Cyclohexanemethanol, 4-(1-methylethyl)-

Endpoint summary

Description of key information

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 408 and in compliance with GLP, test substance was administered via the diet at 300, 1000 and 3000 ppm over a period of 13 weeks to Crl:CD(SD) rats. Control animals received basal diet. This study included a 10-week recovery period. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, sperm analysis, macropathology and histopathology investigations were undertaken. The dose levels for the 90-day study were selected based on the results of a 14-day dietary study, considered to be a supporting study.

It was concluded that dietary administration of test substance to Crl:CD (SD) rats at dietary concentrations 300, 1000 or 3000 ppm for 13 weeks provided clear evidence of systemic exposure but no effects which were deemed to be adverse. There were no test item-related histopathological changes observed for any tissues examined in this study. Plasma biochemistry and urinalysis revealed several slight changes in composition, predominantly in females, which were indicative of adaptations of metabolism/excretion in the liver and kidneys. In the absence of any change in organ weight or any evidence of degenerative or functional change in the liver and kidneys during histopathological evaluation, the slight disturbances of biochemical and urine parameters were considered not to be adverse.

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) was concluded to be 3000 ppm (equivalent to 188 mg/kg bw/day in males and 220 mg/kg bw/day in females).

In a 28-day toxicity study conducted in rodents to evaluate the repeated dose toxicity of the substance.  Doses for the 28-day study were selected based on the results of a 14-day study, considered to be a supporting study.  Oral (gavage) administration of the substance to Crl:CD (SD) rats for 28 days. Change to the liver were considered to be possible alterations to intra-hepatocellular fat metabolism/transport in the high dose animals, however there was no evidence of degenerative liver changes in either sex at any dose level and the findings suggest an adaptive change in the liver and not evidence of adverse toxic effects.  At 300 mg/kg/day, there was also a reduction in sperm velocity and increase in static sperm resulting in an overall reduction in the percentages of motile and progressively motile sperm.  These changes were considered not to be due to a primary toxicity of the substance itself. Instead, the sperm effects are considered to be due to oxidative stress occurring as a result of testicular metabolism of the substance made possible by high plasma levels of the substance and its metabolites following oral gavage administration of a high bolus dose (further discussed in Section 7.8).  Based on the data, the No Observed Adverse Effect Level was concluded to be 100 mg/kg/day.

In the dietary OECD TG 408 study, no adverse effects were observed up to 3000 ppm (eq. to 188 mg/kg bw/day for males and 220 mg/kg bw for females). In the shorter-term OECD TG 407 and 421 reproductive toxicity screening study, both via gavage dose, effects were observed on sperm motility at similar or higher levels. These effects were considered to be specifically associated with bolus dosing.  To confirm that much higher plasma levels occur after oral gavage dosing than after dietary dosing, plasma levels of Mayol were measured during the OECD TG 408 dietary study and the oral gavage OECD TG 414 study. Though the full report is not yet available the evidence indicates that via gavage the peak plasma levels of Mayol are much higher than in the dietary study. These high plasma levels of Mayol show that the oral absorption is fast and high. It is anticipated that at levels < 1 mMol (ca 200 mg/kg bw) Mayol is metabolised/oxidised into its acid and conjugated. At higher levels Mayol as such escapes first pass metabolism in the liver and enters the systemic circulation and direct exposure to the male reproductive system occurs. All organs have the capability to further oxidise substances and also these organs. Compared to other organs the testes is somewhat hypoxic. When significant amounts of substance is oxidised within the testes the oxygen levels may be depleted (oxidative stress) such that it can affect sperm viability. As these effects are only seen at high dose levels that exceed a threshold that cannot be envisgned after exposure to humans, and metabolic overload is anticipated, these effects are not considered as relevant reproductive hazard effects.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 408 without any deviation.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 01-03 December 2015/ signed on 15 February 2016)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1002451619
- Physical state: Colourless liquid
- Expiration date of the lot/batch: 1 January 2017
- Purity test date: 24 November 2015

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

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:CD (SD) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 44 to 50 days
- Weight at study initiation: Males: 226-280 g; Females: 160-216 g
- Housing: Five of the same sex (Main and Recovery phases) in polycarbonate cages with a stainless steel mesh lid.
- Diet: Rat and Mouse No. 1 Maintenance Diet, ad libitum (removed overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (except during urine collection)
- Acclimation period: 15 days before commencement of treatment

DETAILS OF FOOD AND WATER QUALITY:
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated; minimum of 15 air changes per hour
- Photoperiod: 12 hours light : 12 hours dark

IN-LIFE DATES: From: 27 January 2016 To:

Route of administration:

oral: feed

Details on route of administration:

The dietary route of administration was chosen to simulate the conditions of potential human exposure.

Vehicle:

other: Rat and Mouse No. 1 Maintenance Diet, a powdered diet

Details on oral exposure:

DIET PREPARATION
- Method of preparation: On each occasion of the preparation of the premix, the required amount of test substance was added to an equal amount of diet and stirred. An amount of diet equal to the weight of the mixture was added and the mixture was stirred again until visibly homogenous. This doubling up process was repeated until half the final weight of premix was achieved. This mixture was then ground using a mechanical grinder. The weight of the mixture was then made up to the final weight of the premix with diet. The mixture was then mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of this premix were then diluted with further quantities of RM1 diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer. Batches of diet were prepared weekly and stored in amber glass jars until use.
- Frequency of preparation: Weekly, in advance of the first day of feeding.
- Storage of formulation: Frozen (nominally -20 °C) from preparation until required for use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Homogeneity and stability in the diet matrix has been demonstrated (Huntingdon Life Sciences Study No. HIK0023) for the dietary inclusion levels 100 ppm to 7500 ppm Mayol when stored in amber glass jars for 22 days following frozen storage (nominally -20°C) and for 8 days following ambient storage (nominally 15-25 °C).
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 12 of treatment were analyzed for achieved concentration of the test item.

Results: The mean concentrations of test substance in test diets analyzed for Week 1 and Week 12 of the study were within ±12% of nominal concentrations, confirming accurate preparation.

Duration of treatment / exposure:

90 days (13 weeks followed by a 10-week recovery period)

Frequency of treatment:

Continuously. During the recovery period, all animals were given untreated diet.

Dose / conc.:

300 ppm

Dose / conc.:

1 000 ppm

Dose / conc.:

3 000 ppm

No. of animals per sex per dose:

Main phase: 10 animals/sex/dose
Recovery phase: 5 animals/sex/dose for contro and high dose groups only (0 and 3000 ppm)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:The dietary inclusion levels investigated in this study (0, 300, 1000 and 3000 ppm) were selected in conjunction with the Sponsor based on the results of a previous 18-day dietary study in the rat with the test substance at levels of 1500, 3000, 5000 and 7500 ppm (Huntingdon Life Sciences Study No. HIK0023). In that study, administration of the test substance up to 7500 ppm was tolerated with no mortalities and no treatment related clinical signs. At 3000 ppm and above, food intake was lower than control in a dose-dependent manner with a consequently low body weight gain. There was some reduction in sperm motility among males receiving 5000 ppm and above but with no histopathological correlates.
In this study, the high level was selected as 3000 ppm on the basis of the effect on food intake and weight gain, but no impact on sperm motility following 18 days of treatment in the previous study. Higher dose levels were excluded because the OECD 408 guideline indicates that substances administered via the diet should not interfere with normal nutrition, therefore dose levels that caused greater than a 10% reduction in body weight gain were excluded.
The low level was selected as 300 ppm as a 10-fold reduction from the high level, and the intermediate level was selected as 1000 pm as the approximate geometric mean between the low and high level. All levels were expected to be well tolerated in this study, and the levels were anticipated to generate exposure levels of approximately 20, 60 and 180 mg/kg/day, respectively
- Rationale for animal assignment: Randomly allocated on arrival
- Post-exposure recovery period in satellite groups: 10-week recovery period

