- Expiration date of the lot/batch:31-07-2014

- Stability under test conditions: The stability of Methyl Cedryl Ketone and the stability and homogeneity of Methyl Cedryl Ketone in the vehicle were not determined as part of this study.
- Storage condition of test material: Room temperature, in the dark

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Hangzhou Grascent Co., Ltd.; 0100179438
- Expiration date of the lot/batch: 28 February 2021
- Purity: ≥ 75 % (GLC); ≥ 77 % (ketone-test) impurities were not taken into account for calculation of final formulation


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light

- Other information: [3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one = Methyl Cedryl Ketone

Test animals

Species:

rat

Strain:

other: Wistar Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 7-8 weeks old
- Weight at study initiation: males: 181 – 206 g (mean: 193.2 g, ± 20 % = 154.5 – 231.8 g); females: 140 – 170 g (mean: 152.5 g, ± 20 % = 122.0 – 183.0 g)
- Housing: The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: Certificates of food and water are filed for two years at BSL Munich and afterwards archived at Eurofins BioPharma Product Testing Munich GmbH.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22  3 °C
- Humidity (%): 55  10 %
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item formulation or vehicle were administered at a single dose to the animals by oral gavage.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in corn oil. The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed and/or stirred until visual homogeneity was achieved.
Based on the results of stability testing (Eurofins Munich Study No. 191873), the test item formulations were prepared at least every 10 days as given by Eurofins Munich Study No. 191873. The prepared formulation was stored protected from light and at room temperature. Formulates were kept under magnetic stirring during the daily administration.
The application volume for all groups was 4 mL/kg body weight (Table 2). For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was soluble in corn oil.
- Lot/batch no. (if required): MKCH1635 (Sigma Aldrich)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 191873).

Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous according to Eurofins Study No. 191873 (after 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples were taken for substance concentration in study week 1, 5, 9 and in the last week of treatment (16 samples in total).

Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 191874) and until then stored under appropriate conditions based on available stability data. The B-samples were retained at below -15 °C at BSL Munich (test facility) and will be discarded after completion of the final study report.

For a detailed description of the individual findings see Annex 4.

Concentration analysis of formulation samples was determined at three concentrations, 6.25 mg/mL, 20 mg/mL and 62.5 mg/mL in study weeks 1, 5, 9 and in the last week of the study. The mean recoveries observed for the LD dose group was between 94.5 % and 102.2 % of the nominal value, between 84.9% and 100.0 % for the MD dose group and between 91.0 % and 101.4 % of the nominal value for HD dose group. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 99.3 %, 95.7 %, and 96.3 % of the nominal concentration, respectively.

Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %. However, one sample (no. 15, MD week last) did not meet this criterion with a recovery of 84.9 %. The precision of the measurement was high and the validity of the run was confirmed by the quality control samples. Hence the difference appears to be a result of sample preparation, not sample work up.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle (corn oil); Control (C)

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Low dose (LD)

Doses / concentrations 3

Dose / conc.:

80 mg/kg bw/day (nominal)

Remarks:

Mid dose (MD)

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

High dose (HD)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Refer to Supporting, RL1, rat (DRF)/Hangzhou, 2019/Repeated dose toxicity: oral.002

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes; spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes; All animals were observed for clinical signs during the entire treatment period of 90 days. General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

BODY WEIGHT: Yes; The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption was measured weekly during the treatment period.

OPHTHALMOSCOPIC EXAMINATION: Yes; Ophthalmological examination using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period.

HAEMATOLOGY: Yes; Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. The following haematological parameters were examined (Table 3). Coagulation parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes. The following coagulation parameters were examined (Table 4).

CLINICAL CHEMISTRY: Yes; Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examined (Table 5). For an evaluation of test item-related effects on the pituitary-thyroid axis and thyroid hormones, serum samples of all animals were retained at the end of treatment (80 animals) and stored at <-15 °C. T3, T4 and TSH serum levels were determined of main study animals (80 animals; Table 6).

URINALYSIS: Yes; A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. Additionally, urine colour / appearance were recorded. The following parameters (Table 7) were measured using qualitative indicators (Henry Schein Urine Stripes URI 10SL).

FUNCTIONAL OBSERVATIONS: Yes; Once before the first exposure and towards the end of the exposure period but not earlier than in week 11 multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted in all animals.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. One day after the last administration (study day 91) all surviving animals of the treatment period were sacrificed using anaesthesia (ketamine/xylazin) and were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Vaginal smears were examined on the day of necropsy to determine the stage of oestrous cycle.

Organ Weights
The wet weight of the organs (Table 8) of all sacrificed animals was recorded as soon as possible. Paired organs were weighed together. Weight of thyroid/parathyroid glands was measured after fixation. Organ weights of the animal euthanised for animal welfare reasons were not recorded.

HISTOPATHOLOGY: Yes. The following tissues (Table 9) from all animals were preserved in 4 % neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol.

The animal intercurrently euthanised for animal welfare reasons (female animal No. 72 (HD group)) was also subjected to a gross necropsy and the organs preserved for a histopathological examination.
The afore-listed organs (Table 9) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin (HE) staining for the animals of the groups 1 and 4 (control and HD) sacrificed at the end of the treatment period and the animal euthanised before the planned day of sacrifice.