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants.
During the acclimatization and recovery periods, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- The eyes of the animals were examined by means of a binocular indirect ophthalmoscope (at the discretion of the examining veterinary surgeon a slit-lamp biomicroscope.
- Time schedule and dose groups for examinations: Pretreatment - All animals (including spares); Week 12 - All Main phase animals of Groups 1 and 4 (0 and 3000 ppm)
- Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 - All Main phase animals; Recovery Week 10 - All Recovery phase animals. Blood sampling was performed on the morning after overnight collection of urine.
- Anaesthetic used for blood collection: Yes; Animals were held under light general anesthesia induced by isoflurane.
- Animals fasted: Yes; animals, were deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- Parameters:
- Haematology: Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M) and Large unstained cells (LUC), Platelet count (Plt)
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series analyser and appropriate reagent in respect of: Prothrombin time (PT) and Activated partial thromboplastin time (APTT)
- Clinical chemistry: Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer.
Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transferase (gGT), Total bilirubin (Bili), Total bile acids (Bi Ac), Urea, Urea Nitrogen (BUN), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb). Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13 - All Main phase animals; Recovery Week 10 - All Recovery phase animals.
- Metabolism cages used for collection of urine: Yes; animals were placed in an individual metabolism cage, without access to food or water. Urine samples were collected overnight.
- Parameters:
Using manual methods: Appearance - by visual assessment; Volume - using a measuring cylinder; pH - using a pH meter; Specific gravity - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Glucose, Ketones, Bile pigments, Urobilinogen, Blood pigments
Using a Roche P Modular Analyzer: Protein, Sodium, Potassium, Chloride, Creatinine
A microscopic examination of the urine sediment was performed: Epithelial cells, Leucocytes, Erythrocytes, Casts and Other abnormal components

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Sensory reactivity and grip strength assessments were performed on the first five Main phase animals and all Recovery phase animals in Group 1 and 4 (0 and 3000 ppm), and all Main phase animals from Group 2 and 3 (300 and 1000 ppm) during Week 12 of treatment. During Week 12 of treatment, the motor activity on the first five Main phase animals and all Recovery phase animals in Group 1 and 4, and all Main phase animals from Group 2 and 3.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER:
Toxicokinetics: Blood samples were obtained from all Main phase animals for toxicokinetic assessment early in the working day (by 10:30am) on Day 8 and during Week 13 of treatment.

Sacrifice and pathology:

SACRIFICE: Animals were killed by carbon dioxide asphyxiation with subsequent exsanguination. Main study animals were killed following 13 weeks of treatment. Recovery phase animals were killed following 13 weeks of treatment and 10 weeks of recovery. All Main phase and Recovery phase animals were subject to a detailed necropsy.

GROSS PATHOLOGY: Yes; after a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS: For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for main study and recovery animals killed at scheduled intervals.

HISTOPATHOLOGY: Yes
Fixation: Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of those detailed below:
Testes: In modified Davidson’s fluid; Eyes: In Davidson’s fluid.
Histology:
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: Main phase animals of Groups 1 and 4
Abnormalities only: All Main phase animals of Groups 2 and 3
Recovery phase animals: Tissues from Recovery phase animals were retained in fixative pending possible future examination.
Routine staining: Sections were stained with hematoxylin and eosin
Light microscopy: Tissues preserved for examination were examined as follows:
Terminal sacrifice:
All Main phase animals of Groups 1 and 4 (all specified in Table 7.5.1/1)
All Main phase animals of Groups 2 and 3 (Abnormalities only)

Other examinations:

Sperm Analysis: Immediately after scheduled sacrifice of each male, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed. Tissues were collected from the right side for Animal No. 31 assigned to the 1000 ppm group.
The following tests were performed: Sperm motility - all groups; Sperm morphology - Group 1 and 4; Sperm count - Group 1 and 4; Homogenisation-resistant spermatid count - Group 1 and 4; Sperm morphology, sperm count and homogenisation-resistant spermatid count - Group 2 and 3

Statistics:

See "Any other information on materials and methods incl. tables"

Clinical signs:

no effects observed

Description (incidence and severity):

During the treatment and recovery periods, there were no signs observed during the detailed physical examination and arena observation procedures which were related to test substance administration at any dietary inclusion level investigated.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Administration of the test substance was associated with a modest but statistically significant reduction in mean weight gain for males and females given 3000 ppm during the first two weeks of treatment (87% of Control for males and 79% of Control for females). Thereafter, from Week 2-13 of treatment the mean body weight gain of these animals was essentially similar to, or slightly higher than Control, such that overall mean body weight gain during Weeks 0-13 was 98% of Control for males and 96% of Control for females.
- The mean body weight gain of males and females given 300 or 1000 ppm was unaffected by test substance during the treatment period.
- During the 10-week recovery period, there was no evidence of an effect of previous administration with test substance on body weight performance. Mean body weight gain for males in the 3000 ppm group was essentially similar to Controls; for females in this group, mean body weight gain was higher than Control (48 g versus 21 g) but this was considered to be incidental and not related to previous test substance administration.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females given 3000 ppm showed reduced mean food consumption during the majority of the treatment period, compared to Control and pre-treatment values, such that overall mean food intake during Week 1-13 of treatment was 89% of Control. At 1000 ppm, females showed slightly reduced mean food consumptions during Weeks 1-2 of treatment; thereafter mean food consumption was essentially similar to Control. The food intake of females given 300 ppm and for all groups of treated males during the 13-week treatment period was unaffected by test substance administration.
Following the cessation of treatment, the food intake of animals previously given 3000 ppm was essentially similar to Control..