For organs and tissues showing treatment-related changes in the high dose group, these examinations were extended to animals of all other dosage groups.

Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.

The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath Services GmbH, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath Services GmbH, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for histopathology).

Diagnostic Criteria
Histological changes were described, wherever possible, according to distribution, severity and morphologic character. Severity scores were assigned Grade 1 to 5.


Grade 1, Minimal
This corresponds to a histopathologic change ranging from inconspicuous to barely noticeable but so minor, small, or infrequent as to warrant no more than the least assignable grade. For multifocal or diffusely distributed lesions, this grade was used for processes where less than approximately10% of the tissue in an average high-power field was involved.
Grade 2, Slight
This corresponds to a histopathologic change that is a noticeable but not a prominent feature of the tissue. For multifocal or diffusely distributed lesions, this grade was used for processes where between approximately 10% and 25% of the tissue in an average high-power field was involved.
Grade 3, Moderate
This corresponds to a histopathologic change that is a prominent but not a dominant feature of the tissue. For multifocal or diffusely distributed lesions, this grade was used for processes where between approximately 25% and 50% of the tissue in an average high-power field was involved.
Grade 4, Marked
This corresponds to a histopathologic change that is a dominant but not an overwhelming feature of the tissue. For multifocal or diffusely distributed lesions, this grade was used for processes where between approximately 50% and 95% of the tissue in an average high-power field was involved.
Grade 5, Severe
This corresponds to a histopathologic change that is an overwhelming feature of the tissue. For multifocal or diffusely distributed lesions, this grade was used for processes where greater than approximately 95% of the tissue in an average high-power field was involved.




 

Other examinations:

Analysis of alpha2u-globulin in Kidneys
The processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath Services GmbH, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Selected samples were embedded in paraffin, cut at a thickness of 4 µm and stained with HE.

An indirect immunohistochemistry optimised method using the anti-alpha2u-globulin (primary antibody) and BOND Polymer Refine Detection system (containing secondary Rabbit anti mouse IgG, HRP conjugated anti-rabbit Polymer-IgG and the substrate chromogen, 3,3’-Diaminobenzidine tetrahydrochloride hydrate (DAB)) was performed on the kidneys from all the male rats and from females of the control and high dose group using Leica Bond-III system, automated immunohistochemistry stainer.

IHC slides were evaluated using light microscopy and a semi-quantitative method for the analysis of immunostaining. The number and intensity of specifically stained cells was assessed accordingly to their severity and distribution and entered into PathData system: negative (0, no stained cells), minimal (1, rare cells with low staining intensity), slight (2, several cells with low/medium staining intensity), moderate (3, many cells with medium/high staining intensity), marked (4, significant numbers of cells with high staining intensity), diffuse (5, mostly all the cellular structures are immunolabeled).

Histomorphometry by Image Analysis
One immunohistochemistry kidney section from all males in the study and females only from the Control and High Dose groups (plus the positive and negative control blocks), were imaged scanned by a Märzhäuser motorized microscope stage and an Olympus XC30 camera mounted on an Olympus BX43 microscope system at 4x magnification. Quantitative evaluation was made in Olympus imaging and image analysis software cellSens v1.18. The cortex of the kidney was defined as a region of interest. The area of positive anti-alpha2u-globulin immunohistochemistry staining was measured in the region of interest using pixel thresholds by HUE, saturation, and intensity. Illustrative images with measurements are displayed in Figure 1 and Figure 2 (Annex 3).

Arithmetic mean values of the percentage positive area of the cortex were used for further descriptive statistics. Statistical tests were performed using GraphPad Prism 8. The Shapiro-Wilk test for normality was performed. When the data was following normal distribution, the comparisons would be performed with the unpaired t-test. When data did not follow normal distribution, the Mann-Whitney test was used (p-values <0.05 were considered significant).
 

Statistics:

Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next.
The relative organ weights were calculated in relation to the brain weight and in relation to the body weight (measured at necropsy) and are presented as percentage.

All results are reported in a tabular form (summarised in mean or summary tables and/or listed in individual data tables).

Toxicology and pathology data was captured either on paper according to appropriate SOPs or using the validated computerised system Ascentos® System (version 1.3.4, Pathology Data Systems Ltd.).

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Slight to severe salivation was noted in all males of the HD group and in 1/10 of the MD group. Slight to severe salivation was noted in all females of the HD group, in 2/10 of the MD group and in 1/10 in the LD group. Furthermore, moving the bedding observed in all males and all females of the HD group. Clinical symptoms like hairless area and scratch/cut were observed mostly transiently and within the normal background frequency. Female animal no. 72 (HD group) was euthanised in a moribund condition showing severe reduces spontaneous activity, abnormal breathing and nasal discharge. No treatment related clinical signs were recorded in the control group. (Annex 1).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female animal No. 72 (HD group) was euthanised in a moribund condition on study day 14. The animal showed severe reduces spontaneous activity, abnormal breathing and nasal discharge. The remaining animals survived the scheduled period (Annex 1).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight of male and female treated groups and the control group increased during the treatment period (day 1 compared to day 90), however there was statistically significant decrease in the male HD group starting from day 8 to day 90 compared to the control group. Mean body weight in males was decreased between ~6 % (p < 0.05) and ~21 % (p < 0.001) (day 8-90) in HD group when compared to control. In females, the mean body weight was statistically significantly decreased on day 29 and 36 only (~8 % and ~6 % respectively, below control (p < 0.05)). The mean body weights of male and female HD animals were within the range of the historical control data (Annex 5).