Achieved dose: The overall achieved doses during Weeks 1-13 of study at 300, 1000 and 3000 ppm were 19, 62 and 188 mg/kg bw/day for males and 23, 79 and 220 mg/kg bw/day for females, respectively.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No effect was observed and consequently quantitative measurements were not performed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related ophthalmoscopy findings apparent during Week 12 of treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Analysis of haematological parameters during Week 13 of treatment revealed, when compared with Control, a slight decrease in haemoglobin concentration for females given 3000 ppm [14.6, 14.1, 14.3, 13.8** at 0, 300, 1000 and 3000 ppm], and a non-dose dependent slight decrease in haematocrit in all groups of treated females [0.430, 0.410*, 0.417* and 0.407** at 0, 300, 1000 and 3000 ppm]. In addition, all groups of treated females showed a non dose-dependent slight decrease in neutrophil count [1.24, 0.88*, 0.85* and 0.92* at 0, 300, 1000 and 3000 ppm], monocyte count [0.25, 0.18*, 0.13** and 0.16**at 0, 300, 1000 and 3000 ppm] and large unstained cell concentrations [0.05, 0.03*, 0.03* and 0.04* at 0, 300, 1000 and 3000 ppm], and females given 1000 or 3000 ppm also showed a slight decrease in eosinophil concentration [0.12, 0.10, 0.08* and 0.08* at 0, 300, 1000 and 3000 ppm]. Similar differences were not apparent among males. Platelet counts were low for males given 3000 ppm [1000, 934, 970 and 888* at 0, 300, 1000 and 3000 ppm].
- Assessment of clotting times revealed a dose dependent slight prolongation of prothrombin times for females given 1000 or 3000 ppm [20.2, 21.1, 21.8* and 22.1* at 0, 300, 1000 and 3000 ppm].
- All of these modest differences from Control attained statistical significance, however all differences were minor and all values were within the 5-95% confidence limits of the Historical Control Data (HCD) range and generally not dose-related, therefore these minor differences were considered to be of no toxicological importance.
- Following 10 weeks of recovery, further analysis of haematological parameters did not reveal any toxicologically relevant differences from Control.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Biochemical analysis of plasma during Week 13 revealed the following treatment-related differences from Control which attained statistical significance; unless otherwise indicated, values were within the 5-95% confidence limits of the HCD range.
- Females given 3000 ppm showed a slight increase in alkaline phosphatase activity [30, 33, 36, 43** at 0, 300, 1000 and 3000 ppm]. At 1000 or 3000 ppm a non dose-dependent decrease in alanine aminotransferase activity was apparent for females [44, 39, 22*, 23* at 0, 300, 1000 and 3000 ppm], with values outside the HCD range (27 to 62 U/L). Mean urea and blood urea nitrogen levels were slightly increased for females given 1000 or 3000 ppm [5.50, 5.80, 6.43* and 6.49* at 0, 300, 1000 and 3000 ppm]. Males and females given 3000 ppm showed a reduction in cholesterol concentration which was below the HCD range for males [1.58, 1.41, 1.42 and 1.14* at 0, 300, 1000 and 3000 ppm vs HCD range 1.22 to 2.66 mmol/L] and at the lower limit of the HCD range for females [2.33, 2.33, 1.91 and 1.43** at 0, 300, 1000 and 3000 ppm vs HCD range 1.43 to 3.08 mmol/L]. Triglyceride concentrations were also low, and below the HCD range [0.39, 0.43, 0.36 and 0.21** at 0, 300, 1000 and 3000 ppm vs HCD range 0.32 to 1.46 mmol/L] for females given 3000ppm.
- Changes in electrolyte concentrations comprised slightly increased chloride concentrations for males and females given 3000 ppm and for males given 1000 ppm [Males: 101, 102, 102* and 102* / Females: 101, 102, 102 and 103** at 0, 300, 1000 and 3000 ppm]. In addition, phosphorus concentrations were slightly increased for males at 3000 ppm and slightly decreased for all groups of treated females, although in the absence of a dose response [Males: 1.86, 1.90, 1.96 and 2.00* / Females: 1.67, 1.40*, 1.53* and 1.39** at 0, 300, 1000 and 3000 ppm]. Calcium concentrations were slightly low for females given 1000 or 3000 ppm, with a dose response apparent, and values being at the limit of or below the HCD range [2.61, 2.57, 2.51** and 2.38** at 0, 300, 1000 and 3000 ppm vs HCD range 2.51 to 2.79 mmol/L].
- At 3000 ppm, females showed a reduction in total protein and albumin concentrations with values at the limit of or below the HCD range [Total protein: 68, 68, 65 and 62** vs HCD range 63 to 78 g/L / Albumin: 38, 40, 38 and 35* at 0, 300, 1000 and 3000 ppm vs HCD range 35 to 43 g/L].
- Following 10 weeks of recovery, further analysis did not reveal any toxicologically relevant differences from Control, indicating that full reversibility of all previously noted changes had occurred. Females previously given 3000 ppm showed slightly low glucose concentrations, however in the absence of a similar effect during the treatment period, this finding was considered incidental and unrelated to previous test substance administration.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Analysis of urine collected during Week 13 revealed, when compared with Control, a statistically significant but non dose-dependent slight decrease in urinary protein output for females given 1000 or 3000 ppm; values were, however, within the HCD range for animals of this age and strain and therefore considered to be of no toxicological importance. Microscopic examination of the urine sediment did not reveal any abnormalities at any dietary level investigated.
- There were no toxicologically important changes apparent in the urine in Week 10 of the recovery period.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor activity: Motor activity scores for males and females during Week 12 of treatment showed some intra and inter group variation, with differences in occasional 6-minute recording periods attaining statistical significance; however overall there was no evidence of an effect of treatment with test substance.
Sensory reactivity and grip strength: Sensory reactivity observations and grip strength values for all groups of animals during Week 12 of treatment were considered unaffected by test substance administration.
All groups of treated males showed a marginal increase in mean forelimb and hindlimb grip strength when compared to Control. Differences did not attain statistical significance, and in the absence of a clear dose response relationship or similar effects on grip strength in these groups of females, these minor differences in grip strength were considered incidental and unrelated to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Analysis of organ weights after 13 weeks of treatment with the test substance revealed a slight increase in mean absolute (+17%) and adjusted (+20%) adrenal weights for females given 3000 ppm when compared to Controls, with statistical significance attained for the mean adjusted adrenal weights. The mean absolute and adjusted adrenal weights for females given 3000 ppm was comparable to that of control following 10 weeks of recovery, suggesting a reversibility of the minor change seen at the end of treatment period.
Following 10 weeks of recovery, females previously given 3000 ppm showed low mean absolute (-27%) and adjusted (-38%) thymus weights when compared to Controls, with statistical significance attained for the mean adjusted thymus weights; in the absence of a similar difference at the end of the treatment period, or in the males at the end of the treatment or recovery periods, this difference was considered to be incidental and not related to previous test substance administration.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination performed after 13 weeks of treatment and after 10 weeks of recovery revealed no test item related lesions.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related histopathological findings observed among the tissues evaluated at the end of the treatment period. Consequently, histopathological evaluation of the tissues retained at the end of the recovery period was not undertaken.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Sperm Analysis: No effects on sperm motility, morphology or sperm counts were apparent following 13 weeks treatment at dietary concentrations up to 3000 ppm or following 10 weeks of recovery.
At the end of the treatment period, a slight but statistically significant lowering of testicular spermatid counts was observed in males treated at 3000 ppm when compared with the Control group; all individual values were within the concurrent Control range, and no effect of test substance administration was inferred.

Key result

Dose descriptor:

NOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

other: Sperm motility, morphology and sperm counts were unaffected.

Remarks on result:

other: equivalent to 188 mg/kg bw/day in males and 220 mg/kg bw/day in females

Key result

Critical effects observed:

no

The systemic toxic potential of the test substance, an industrial chemical, was assessed by dietary administration to Crl:CD(SD) rats over a period of 13 weeks. Groups of male and female rats received the test substance at constant dietary concentrations of 0, 300, 1000 or 3000 ppm. Administration of the test item at concentrations up to and including 3000 ppm (equivalent to 188 mg/kg/day in males and 220 mg/kg/day in females) was well tolerated, no premature deaths occurred, there were no test article-related clinical signs observed or adverse effects on body weight, food consumption, sensory reactivity, grip strength, motor activity, ophthalmoscopy, haematology, blood chemistry, urinalysis, organ weights, sperm analysis or macropathology. There were no target organs identified during the micropathological evaluations.

In males and females given 3000 ppm low body weight gain was observed during the first two weeks of treatment. During the full 13-week dosing period, the weight gain of these animals 2-4% lower than Controls, and as there was no impact on the clinical condition of these animals and the weight gain observed in the 10-week recovery period was essentially similar to or slightly higher than Control, the lower weight gain observed during the treatment period was considered not to be adverse.  

Females given the test substance at 3000 showed low food consumption throughout the majority of the treatment period when compared to Control and pre-treatment values; mean food intake was also low for females given 1000 ppm during the first two weeks of treatment. In the absence of any change in clinical condition of these females, or any toxicologically important effects on body weight performance, these differences in food intake were considered not to be adverse at the magnitude observed. Anticipated food consumption was apparent in test and control animals following the cessation of treatment. 

At the end of the dosing period several minor test item-related changes in blood plasma and urine composition were apparent, primarily in females given 1000 or 3000 ppm, that indicated effects on the liver and kidneys, although there were no changes in the weight of these organs at terminal necropsy. Plasma indicators of liver effects comprised changes in enzyme activity and concentrations of proteins, cholesterol and triglycerides. Markers of kidney effects comprised slight increases in urea and blood urea nitrogen, and slightly increased chloride and phosphorus concentrations and slightly decreased calcium concentrations in blood plasma, and low total protein output in the urine. Despite these indicators of effects on the liver and kidney, there were no changes in the general clinical condition of the animals and no macropathological or micropathological abnormalities in either of these organs. Assessments conducted following the 10-week off-dose period showed that full recovery was evident for the blood chemistry and urinalysis changes. It was therefore concluded that these changeswere a normal adaptive metabolism/excretion response in the liver and kidneys; such disturbances of biochemical and urine parameters following administration of a xenobiotic in the absence of corroborative pathology or functional change of the organs is considered not to be adverse. 

Some slight changes in red cell parameters were apparent, predominantly among females, at the end of the dosing period. All of the differences were small, were within the Historical Control Data range, had no impact on the general health of the animals, had no micropathological correlate and showed full recovery following the 10-week off-dose period, and were therefore considered not to be adverse. 

Females given 3000 ppm showed slightly high adjusted adrenal gland weights at the end of the treatment period. In the absence of any macropathological or micropathological changes in the adrenal glands, this minor difference was considered to of no toxicological importance and not adverse.   

Conclusions:

It was concluded that dietary administration of the test substance to Crl:CD (SD) rats at dietary concentrations 300, 1000 or 3000 ppm for 13 weeks provided clear evidence of systemic exposure but no effects that were deemed to be adverse. There were no test item-related histopathological changes observed for any tissues examined in this study. Plasma biochemistry and urinalysis revealed several slight changes in composition, predominantly in females, which were indicative of adaptations of metabolism/excretion in the liver and kidneys. In the absence of any change in organ weight or any evidence of degenerative or functional change in the liver and kidneys during histopathological evaluation, the slight disturbances of biochemical and urine parameters were considered not to be adverse. The No Observed Adverse Effect Level (NOAEL) was concluded to be 3000 ppm (equivalent to 188 mg/kg/day in males and 220 mg/kg/day in females).

Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 408 and in compliance with GLP, test substance was administered via the diet at 300, 1000 and 3000 ppm over a period of 13 weeks to Crl:CD(SD) rats. Control animals received basal diet. This study included a 10-week recovery period. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, sperm analysis, macropathology and histopathology investigations were undertaken.

 

Dietary administration of the test substance at concentrations up to and including 3000 ppm (equivalent to 188 mg/kg/day for males and 220 mg/kg/day for females) for 13 weeks was well tolerated. There were no premature deaths, no test item-related signs observed during the detailed physical examination and arena observations, and sensory reactivity observations, grip strength, motor activity scores and ophthalmoscopy assessments for all groups of animals during Week 12 were unaffected by treatment.

Males and females given 3000 ppm showed statistically significantly reduced body weight gain during Week 0-2 of treatment, such that overall mean weight gain during the 13-week dosing period was 96-98% of Control, although statistical significance was not attained for the difference in overall mean weight gain; the mean weight gain of males and females given 300 or 1000 ppm was unaffected. Following the cessation of treatment, the mean body weight gain of Recovery phase males and females which had previously received 3000 ppm was unaffected by previous test substance administration.  

During the first two weeks of treatment, mean food consumption was reduced, when compared to Control and pre-treatment values for females given 1000 or 3000 ppm; this reduction in food intake continued during the remainder of the treatment period for females given 3000 ppm. The food intake of females given 300 ppm and for all groups of treated males during the 13-week treatment period was unaffected by test substance administration, and the food intake of animals previously given 3000 ppm was essentially similar to Control throughout the 10-week recovery period.

Analysis of haematological parameters in Week 13 revealed, when compared to Controls, the following statistically significant difference: reduced haemoglobin concentrations in females given 3000 ppm; reduced haematocrit in all groups of treated females; a non dose-dependent slight decrease in neutrophil, monocyte and large unstained cell concentrations in all groups of treated females; a slight decrease in eosinophil concentrations in females given 1000 or 3000 ppm; reduced platelet counts for males given 3000 ppm; a dose dependent slight prolongation of prothrombin times for females given 1000 or 3000 ppm. All values were within the Historical Control Data (HCD) range and similar differences were not evident following 10 weeks of recovery, indicating full reversibility of these minor changes. 

During Week 13, biochemical analysis of plasma revealed the following statistically significant differences when compared to Controls: a trend towards slightly reduced alanine aminotransferase activity in females given 1000 or 3000 ppm; slightly increased alkaline phosphatase activity in females given 3000 ppm; slightly high plasma urea and blood urea nitrogen concentrations in females given 1000 or 3000 ppm; reduced cholesterol concentrations for males and females at 3000 ppm; reduced triglyceride concentrations for females at 3000 ppm; slightly increased chloride concentrations for both sexes given 3000 ppm and for males given 1000 ppm; slightly increased phosphorus concentrations for males at 3000 ppm and slightly decreased for all groups of treated females; slightly reduced calcium concentrations for females given 1000 or 3000 ppm; reduced total protein associated with reduced albumin concentration for females given 3000 ppm. All of these minor differences showed full recovery after 10 weeks off-dose.

Analysis of urine collected during Week 13 of treatment revealed a slight, non-dose dependent decrease in urinary protein output in females given 1000 or 3000 ppm. Values were within the HCD range and a similar difference was not apparent 10 weeks after the cessation of dosing. Microscopic examination of the urine sediment did not reveal any abnormalities at any dietary level investigated.

The analysis of organ weights following 13 weeks of treatment indicated a slight increase in mean absolute and adjusted adrenal gland weights for females given 3000 ppm. There were no test item-elated changes in organ weights following 10 weeks of recovery.

Sperm motility, morphology and sperm counts were unaffected by test substance administration at all dietary concentrations investigated.

There were no macroscopic abnormalities detected at scheduled termination after 13 weeks of treatment or after 10 weeks of recovery that were attributable to treatment with test substance. Histopathological evaluation of the retained tissues of the 3000 ppm animals killed after
13 weeks of treatment did not reveal any test item-related micropathological changes.

Conclusion

It was concluded that dietary administration of the test substance to Crl:CD (SD) rats at dietary concentrations 300, 1000 or 3000 ppm for 13 weeks provided clear evidence of systemic exposure but no effects that were deemed to be adverse. There were no test item-related histopathological changes observed for any tissues examined in this study. Plasma biochemistry and urinalysis revealed several slight changes in composition, predominantly in females, which were indicative of adaptations of metabolism/excretion in the liver and kidneys. In the absence of any change in organ weight or any evidence of degenerative or functional change in the liver and kidneys during histopathological evaluation, the slight disturbances of biochemical and urine parameters were considered not to be adverse. The No Observed Adverse Effect Level (NOAEL) was concluded to be 3000 ppm (equivalent to 188 mg/kg/day in males and 220 mg/kg/day in females).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 56 to 62 days old
- Weight at study initiation: 306 g to 396 g (males) and 196 g to 258 g (females)
- Housing: Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals (i.e., 5 of the same sex). Each sex was allocated separately.
- Diet (e.g. ad libitum): Rat and Mouse No. 1 Maintenance Diet; the diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Ad libitum; however, removed overnight before blood sampling for haematology or blood chemistry.
- Water (e.g. ad libitum): Ad libitum; potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: 27 Days before commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 ºC
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark

IN-LIFE DATES: From: 14 November 2012 (animal arrival) To: 8 January 2013 (day of necropsy)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the required amount of the substance and magnetically stirred until all of the test material had dissolved. The remaining amount of vehicle was added to make up the required total volume. The formulation was then returned to the container and mixed using a high shear homogeniser to produce a homogeneous suspension. Remaining concentrations were formulated in ascending order using the same method.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Specimen formulations (typically 400 mL) were prepared at concentrations of 1 and 250 mg/mL and equally split between four amber glass screw-capped bottles. Stability was assessed following storage at ambient temperature (nominally 21°C) for 0, 1 and 2 hours (continually stirred) and 1 day (Bottle 1), and refrigeration (nominally 2 to 8 °C) for 1 day, 8 days and 15 days (Bottles 2, 3 and 4). Prior to initial sampling on each day, the formulation was mixed by 20-fold inversion and magnetically stirred for a minimum of five minutes.
Samples of each formulation prepared for administration in Weeks 1 and 4 of treatment were analysed for achieved concentration of the test substance.