Overall, the body weight change (day 1-90) of the treated group of males showed a decrease in body weight with dose dependency and statistical significance only in the HD group. The change was a statistically significant decrease in the male treated groups (LD -~8 %, MD -~11 % and HD -~48 % (p< 0.001) respectively, below control). In females, the overall body weight change (day 1-90) was decreased in HD group (~16%) without statistical significance. The weekly body weight change during the treatment period showed statistical significance decrease in the male HD group in week 1, 3, 4, 7-9 (~48 %, ~53 %, ~59 %, ~154 %, ~64 %, ~105 % respectively, below control) and an increased change without statistical significance was found in the male HD group in week 11 (~431 % above control). In females, a decreased body weight change with statistical significance was found in the HD group on week 4 (~120 % above control). The statistical significances in the female HD groups were not considered to be an effect of the test item and there were no dose dependent changes noted between the treated groups of females (Annex 1).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males of the HD group, decrease in mean food consumption (n=2 cages) was observed on week 5, 9, 11 (~11 %, ~34 % and ~11 % respectively, below control) and in LD group lower mean food consumption was observed on week 5 (~15 % below control) and higher consumption on week 7 (~55 % above control) when compared to control. In MD male, higher mean food consumption was observed on week 3 (~16 % above control) when compared to control. In the female HD group, decrease in mean food consumption was noted on week 1, 4-5, 12-13 (~15 %, ~16-~17 % and ~10-~13 % respectively, below control) and higher mean food consumption on week 9-10 (~54-~57 % above control) when compared to control. Single incidence of lower mean food consumption was observed in LD group on week 1, 8 and 12 (~10 %, ~15 % and ~10 %, below control) and in MD group on week 7 (~12 % below control) when compared to control.

Overall, the mean total food consumption (day 1-90) was found to be comparable in all male and female dose groups when compared to the control group. (Annex 1)..

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmologic findings were observed in any of the animals of this study.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In haematology a statistically significant decrease in male and female HD animals in RBC (~10 % (p < 0.001) and ~7 % (p < 0.05) below controls), in male and female HD animals in HGB (~7 % (p < 0.01) and ~6 % (p < 0.01) below controls), in male HD and in female MD and HD animals in HCT (~11 % (p < 0.001), ~6 % (p < 0.05) and ~9 % (p < 0.001), respectively, below controls) and in male MD in MCV (~3 % (p < 0.05) below control) were found.

A statistically significant increase in male (~2 % (p < 0.05) above control) and female MD (~3 % (p < 0.01) above control) animals and male (~4 % (p < 0.001) above control) and female (~3 % (p < 0.001) above control) HD animals in MCHC, in male HD and in female MD and HD animals in RET (~48 % (p < 0.05), ~34 % (p < 0.01) and ~106 % (p < 0.001), respectively, above controls), in male HD and in female MD and HD animals in PLT (~37 % (p < 0.001), ~21 % (p < 0.01) and ~29 % (p < 0.001), respectively, above controls), in male LD in LYM (~11 % (p < 0.01) below control) and NEUT ( ~46 % (p < 0.01) above control) were found. All values were within the range of the historical control data except for the PLT in male HD group which was slightly above (Annex 1).

In blood coagulation a statistically significant increase in male HD animals in PT (~ 11 % (p < 0.001) above control) was detected (Annex 1). This value lies within the historical control data.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the observation period a statistically significant decrease in male MD and female MD and HD animals in ALAT (~43 % (p < 0.001), ~44 % (p < 0.001) and ~45 % (p < 0.001), respectively, below controls), in male LD, MD and HD animals in Gluc (~15 % (p < 0.05), ~25 % (p < 0.001) and ~42 % (p < 0.001), respectively, below control), in male and female HD animals in Crea (~22 % (p < 0.05) and ~34 % (p < 0.001) below controls), in male HD animals in TP (~ 8 % (p < 0.001) below control), in male MD and HD animals in ALB (~5 % (p < 0.05) and ~8 % (p < 0.001) below control), in male LD, MD and HD animals in CHOL (~33 % (p < 0.001) and in HD animals ~29% (p < 0.001), respectively, below control), in male HD animals in TBA (~59 % (p < 0.05) above control), in male LD, MD and HD and in female MD animals in TG (~35 % (p < 0.01), ~41 %(p < 0.001), ~34% (p < 0.01) and ~48 % (p < 0.001), respectively, below control) and in male LD, MD, HD animals in HDL and LDL (~27 % (p < 0.001), ~26 % (p < 0.001). ~20 % (p < 0.01), ~43 % (p < 0.001), ~27 % (p < 0.001) and ~47 % (p < 0.001), respectively, below controls) were found. A statistically significant increase in male MD and HD and female LD, MD and HD animals in TBIL (~68 % (p < 0.01), ~331 % (p < 0.001), ~25 % (p < 0.05), ~90 % (p < 0.001) and ~209 % (p < 0.001), respectively, above controls), in male HD animals in Urea (~ 38 % (p < 0.01) above control) and in female HD animals in K (~12 % (p < 0.05) above control) were found (Annex 1).

A statistically significant decrease was found in male HD animals only in T3 (p < 0.01) and T4 (p < 0.001) hormone analysis. No statistically significant difference was found in the TSH hormone in male and female animals when compared to the control (Annex 1)..

All values were within the range of the historical control data except for TBIL in male and female MD and HD groups which were above.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The test item had no toxicologically relevant effect on all urinary parameters analysed, on the day of necropsy (study day 91) with exception of an increase in specific gravity in female MD and HD animals (Annex 1).

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test item related effects were observed in any parameter of the functional observation battery at the end of the treatment period when compared to observations before treatment except in study week 13 (Annex 1 & 2).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In male animals a statistically significant higher mean brain organ/body weight ratio was seen for the HD group (~26 % (p < 0.001) above control). A statistically significant higher mean spleen organ/body weight ratio was seen for the HD group (~38 % (p < 0.001) above control). A statistically significant lower mean thymus weight and organ/brain weight ratio was seen for the HD group (~25 % (p < 0.01) and ~22 % (p < 0.05) below control). A statistically significant higher mean testes organ/body weight ratio was seen for the HD group (~30 % (p < 0.001) above control). A statistically significant higher mean kidney weight, mean organ/body weight ratio and mean organ/brain ratio was seen for the HD group (~25 % (p < 0.001), ~62 % (p < 0.001) and ~29 % (p < 0.001), respectively, above control) and a mean organ/body weight ratio was seen for the MD group (~21% (p < 0.001) above control). A statistically significant lower mean pituitary gland weight and mean organ/brain weight ratio was seen for the HD group (~27 % (p < 0.01) and ~25 % (p < 0.01) below control). A statistically significant lower mean heart weight and mean organ/brain weight ratio was seen for the HD group (~19 % (p < 0.001) and ~17 % (p < 0.01) below control). A statistically significant lower mean epididymides weight and mean organ/brain weight ratio was seen for the HD group (~24 % (p < 0.01) and ~22 % (p < 0.01) below control). A statistically significant lower mean prostate weight and mean organ/brain weight ratio was seen for the HD group (~30 % (p < 0.001) and ~28 % (p < 0.001) below control). A statistically significant higher mean liver weight, mean organ/body weight ratio and mean organ/brain ratio was seen for the MD and HD groups (~20 % (p < 0.001), ~26 % (p < 0.001) and ~19 % (p < 0.01), ~35 % (p < 0.001), ~75 % (p < 0.001) and ~39 % (p < 0.001), respectively, above control) (Annex 1).

In female animals statistically significant higher mean spleen weight, mean organ/body weight ratio and mean organ/brain ratio was seen for the HD group (~21 % (p < 0.01), ~31 % (p < 0.001) and ~22 % (p < 0.01), respectively, above control) and a mean organ/body weight ratio and a mean organ/brain weight ratio was seen for the MD group (~16 % (p < 0.05) and ~15% (p < 0.05) above control). A statistically significant higher mean kidney weight, mean organ/body weight ratio and mean organ/brain ratio was seen for the MD and HD group (~12 % (p < 0.001), ~ 14 % (p < 0.01), ~14 % (p < 0.001), ~14 % (p < 0.001), ~24 % (p < 0.001) and ~16 % (p < 0.001), respectively, above control) and a mean weight and a mean organ/brain weight ratio was seen for the LD group (~8 % (p < 0.05) and ~11 % (p < 0.05) above control). A statistically significant lower mean adrenal gland weight was seen for the MD and HD group (~14 % (p < 0.05) and ~ 18 % (p < 0.01) below control) and lower mean organ/brain weight ratio was seen for the HD group (~16 % (p < 0.05) below control). A statistically significant higher mean liver weight, mean organ/body weight ratio and mean organ/brain ratio was seen for the LD, MD and HD group (~15 % (p < 0.01), ~14 % (p < 0.01), ~18 % (p < 0.05), ~28 % (p < 0.001), ~31 % (p < 0.001), ~31 % (p < 0.001), ~56 % (p < 0.001), ~69 % (p < 0.001) and ~58 % (p < 0.001), respectively, above control). (Annex 1).

All values were within the range of the historical control data except for the liver and kidneys mean weight in male HD group and the liver mean weight in female MD and HD groups which were above
 

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of the animals at necropsy listed in male animal no. 31 (HD group) a depressed, dark, glandular mucosa surface of the stomach, in male animal no. 19 (LD group) an abnormal dark red color in the thymus, in male animal no. 26 (MD group) abnormal spotted color in the thymus, in female animals no. 67 (MD group) a diaphragmal herniation in the liver, in animal no. 71 (HD group) an abnormal white color in the ovaries, in female animal no. 50 (C group) a fluidfilled uterus .

The female animal no. 72 (HD group) euthanized on study day 14 listed a dark red fluid filled thoracic cavity and a cyst (0.2 – 0.6 cm) in the uterus (Annex 1).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Refer to Annex 3 for figures. Tables are presented below.

Liver
Half of the HD male rats and one female presented minimal foci of single cell death often associated with multifocal, hepatocellular fatty change. The fatty change was multifocal and characterized by hepatocytes presenting variable-sized, clear, well-defined vacuoles within the cytoplasm (Table 3). Hepatocellular fatty change was also detected in the control dose and intermediate LD and MD groups from both sexes, but not associated with further degenerative changes in hepatocytes.