Duration of treatment / exposure:

Once/day for 28 days. Animals were sacrificed on Day 29.

Frequency of treatment:

Once/day.

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:

The dose levels for investigation in this 4-week toxicity (OECD407) study (0, 30, 100 and 300 mg/kg/day) were selected in conjunction with the Sponsor based on the results of a 14-day preliminary study (Huntingdon Life Sciences Study Number HIK0015). In that study, oral dose levels of 50, 150, 300, 500, 750 and 1000 mg/kg/day were investigated. The doses of 750 and 1000 mg/kg/day were found to exceed the maximum tolerated dose, with a marked decline in clinical condition observed after 3 or 4 doses, requiring premature sacrifice of the animals, and these doses were considered unsuitable for further investigation. The dose levels up to and including 500 mg/kg/day were well tolerated for the full 14-day period with no unscheduled deaths. Treatment related effects were limited to a suggestion of slightly low weight gain in females receiving 500 mg/kg/day, slightly increased liver weight in males and females at 300 or 500 mg/kg/day, an absence of motile sperm in all males receiving 500 mg/kg/day and one out of five males at 300 mg/kg/day, with a reduction in epididymal sperm counts apparent at 500 mg/kg/day.

In view of the sperm effects detected at 500 mg/kg/day in the preliminary study, a dose level of 300 mg/kg/day (threshold for Category 2 classification (EEC 1272/2008 and UN GHS systems)) was selected as the high dose level for the current OECD407 study and a dose of 30 mg/kg/day (threshold for Category 1 classification (EEC 1272/2008 and UN GHS systems)) was selected as the low dose level. The intermediate dose level of 100 mg/kg/day was selected as the approximate geometric mean between the low and high dose levels.

Positive control:

Not required per OECD Test Guideline No. 407.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Inspected visually at least twice daily for evidence of ill-health or reaction to treatment. For signs associated with dosing, detailed observations were recorded twice weekly (start and middle of each week) at the following times: (a) pre-dose observation; (b) at the end of dosing each group; (c) 1 to 2 hours after completion of dosing of all groups; and (d) as late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour. Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.


BODY WEIGHT: Yes
- Time schedule for examinations: One week before treatment commenced (Day -7), on the day that treatment commenced (Day 1), twice weekly throughout the study (last scheduled bodyweight was recorded on Day 28) and before necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g/animal/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Assessed by daily qualitative visual observation.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After overnight withdrawal of food on Day 29
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After overnight withdrawal of food on Day 29
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All animals

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before dosing during Week 4 of treatment.
- Dose groups that were examined: All animals
- Battery of functions tested: Sensory reactivity, grip strength, approach and touch response, auditory startle reflex, tail pinch response, and motor activity.

Sacrifice and pathology:

Animals were killed following completion of 4 weeks of treatment on Day 29 of study.

ORGAN WEIGHTS: Yes
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Sperm analysis was conducted for all male animals in each group as follows:
(a) Sperm sampling: Immediately after sacrifice the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed;
(b) Sperm motility: The percentages of motile and progressively motile sperm and sperm motion parameters were reported;
(c) Sperm count: The concentration (million/g) and total number of sperm in the left cauda epididymis were reported; and
(d) Homogenisation-resistant spermatids count: The concentration (Million/g) and total number of spermatids in the left testis were reported.

Statistics:

Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data.

Significant differences between control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level; treated groups compared with control using Dunnett’s test, Shirley’s test, or Williams’ test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation in all males and females receiving 300 mg/kg/day and one male receiving 100 mg/kg/day and chin rubbing in females receiving 300 mg/kg/day were noted but, according to the study authors, are commonly seen in studies where the route of administration is oral gavage and are considered to reflect distaste of the test formulations and are not indicative of an adverse effect of treatment with the substance.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

Daily visual assessment of water intake indicated that animals receiving 300 mg/kg/day generally consumed more water than the other study groups (including controls) from Day 5 of treatment until scheduled termination. However, this finding was considered likely related to the increased incidence of post-dosing salivation in animals of this group.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Minor inter-group differences from controls were apparent which attained statistical significance, including red cell distribution width and eosinophil concentrations for females at 300 mg/kg/day, and basophil and large unstained cell concentrations for males at 100 or
300 mg/kg/day. All values were within the historical control data range, and the observed differences lacked either dose-relationship, concomitant changes in other related haematological parameters or microscopic correlates, and were considered attributable to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Slight increase in aspartate amino-transferase activity in males and females receiving 300 mg/kg/day and urea concentrations of females receiving 300 mg/kg/day. In addition, glucose concentrations were slightly low in all groups of treated males compared with controls, although there was no dose response apparent. Low cholesterol concentrations were apparent in males and females at 300 mg/kg/day compared to controls and sodium concentrations were marginally low among females receiving 300 mg/kg/day. Assessment of bile acid concentrations revealed a high degree of inter and intra-group variability, however there was a high group mean concentration among females receiving 300 mg/kg/day as a result of two individual animals (Numbers 36 and 37) which had high concentrations. All of these differences from control values were, however, within the historical control data range.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

High mean motor activity scores noted in females receiving 300 mg/kg/day for both high and low beam breaks (rearing and cage floor activity, respectively) during the first and third 6-minute recording periods attaining statistical significance and resulting in high total scores (differences were attributable to high activity levels for 2 females).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased body weight-adjusted kidney weights in males receiving 30 mg/kg/day (1.16-times the control values) and in males and females receiving 100 or 300 mg/kg/day (≤ 1.17-times the control values); there was no dose response apparent among males. In addition, increased body weight-adjusted liver weights were evident in males receiving 300 mg/kg/day (1.31-times the control values) and in all groups of treated females (1.29- to 1.79-times the control values). In males receiving 100 or 300 mg/kg/day, slightly increased body weight-adjusted testis weights were also apparent (≤ 1.12-times the control values).

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no abnormalities detected which were considered indicative of an adverse effect of the administration of the substance. All macroscopic findings were of the type encountered normally in young Crl:CD(SD) rats at these laboratories.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with the substance were seen in the liver. These changes consisted of a dose-dependent increase in the incidence and/or severity of periportal vacuolation of hepatocytes in rats given the substance; the incidence and severity of this lesion was greater in females than in males. Four of the 5 control females also had this finding at minimal severity. The nature and incidence of all other findings were consistent with the commonly seen background of microscopic changes.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Computer assisted sperm analysis conducted after 4 weeks of treatment revealed a statistically significant reduction in sperm velocity and amplitude of lateral head displacement at 300 mg/kg/day when compared to controls. These differences were associated with an increase in the percentage of static sperm and a marked decrease in the percentage of motile and progressively motile sperm in 2 of the 5 males (Numbers 11 and 12), resulting in an overall group mean of 0.74-times the control values and 0.70-times the control values for motile and progressively motile sperm, respectively; these differences did not, however, attain statistical significance. Sperm motility was unaffected by treatment at 30 or 100 mg/kg/day. There was no evidence of an effect of treatment on epididymal or testicular spermatid concentrations (millions/g) and total sperm number at any dose level investigated.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on liver and sperm findings at the higher dose level of 300 mg/kg/day,

Critical effects observed:

no

Histopathology

Summary of treatment related findings in the liver for animals killed after 4 weeks of treatment:

 

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	30	100	300	0	30	100	300
Hepatocyte Vacuolation, Periportal
Minimal	0	1	1	0	4	1	0	0
Slight	0	0	0	2	0	1	2	0
Moderate	0	0	0	0	0	1	0	2
Marked	0	0	0	0	0	0	1	3
Total	0	1	1	2	4	3	3	5
Number of tissues examined	5	5	5	5	5	5	5	5