Several HD males and females also presented multifocal, well-circumscribed, non-encapsulated, small (up to 500 µm in diameter) eosinophilic foci, where clusters of hepatocytes showed an enlarged, light, pale pink cytoplasm. Multifocally, most of the HD males and one female at presented enlarged nuclei (minimal to slight karyomegaly), with coarse chromatin and prominent nucleoli.

Most of the HD males and females presented with a proliferation of bile ducts (hyperplasia) at portal spaces which exceeded in severity and frequency the presence of this finding in the control group. This finding in MD males and females was similar in severity and frequency to the control group.

Several treated HD males and females showed induced centrilobular hepatocellular hypertrophy, characterized by hepatocytes with enlarged and homogeneous cytoplasm and compression of sinusoidal spaces.

Thyroid gland
The thyroid gland from all the HD males and most of the females presented diffuse, bilateral, areas of follicular cell hypertrophy together with hypercellularity and increase in number of follicles (follicular hyperplasia), with a predominance of follicles in reabsorption phase (Table 5). The follicular epithelium presented intracytoplasmic vacuolation, variable height (up to 10 µm), with occasional cellular crowding and minimal papillary protrusion into the lumen. This lesion was accompanied by a colloid alteration: paler, basophilic colour of the colloid with pyknotic clumps. The association of follicular hypertrophy with hyperplasia led to include them in the same diagnosis. These findings were not present in the LD and MD males and females.

Kidney
The kidneys of the following groups were analysed with an optimized immunohistochemistry technique: all control, LD, MD and HD males, all control and HD females.

Anti-alpha2u-globulin IHC was validated by specific positive immunolabeling in the positive control slide (Figure 9); in contrast to the lack of positive immunolabelling for the negative IHC control slide (Figure 10). There was a very mild, unspecific, background immunolabeling observed in the glomeruli, apical border of the renal tubules, interstitium and some vessels from all the animals/groups.

In most of the males from the control group and all treated groups, there were large deposits of an amorphous, acellular, bright, eosinophilic, up to 40 µm, proteinaceous deposits (hyaline droplets), which were distorting the cytoplasm from the tubular cells of the proximal segment in the renal cortex or intraluminal. In addition, in males from all treated groups, there were abundant, smaller (up to 5 µm), eosinophilic, parallel, round to elongated, proteinaceous bodies (eosinophilic bodies) at the basal levels of the renal epithelium in the cortex, which exceeded in frequency and severity the background levels shown in control group (Figure 6 to Figure 8, and Figure 11 to Figure 15, Table 4). These proteinaceous deposits were not observed in any female group (Figure 16, Figure 17, Table 4). All HD males presented an increase in mineralization in the tubular lumen, mostly in the outer medulla, compare to the control group. Tubular basophilia in the renal cortex exceeded background levels from the control group in males exposed to ≥ LD and HD females. The presence of intratubular casts appeared with more frequency or severity in LD and MD male groups compared to females, and to males from control or high dose groups.

Immunohistochemistry results showed positive immunolabelling against alpha2u-globulin in all males from the control and treated groups, whereas no specific immunolabelling was observed in females. The positive alpha2u-globulin immunolabelling was greater at LD and MD compared to the control group and high dose groups. The alpha2u-globulin positive signal in the intermediate dose groups was found targeting most of the proteinaceous deposits: the hyaline droplets and elongated, eosinophilic bodies. In high dose group, most of the hyaline droplets were alpha2u-globulin positive whereas a large fraction of the elongated proteinaceous eosinophilic bodies was not alpha2u-globulin immunoreactive (Figure 15).

The individual image analysis data are presented in Table 6 and the group arithmetic means are summarized in Table 7.

The “Mean % Positive alpha2u-globulin Area (%)” values from the groups followed a normal distribution and unpaired T-test was applied to investigate the statistical significance level. Medium dose group was the only exception were values were not following a normal distribution, where Mann-Whitney test was applied (Table 8-Table 10). Male groups presented specific positive immunolabelling against alpha2u-globulin, higher at LD and MD, followed by the control and then the high dose group. The increase in alpha2u-globulin positive vs Control group was statistically significant in LD males but was statistically non-significant in MD males (Table 10). The decrease in alpha2u-globulin immunolabelling of the high dose group vs control was also considered statistically significant. Female control and high dose group did not show positive specific immunolabelling against alpha2u-globulin, and the detection of the positive signal were associated with background/non-specific stain. The changes in males were considered characteristic early changes associated with alpha2u-globulin nephropathy.

Illustrative images showing the difference between the groups can be observed in Figures 20-24.

 

Histopathological findings: neoplastic:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

25 other: mg/kg bw/day; In males only (alpha-2u-globulin-mediated nephropathy)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

no

Any other information on results incl. tables

Table 3: Incidence and mean severity of relevant microscopic findings in the Liver, only survivors

Findings in the Liver	Treatment Period Dose (mg/kg bw/day)
	0		25		80			250
Microscopic findings	Affected / Mean Grade
Number of animals	10 (M)	10 (F)	10 (M)	10 (F)	10 (M)	10 (F)	10 (M)		9 (F)
Fatty change	5/1.4	4/1.0	9/1.0	9/1.0	8/1.1	5/1.2	8/1.4		5/1.2
Single cell death, hepatocell.	-	-	-	-	-	-	5/1.0		1/1.0
Karyomegaly, hepatocellular	-	-	-	-	-	-	8/1.3		1/1.0
Hyperplasia, bile duct	2/1.0	2/1.0	-	-	2/1.0	1/1.0	9/1.2		7/1.3
Eosinophilic foci, hepatocell.	-	-	-	-	-	-	3/1.0		5/1.4
Hypertrophy, hepatocellular	-	-	-	-	-	-	8/1.6		8/1.5

Table 4: Incidence and mean severity of microscopic findings in the kidneys, only survivors.

Findings in the Kidneys	Treatment Period Dose (mg/kg bw/day)
	0		25		80			250
Microscopic findings	Affected / Mean Grade
Number of animals	10 (M)	10 (F)	10 (M)	10 (F)	10 (M)	10 (F)	10 (M)		9 (F)
Hyaline droplets	9/1.4	-	10/1.5	-	10/1.7	-	10/1.2		-
Eosinophilic bodies	3/1.0	-	10/2.6	-	10/2.8	-	10/2.5		-
Cast, intratubular	-	-	3/1.0	-	2/2.0	1/1.0	1/1.0		-
Tubular basophilia	7/1.1	4/1.0	9/1.6	5/1.0	8/1.6	5/1.0	10/2.0		8/1.6
Mineralization, tubular	2/1.5	-	2/1.0	-	1/1.0	1/1.0	10/1.4		1/1.0

Table 5: Incidence and mean severity of relevant microscopic findings in the thyroid gland, only survivors

Findings in the Thyroid Gland	Treatment Period Dose (mg/kg bw/day)
	0		25		80			250
Microscopic findings	Affected / Mean Grade
Number of animals	10 (M)	10 (F)	10 (M)	10 (F)	10 (M)	10 (F)	10 (M)		9 (F)
Hypertrophy/hyperplasia, follicular cell	-	-	-	-	-	-	10/2.9		8/3.0

Table 6: Individual sample data of kidney cortex area, positive alpha2u-globulin

area and percentage positive alpha2u-globulin area of the kidney cortex area.

Group Name	Sex	Animal No.	Kidney Cortex Area (µm²)	Positive alpha2u-globulin Area (µm²)	Positive alpha2u-globulin
					Area (%)
Control	Male	1	38728731.8	9460.1	0.02%
		2	52790768.2	432766.31	0.82%
		3	51295307.1	13129.93	0.03%
		4	17877970.9	53187.85	0.30%
		5	27275907.3	73469.01	0.27%
		6	46696084	139710.65	0.30%
		7	48381523.7	462873.03	0.96%
		8	55406655.5	386263.34	0.70%
		9	45352378.5	197341.13	0.44%
		10	57575647.3	1051877.6	1.83%
Low Dose	Male	11	54669618.5	1462280.3	2.68%
		12	53195422.6	1908886.6	3.59%
		13	65788617.6	174459.87	0.27%
		14	64408455.5	644023.49	1.00%
		15	46226033.1	423896.27	0.92%
		16	46034508.8	432162.91	0.94%
		17	52474357.4	872893.22	1.66%
		18	51542463.2	539665.45	1.05%
		19	47355426.3	1353946	2.86%
		20	38237244.3	644802	1.69%
Medium Dose	Male	21	62940651.3	1988745.4	3.16%
		22	51948876.4	293246	0.56%
		23	54768511.8	1306948.2	2.39%
		24	62477755.5	676534.26	1.08%
		25	52889692	257855.32	0.49%
		26	62401241	311629.43	0.50%
		27	68802853.2	218075.31	0.32%
		28	57998574.7	341818	0.59%
		29	56781173.9	430029.16	0.76%
		30	49987778.4	481610.5	0.96%
High Dose	Male	31	57686591.3	58869.62	0.10%
		32	47696395.4	50816.16	0.11%
		33	59245111.5	53404.84	0.09%
		34	52794586.5	36237.71	0.07%
		35	58219756.4	43742.97	0.08%
		36	52971758.8	10305.23	0.02%
		37	54976253.3	62035.04	0.11%
		38	46124817.6	24826.58	0.05%
		39	52240314.1	56701.6	0.11%
		40	57686893.9	78374.18	0.14%
Control	Female	41	29202716.6	11024.73	0.04%
		42	26448723.3	10097.75	0.04%
		43	27261753.3	2310.78	0.01%
		44	32978699.7	8205.73	0.03%
		45	34184512.4	9166.97	0.03%
		46	37767933.9	40661.3	0.11%
		47	30729441.8	23543.66	0.08%
		48	28784938	22148.44	0.08%
		49	37395198.3	10817.25	0.03%
		50	37017844.9	1157.29	0.00%
High Dose	Female	71	34213482.7	16202.09	0.05%
		72	34646437.5	7581.4	0.02%
		73	37407498.3	11913.64	0.03%
		74	34578003.1	20338.27	0.06%
		75	31793076.9	4351.27	0.01%
		76	43829677.2	11788.01	0.03%
		77	35070895.3	10406.11	0.03%
		78	44861389.7	10874.36	0.02%
		79	31183736	29033.18	0.09%
		80	37159933	14548	0.04%
Negative control	Male	na	48854635.3	6334.65	0.01%
Positive control	Male	na	33208218.5	112874.04	0.34%

 Na= not applied

Table 7: Summarized group arithmetic mean of kidney cortex area,

positive alpha2u-globulin area and percentage positive alpha2u-globulin

area of the kidney cortex area.