 

Conclusions:

A 28-day toxicity study was conducted in rodents to evaluate the repeated dose toxicity of the substance. Doses for the 28-day study were selected based on the results of a 14-day oral gavage study, considered to be a supporting study. Oral (gavage) administration of the substance to Crl:CD (SD) rats for 28 days affected the liver and sperm motility. The liver changes were suggestive of possible alterations to intra-hepatocellular fat metabolism/transport in the high dose animals, however there was no evidence of degenerative liver changes in either sex at any dose level and the findings suggest an adaptive change in the liver and not a significant toxicity. At 300 mg/kg/day, there was also a reduction in sperm velocity and increase in static sperm resulting in an overall reduction in the percentages of motile and progressively motile sperm. These changes were considered not to be due to a primary toxicity of the substance itself. Instead, the sperm effects are considered to be due to oxidative stress occurring as a result of testicular metabolism of the substance made possible by high plasma levels of the substance and its metabolites following oral gavage administration of a high bolus dose (further discussed in Section 7.8). Based on the data, the No Observed Adverse Effect Level was concluded to be 100 mg/kg/day.

Executive summary:

The 28-day oral gavage toxicity study was conducted in Crl:CD (SD) rats to evaluate the potential systemic toxicity of the substance. This GLP-compliant study was designed and conducted according to the OECD Testing Guideline No. 407. Three groups, each comprising five male and five female Crl:CD (SD) rats received the substance at oral gavage doses of 0 (corn oil vehicle), 30, 100 or 300 mg/kg/day at a volume-dose of 4 mL/kg bodyweight. Dose levels for the 28-day study were selected based on a 14-day oral gavage toxicity study in the same strain of rats, which was considered a supporting study and is included in this dossier (Huntingdon Life Sciences, 2013). 

 

During the 28-day study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food and water consumption, haematology (peripheral blood), blood chemistry, organ weight, sperm analysis, macropathology and histopathology investigations were undertaken. As expected, treatment with the substance at doses up to and including 300 mg/kg/day was well tolerated; there were no mortalities and no treatment-related clinical signs observed. Salivation, chin-rubbing, and increased water consumption at the high dose were consistent with observations for orally-gavaged substances that are unpalatable to rats. Neurobehavioural assessments conducted during Week 4 of treatment revealed a slight increase in rearing and cage-floor locomotor activity among females receiving 300 mg/kg/day that was attributed to the activity of two females at this dose level. The biological significance of this finding is uncertain in the absence of similar effects during detailed arena observations and routine clinical observations, or any histopathological changes in the brain, spinal cord, nerves or muscles. There was no effect of treatment on sensory reactivity or grip strength at any dose level investigated. There were no adverse effects on bodyweight and mean food consumption.

 

The haematological investigation conducted after four weeks of treatment revealed no treatment-related changes. Test article-related biochemical changes in the blood plasma included a slight increase in aspartate amino-transferase (AST) concentrations in males and females receiving 300 mg/kg/day, elevated bile acid and slightly high urea concentrations among females at 300 mg/kg/day, slightly low glucose concentrations in all groups of treated males, slightly low cholesterol concentrations in males and females at 300 mg/kg/day and marginally low sodium concentrations among females receiving 300 mg/kg/day. All of these statistical differences from Control values were within the Historical Control Data range.

 

There were no treatment-related abnormalities detected at macroscopic examination after 28 days of treatment. Histopathological changes that were attributable to treatment occurred in the liver, where there was an increase in the incidence and/or severity of periportal hepatocyte vacuolation; the severity of this lesion was greater in females than in males, with all female dose groups affected in a dose-dependent manner, whereas only males given 300 mg/kg/day were affected. The only other hepatic change was an increase in liver weights in males at the high dose and in groups of treated females. Importantly, there was no evidence of any degenerative liver changes. Moreover, all biochemistry changes that might have been related to the liver changes are considered non-adverse, given that the values fell within the Historical Control Data range and were unaccompanied by liver necrosis or indications of degeneration. Biochemistry changes such as alterations in AST and cholesterol are of limited diagnostic utility in the absence of histological changes such as necrosis or hepatic degeneration (Ennulat et al., 2010). With regard to the periportal vacuolation, no further description or details were provided in the study report. It is important to note that hepatic macrovesicular vacuolation is considered a possible physiological adaptation to address an imbalance between hepatocyte uptake of lipids from the blood and secretion of the lipoproteins that carry the lipids (Thoolen et al., 2010). Taken altogether, while these liver changes were suggestive of possible alterations to intra-hepatocellular fat metabolism/transport in the high dose animals, as there was no evidence of any degenerative liver changes, the overall liver changes are considered likely to be adaptive and non-adverse. Hepatocyte vacuolation without evidence of degenerative or functional change reflected as a disturbance of biochemical parameters is considered not to be adverse. As there was no evidence of degenerative or functional changes at low- and mid-dose levels, the morphological change of hepatocyte vacuolation observed at 30 or 100 mg/kg/day in female rats was considered by the Study Director not to be adverse.  Similarly, given the low magnitude of the biochemical changes (falling within the Historical Control data range) and the absence of degenerative changes in the liver, the findings at 300 mg/kg bw/day are also considered not to be adverse.

 

Computer assisted sperm analysis revealed a reduction in sperm velocity and amplitude of lateral head displacement, with an associated increase in static sperm and a decrease in the percentage of motile and progressively motile sperm at 300 mg/kg/day. Sperm motility was unaffected by treatment at 30 or 100 mg/kg/day, and there was no evidence of an effect of treatment on epididymal or testicular spermatid concentrations (millions/g) and total sperm at any dose level investigated.

 

Kidney weights were increased in males at 30 mg/kg/day and in males and females at 100 and 300 mg/kg/day, unaccompanied by any histological change, and liver weights were increased in males at 300 mg/kg/day and all groups of treated females compared to respective Control groups. Males receiving 100 or 300 mg/kg/day also had slightly increased testis weights compared to Controls, but there were no microscopic correlates for the effects on sperm motility or testis weights.

 

Based only on the results from this oral gavage study, the Study Director concluded that the No Observed Adverse Effect Level was 100 mg/kg/day.

 

With regard to target organs of the substance, it should be noted that the effects on sperm motility observed in this study are considered to fall within the category of data contributing to an understanding of the classification of the substance for reproductive toxicity and is not further addressed here, other than to note that the effects on sperm motility are considered not to be a primary toxicity of the substance itself. Instead, the effects are considered to be due to oxidative stress occurring as a result of testicular metabolism of the substance made possible by high plasma levels of the substance and its metabolites following oral gavage administration of a high bolus dose (see IUCLID Section 7.8). The lack of any changes in testicular tissues supports this conclusion.

 

Therefore, the discussion of possible target organs for the substance should consider the findings in the liver. While it is acknowledged that the liver changes at the dose of 300 mg/kg/day were suggestive of possible alterations to intra-hepatocellular fat metabolism/transport in the high dose animals, there was no evidence of degenerative liver changes in either sex at any dose level.  Moreover, as an adaptive change, it seems likely that the vacuolation would be reversible. Based on the results and taking into consideration the “slight” nature of some of the changes (e.g., the clinical chemistry findings were values that were within the Historical Control Data range), that the liver findings may represent an adaptive change, and the lack of any degenerative changes in the liver, it is considered that the liver findings at 300 mg/kg bw/day do not represent a significant toxicity. 

 

Based on the liver and sperm findings at the high dose level in this study, the NOAEL for oral gavage administration of the substance in this general toxicity study in rats is concluded to be 100 mg/kg/day.