Group Name	Sex	Animal No.	Mean Kidney Cortex Area (µm²)	Mean Positive anti-α2 globulin	Mean % Positive anti-α2 globulin	Standard deviation
				Area (µm²)	Area (%)
C	Male	1-10	44138097.42	282007.892	0.57%	0.54%
LD	Male	11-20	53726750.24	770748.68	1.44%	1.05%
MD	Male	21-30	580997108.1	6306491.56	1.08%	0.94%
HD	Male	31-40	539642478.8	475313.92	0.09%	0.03%
C	Female	41-50	51717268.53	38906.14	0.08%	0.03%
HD	Female	71-40	36474412.97	13703.63	0.04%	0.02%

Table 8: Column Statistics for Normality Test of Male Groups.

Group	C	LD	MD	HD
Number of values	10	10	10	10
Minimum	0,02400	0,2650	0,3170	0,01900
25% Percentile	0,2083	0,9335	0,4963	0,06525
Median	0,3670	1,355	0,6730	0,09600
75% Percentile	0,8543	2,721	1,409	0,1100
Maximum	1,827	3,588	3,160	0,1360
Range	1,803	3,323	2,843	0,1170
Mean	0,5652	1,664	1,081	0,08740
Std. Deviation	0,5435	1,055	0,9385	0,03395
Std. Error of Mean	0,1719	0,3336	0,2968	0,01074
Lower 95% CI of mean	0,1764	0,9093	0,4092	0,06311
Upper 95% CI of mean	0,9540	2,419	1,752	0,1117
Sum	5,652	16,64	10,81	0,8740
Shapiro-Wilk normality test	C	LD	MD	HD
W	0,8613	0,9145	0,7488	0,9546
P value	0,0791	0,3135	0,0034	0,7235
Passed normality test (alpha≥0.05)?	Yes	Yes	No	Yes

Table 9:  Column Statistics for Normality Test of Female Groups.

Group	C	HD
Number of values	10	10
Minimum	0,003000	0,01400
25% Percentile	0,02075	0,02350
Median	0,03350	0,03100
75% Percentile	0,07700	0,05000
Maximum	0,1080	0,09300
Range	0,1050	0,07900
Mean	0,04300	0,03870
Std. Deviation	0,03365	0,02307
Std. Error of Mean	0,01064	0,007297
Lower 95% CI of mean	0,01893	0,02219
Upper 95% CI of mean	0,06707	0,05521
Sum	0,4300	0,3870
Shapiro-Wilk normality test	C	HD
W	0,9008	0,8542
P value	0,2234	0,0651
Passed normality test (alpha≥0.05)?	Yes	Yes

Table 10: P values.

Groups compared: Control - High dose	p-value	Significance
Control Male-Low dose male	0,0090	Yes
Control Male-Medium dose male	0,0753	No
Control Male-High dose male	0,0125	Yes
Control female- High dose Female	0,7428	No

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study and based on the pathology evaluation, the NOAEL for a repeated exposure to [3R-(3alpha,3abeta,7beta,8aalpha)] -1 - (2,3,4,7,8,8a-hexahydro- 3,6,8,8-tetramethyl- 1H-3a, 7-methanoazulen-5-yl) ethan-1-one by oral gavage over a period of 90 days in male and female Wistar rats, can be established at 80 mg/kg bw/day.

Executive summary:

In a repeated dose toxicity study (191870), [3R-(3alpha,3abeta,7beta,8aalpha)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a, 7-methanoazulen-5-yl) ethan-1-one was administered to Wistar Crl: WI(Han) rats (10/sex/dose) by gavage in corn oil at dose levels of 0, 25 (LD), 80 (MD) and 250 (HD) mg/kg bw/day daily for 90 days.

 

Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.

The body weight of HD male animals was statistically significantly reduced from study day 8 until the end of treatment. The body weight change of HD male animals was statistically significantly reduced compared to the control group. The body weight of HD female animals was statistically significantly reduced from study day 29 and 36 only. The body weight change of HD female animals was not statistically significantly reduced compared to the control group. The mean body weights of male and female HD animals were within the range of the historical control data.

No treatment related or toxicologically relevant effects on body weight gain, food consumption, clinical observations, functional observation battery, ophthalmology, haematological parameters, blood coagulation, clinical biochemistry, urine, thyroid hormones or reproductive organs were observed during the treatment period. The male HD PLT count, male and female MD and HD TBIL and male HD mean kidney weight and male HD and female MD and HD mean liver weights were above the historical control data and are considered to be test item related.

Macroscopic findings during necropsy were considered to be incidental and not test item related.