The liver morphology changes together with the minor clinical chemistry changes are not considered to meet the criteria for classification, based on the following considerations:

--there were no degenerative changes observed in the liver; and

--the changes observed (hepatocyte vacuolation) were not indicative of significant functional changes in the liver, nor significant organ damage, nor necrosis, fibrosis or granuloma, nor a morphological change that provides evidence of marked organ dysfunction, nor was there evidence of cell death (see criteria for classification Category 2, in Annex 1, Section 3.9.2.7.3). 

 

The liver-related clinical chemistry findings themselves are not considered to support classification for specific target organ toxicity following repeated exposure (STOT-RE) as these were “small changes in clinical biochemistry” that may be of “doubtful or minimal toxicological importance” (see Annex 1, Section 3.9.2.8).

 

In summary, it is considered that the liver findings observed following 28 days of oral dosing with the substance do not clearly indicate a significant toxicity, and consequently the substance does not meet the criteria for classification of STOT-RE. 

 

References

*Ennulat D, Magid-Slav M, Rehm S, Tatsuoka KS. Diagnostic performance of traditional hepatobiliary biomarkers of drug-induced liver injury in the rat. Toxicol Sci. 2010;116(2):397-412.

*Huntingdon Life Sciences. Mayol: Preliminary toxicity study by oral gavage administration to Crl:CD(SD) rats for 14 days. Report No. HIK0015. 2013. Huntingdon Life Sciences, Eye, Suffolk, UK. April 10, 2013.

*Thoolen B, Maronpot RR, Harada T, Nyska A, Rousseaux C, Nolte T, et al. Proliferative and nonproliferative lesions of the rat and mouse hepatobiliary system. Toxicol Pathol. 2010;38(suppl):5S-81S.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

188 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The information is reliable and consistent with the database as a whole.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The key study was a repeated dose toxicity study performed in accordance with OECD test guideline No. 408 and in compliance with GLP, test substance was administered via the diet at 300, 1000 and 3000 ppm over a period of 13 weeks to Crl:CD(SD) rats. Control animals received basal diet. This study included a 10-week recovery period. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, sperm analysis, macropathology and histopathology investigations were undertaken.

 

Dietary administration of the test substance at concentrations up to and including 3000 ppm (equivalent to 188 mg/kg/day for males and 220 mg/kg/day for females) for 13 weeks was well tolerated. There were no premature deaths, no test item-related signs observed during the detailed physical examination and arena observations, and sensory reactivity observations, grip strength, motor activity scores and ophthalmoscopy assessments for all groups of animals during Week 12 were unaffected by treatment.

Males and females given 3000 ppm showed statistically significantly reduced body weight gain during Week 0-2 of treatment, such that overall mean weight gain during the 13-week dosing period was 96-98% of Control, although statistical significance was not attained for the difference in overall mean weight gain; the mean weight gain of males and females given 300 or 1000 ppm was unaffected. Following the cessation of treatment, the mean body weight gain of Recovery phase males and females which had previously received 3000 ppm was unaffected by previous test substance administration.  

During the first two weeks of treatment, mean food consumption was reduced, when compared to Control and pre-treatment values for females given 1000 or 3000 ppm; this reduction in food intake continued during the remainder of the treatment period for females given 3000 ppm. The food intake of females given 300 ppm and for all groups of treated males during the 13-week treatment period was unaffected by test substance administration, and the food intake of animals previously given 3000 ppm was essentially similar to Control throughout the 10-week recovery period.

Analysis of haematological parameters in Week 13 revealed, when compared to Controls, the following statistically significant difference: reduced haemoglobin concentrations in females given 3000 ppm; reduced haematocrit in all groups of treated females; a non dose-dependent slight decrease in neutrophil, monocyte and large unstained cell concentrations in all groups of treated females; a slight decrease in eosinophil concentrations in females given 1000 or 3000 ppm; reduced platelet counts for males given 3000 ppm; a dose dependent slight prolongation of prothrombin times for females given 1000 or 3000 ppm. All values were within the Historical Control Data (HCD) range and similar differences were not evident following 10 weeks of recovery, indicating full reversibility of these minor changes. 

During Week 13, biochemical analysis of plasma revealed the following statistically significant differences when compared to Controls: a trend towards slightly reduced alanine aminotransferase activity in females given 1000 or 3000 ppm; slightly increased alkaline phosphatase activity in females given 3000 ppm; slightly high plasma urea and blood urea nitrogen concentrations in females given 1000 or 3000 ppm; reduced cholesterol concentrations for males and females at 3000 ppm; reduced triglyceride concentrations for females at 3000 ppm; slightly increased chloride concentrations for both sexes given 3000 ppm and for males given 1000 ppm; slightly increased phosphorus concentrations for males at 3000 ppm and slightly decreased for all groups of treated females; slightly reduced calcium concentrations for females given 1000 or 3000 ppm; reduced total protein associated with reduced albumin concentration for females given 3000 ppm. All of these minor differences showed full recovery after 10 weeks off-dose.

Analysis of urine collected during Week 13 of treatment revealed a slight, non-dose dependent decrease in urinary protein output in females given 1000 or 3000 ppm. Values were within the HCD range and a similar difference was not apparent 10 weeks after the cessation of dosing. Microscopic examination of the urine sediment did not reveal any abnormalities at any dietary level investigated.

The analysis of organ weights following 13 weeks of treatment indicated a slight increase in mean absolute and adjusted adrenal gland weights for females given 3000 ppm. There were no test item-elated changes in organ weights following 10 weeks of recovery.

Sperm motility, morphology and sperm counts were unaffected by test substance administration at all dietary concentrations investigated.

There were no macroscopic abnormalities detected at scheduled termination after 13 weeks of treatment or after 10 weeks of recovery that were attributable to treatment with test substance. Histopathological evaluation of the retained tissues of the 3000 ppm animals killed after 13 weeks of treatment did not reveal any test item-related micropathological changes.

Conclusion

It was concluded that dietary administration of the test substance to Crl:CD (SD) rats at dietary concentrations 300, 1000 or 3000 ppm for 13 weeks provided clear evidence of systemic exposure but no effects that were deemed to be adverse. There were no test item-related histopathological changes observed for any tissues examined in this study. Plasma biochemistry and urinalysis revealed several slight changes in composition, predominantly in females, which were indicative of adaptations of metabolism/excretion in the liver and kidneys. In the absence of any change in organ weight or any evidence of degenerative or functional change in the liver and kidneys during histopathological evaluation, the slight disturbances of biochemical and urine parameters were considered not to be adverse. The No Observed Adverse Effect Level (NOAEL) was concluded to be 3000 ppm (equivalent to 188 mg/kg/day in males and 220 mg/kg/day in females).

A 28-day oral gavage toxicity study was conducted in Crl:CD (SD) rats to evaluate the potential systemic toxicity of the substance (Huntingdon Life Sciences, 2013a). This GLP-compliant study was designed and conducted according to the OECD Testing Guideline No. 407. Three groups, each comprising five male and five female Crl:CD (SD) rats received the substance at oral gavage doses of 0 (corn oil vehicle), 30, 100 or 300 mg/kg/day at a volume-dose of 4 mL/kg bodyweight. Dose levels for the 28-day study were selected based on a 14-day oral gavage toxicity study in the same strain of rats (Huntingdon Life Sciences 2013b). In the 14-day study, the daily gavage administration of the substance at doses up to and including 500 mg/kg/day was well tolerated. However, terminal investigations revealed increased liver weights in males and females receiving 300 or 500 mg of the substance/kg/day, with the liver of one female receiving 500 mg/kg/day being macroscopically enlarged (despite the increased dose levels, the magnitude of these liver weight increases were comparable between the 2 groups of males and also comparable between the 2 groups of females). Additionally, computer assisted sperm analysis (CASA) revealed treatment-related effects on sperm motility, sperm concentration, and total number of epididymal spermatids at dose levels of 300 and/or 500 mg/kg/day.  Testicular sperm numbers were unaffected at all dose levels. Although it was considered likely that a high dose level of 500 mg/kg/day would be tolerated for the duration of the OECD 407 28-day toxicity study, in view of the sperm effects detected in this preliminary study, and taking into consideration the longer period of dosing in the 28-day study, the high dose level for the OECD 407 study was set below 500 mg/kg/day.