Under the conditions of this study, after a repeated exposure to [3R-(3alpha,3abeta,7beta,8aalpha)] -1 - (2,3,4,7,8,8a-hexahydro- 3,6,8,8-tetramethyl- 1H-3a, 7-methanoazulen-5-yl) ethan-1-one by oral gavage over a period of 90 days in Wistar rats, there were induced findings in the liver, thyroid and kidney in HD males and females. In the liver, the fatty change associated with single cell death observed in some HD males and one female, was considered an adverse finding caused by the Test Item. In the intermediate doses (LD and MD), there was also a subtle increase in severity or frequency levels of fatty changes compare to control group but in absence of other degenerative changes, therefore it was considered an adaptative finding at these dosages. The karyomegaly observed in most of the males and in one HD female was also deemed to be a treatment induced adaptative finding. Karyomegaly is considered often a reflection of hepatocyte polyploidy that occurs when there is duplication of nuclear material in the absence of cytokinesis. Also, in several HD males and females, the presence of well-defined, eosinophilic foci of hepatocellular alteration and the increase in frequency and severity of bile duct hyperplasia were deemed to be induced by the treatment. The eosinophilic foci are considered to represent a localized proliferation of hepatocytes that are phenotypically different from surrounding hepatocyte parenchyma and could represent preneoplastic lesion. Hepatocellular hypertrophy observed in some HD males and females was deemed to be an adaptative Test Item induced effect, which would probably recover after withdrawn of the treatment. This often occurs secondary to increase in microsomal enzymes following exposure to enzyme inducing xenobiotics.

In the thyroid gland, the follicular hypertrophy associated with alterations in the colloid in HD males and females only was considered a test item-related change. These findings were not present in the LD and MD males and females. In rats, liver changes (hepatocellular hypertrophy) may induce an increase in thyroid hormones degradation and an increased in TSH leading to thyroid gland hypertrophy. This phenomenon is more rat specific and of less toxicologic relevance in humans. Both, follicular cell hyperplasia and hypertrophy are expected to return to normal if the stimulus is withdrawn. A statistically significant decrease in T3 (p < 0.01) and T4 (p < 0.001) was found in HD male animals only. However, all values were within the range of the historical control data. There were no statistically significant changes in the level of TSH in male or females at any dose and no statistically significant changes in (para)thyroid weight in males or females at any dose group.

In the kidney of the males from all the groups, the hyaline droplets and the elongated, eosinophilic bodies at the basal level of the renal epithelium were observed. Both were considered proteinaceous deposits accumulated in the cytoplasm from renal tubular epithelial cells. On haematoxylin and eosin (HE) pathology evaluation, there was an increase (in frequency or severity) of proteinaceous deposits in all treated groups compare to the control group, especially for the elongated eosinophilic bodies at the basal area. This was considered a Test Item induced effect. Tubular basophilia in HD females, in absence of further degenerative changes, was considered in this case most likely an adaptative response.

An indirect immunohistochemistry optimized method using the anti-alpha2u-globulin (primary antibody) and BOND Polymer Refine Detection system (containing secondary Rabbit anti mouse IgG, HRP conjugated anti-rabbit Polymer-IgG and the substrate chromogen, 3,3’-Diaminobenzidine tetrahydrochloride hydrate (DAB)) was performed on the kidneys from all the male rats and from females of the control and high dose group using Leica Bond-III system, automated immunohistochemistry stainer. Anti-alpha2u-globulin IHC was validated by specific positive immunolabeling in the positive control slide; in contrast to the lack of positive immunolabelling for the negative IHC control slide.

With immunohistochemistry, alpha2u-globulin positive signal was detected on the proteinaceous deposits (i.e. hyaline droplets and the elongated, eosinophilic bodies) in all the male groups, which demonstrated the accumulation of this protein in renal tubules. After image analysis evaluation, the increase in the percentage of positive alpha2u-globulin signal in the intermediate doses compared the controls, revealed an induced increase in accumulation of alpha2u-globulin related to the treatment at these dosages, however these differences were found to be statistically different only at the LD compared to control group. In comparative evaluation, image analysis showed that the HD male group presented a decrease in the presence of alpha2u-globulin-positive signal compare to the control group and intermediate doses. The most likely hypothesis is that the induced hepatic damage at the high dose, may impair the hepatic production of alpha2u-globulin decreasing progressively the circulating level, and therefore decreasing the renal accumulation. There may also be an overloading of the metabolic pathway and therefore a decrease in the reduction of MCK and subsequent conjugation with glucuronic acid and/or alpha2u-globulin. The glucuronic pathway can be overloaded for non-acidic substances (Howell et al., 1986); possibly the alpha-2u pathway can become overloaded also.

Alpha2u-globulin is a circulating low molecular protein which is synthesized in the liver of male rats only, under androgen regulation on physiological conditions. This explains the absence of accumulation of alpha2u-globulin in the female groups and the presence of alpha2u-globulin in the male control group. Furthermore, alpha2u-globulin accumulation is a male- and rat- specific entity, which will not be translated to humans. Many xenobiotics bind to circulating alpha2u-globulin and decrease effectiveness of lysosomal degradation in the proximal tubules resulting in greater accumulation. The increase in tubular basophilia in LD and MD males exceeded the background levels from the control group. This, and the presence of tubular hyaline casts in few treated males were considered characteristic early changes associated with alpha2u-globulin nephropathy. This effect is not relevant for the human health risk assessment.

Under the conditions of this study and based on the pathology evaluation, the NOAEL for a repeated exposure to [3R-(3alpha,3abeta,7beta,8aalpha)] -1 - (2,3,4,7,8,8a-hexahydro- 3,6,8,8-tetramethyl- 1H-3a, 7-methanoazulen-5-yl) ethan-1-one by oral gavage over a period of 90 days in male and female Wistar rats, can be established at 80 mg/kg bw/day.