 

During the 28-day study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food and water consumption, haematology (peripheral blood), blood chemistry, organ weight, sperm analysis, macropathology and histopathology investigations were undertaken. As expected, treatment with the substance at doses up to and including 300 mg/kg/day was well tolerated; there were no mortalities and no treatment-related clinical signs observed. Salivation, chin-rubbing, and increased water consumption at the high dose were consistent with observations for orally-gavaged substances that are unpalatable to rats. Neurobehavioral assessments conducted during Week 4 of treatment revealed a slight increase in rearing and cage-floor locomotor activity among females receiving 300 mg/kg/day that was attributed to the activity of two females at this dose level. The biological significance of this finding is uncertain in the absence of similar effects during detailed arena observations and routine clinical observations, or any histopathological changes in the brain, spinal cord, nerves or muscles. There was no effect of treatment on sensory reactivity or grip strength at any dose level investigated. There were no adverse effects on bodyweight and mean food consumption.

 

The haematological investigation conducted after four weeks of treatment revealed no treatment-related changes. Test article-related biochemical changes in the blood plasma included a slight increase in aspartate amino-transferase (AST) concentrations in males and females receiving 300 mg/kg/day, elevated bile acid and slightly high urea concentrations among females at 300 mg/kg/day, slightly low glucose concentrations in all groups of treated males, slightly low cholesterol concentrations in males and females at 300 mg/kg/day and marginally low sodium concentrations among females receiving 300 mg/kg/day. All of these statistical differences from Control values were within the Historical Control Data range.

 

There were no treatment-related abnormalities detected at macroscopic examination after 28 days of treatment. Histopathological changes that were attributable to treatment occurred in the liver, where there was an increase in the incidence and/or severity of periportal hepatocyte vacuolation; the severity of this lesion was greater in females than in males, with all female dose groups affected in a dose-dependent manner, whereas only males given 300 mg/kg/day were affected. The only other hepatic change was an increase in liver weights in males at the high dose and in groups of treated females. Importantly, there was no evidence of any degenerative liver changes. Moreover, all biochemistry changes that might have been related to the liver changes are considered non-adverse, given that the values fell within the Historical Control Data range and were unaccompanied by liver necrosis or indications of degeneration. Biochemistry changes such as alterations in AST and cholesterol are of limited diagnostic utility in the absence of histological changes such as necrosis or hepatic degeneration (Ennulat et al., 2010). With regard to the periportal vacuolation, no further description or details were provided in the study report. It is important to note that hepatic macrovesicular vacuolation is considered a possible physiological adaptation to address an imbalance between hepatocyte uptake of lipids from the blood and secretion of the lipoproteins that carry the lipids (Thoolen et al., 2010). Taken altogether, while these liver changes were suggestive of possible alterations to intra-hepatocellular fat metabolism/transport in the high dose animals, as there was no evidence of any degenerative liver changes, the overall liver changes are considered likely to be adaptive and non-adverse. Hepatocyte vacuolation without evidence of degenerative or functional change reflected as a disturbance of biochemical parameters is considered not to be adverse. As there was no evidence of degenerative or functional changes at low- and mid-dose levels, the morphological change of hepatocyte vacuolation observed at 30 or 100 mg/kg/day in female rats was considered by the Study Director not to be adverse.  Similarly, given the low magnitude of the biochemical changes (falling within the Historical Control data range) and the absence of degenerative changes in the liver, the findings at 300 mg/kg bw/day are also considered not to be adverse.

 

CASA revealed a reduction in sperm velocity and amplitude of lateral head displacement, with an associated increase in static sperm and a decrease in the percentage of motile and progressively motile sperm at 300 mg/kg/day. Sperm motility was unaffected by treatment at 30 or 100 mg/kg/day, and there was no evidence of an effect of treatment on epididymal or testicular spermatid concentrations (millions/g) and total sperm at any dose level investigated.

 

Kidney weights were increased in males at 30 mg/kg/day and in males and females at 100 and 300 mg/kg/day, unaccompanied by any histological change, and liver weights were increased in males at 300 mg/kg/day and all groups of treated females compared to respective Control groups. Males receiving 100 or 300 mg/kg/day also had slightly increased testis weights compared to Controls, but there were no microscopic correlates for the effects on sperm motility or testis weights.

 

It should be noted that the effects on sperm motility are considered to be due to oxidative stress occurring as a result of testicular metabolism of the substance made possible by high plasma levels of the substance and its metabolites following oral gavage administration of a high bolus dose (see IUCLID Section 7.8). An overloading of the liver, emphasized with oral gavage dosing, results in the substance and/or its non-conjugated metabolites entering into the circulation, resulting in metabolism in the testes. As the oxygen required for metabolism is in short supply in these tissues, oxidative stress may have occurred in the testes and resulted in sperm effects. The high gavage doses (>100 mg/kg/day), fast oral absorption, and distribution lead to secondary effects in the testis. The lack of any changes in testicular tissues supports the conclusion that the sperm effects are not due to direct injury to this organ.

 

Regarding the liver, while it is acknowledged that the hepatic changes at the oral gavage dose level of 300 mg/kg/day were suggestive of possible alterations to intra-hepatocellular fat metabolism/transport in the high dose animals, there was no evidence of degenerative liver changes in either sex at any dose level.  Moreover, as an adaptive change, it seems likely that the vacuolation would be reversible. A subsequent study using the oral dietary route of administration in the same strain of rats for a total of 18 days showed a slight, reversible, increase in adjusted, but not absolute, liver weights, with no treatment-related macroscopic changes (Huntingdon Life Sciences, 2014; see Section 7.8.1 Repeated dose toxicity: 14-day oral (dietary).002). The liver weight changes in this subsequent dietary study were smaller in magnitude to those seen in the initial 14-day oral gavage study, and was not accompanied by any macroscopic evidence of liver changes. While it is acknowledged that the exposure duration was shorter than in the 28-day study, the reversibility of the liver changes shown in the 14-day dietary study supports the likely reversibility of liver changes observed with the substance. Based on the results and taking into consideration the “slight” nature of some of the changes (e.g., the clinical chemistry findings were values that were within the Historical Control Data range), it is concluded that the liver findings may represent an adaptive change. Given the lack of any degenerative changes in the liver, it is considered that the liver findings at 300 mg/kg/day do not represent a significant toxicity. 

 

References

*Ennulat D, Magid-Slav M, Rehm S, Tatsuoka KS. Diagnostic performance of traditional hepatobiliary biomarkers of drug-induced liver injury in the rat. Toxicol Sci. 2010;116(2):397-412.

*Huntingdon Life Sciences. Mayol: Toxicity study by oral (gavage) administration to Crl:CD(SD) rats for 4 weeks. Report No. HIK0016. 2013a. Huntingdon Life Sciences, Eye, Suffolk, UK. April 25, 2013.

*Huntingdon Life Sciences. Mayol: Preliminary toxicity study by oral gavage administration to Crl:CD(SD) rats for 14 days. Report No. HIK0015. 2013b. Huntingdon Life Sciences, Eye, Suffolk, UK. April 10, 2013.

*Huntingdon Life Sciences. Mayol: 14-Day investigative toxicity study by dietary administration to Crl:CD(SD) rats with a 4-week recovery period Report No. HIK0023. 2013b. Huntingdon Life Sciences, Eye, Suffolk, UK. February 20, 2014

*Thoolen B, Maronpot RR, Harada T, Nyska A, Rousseaux C, Nolte T, et al. Proliferative and nonproliferative lesions of the rat and mouse hepatobiliary system. Toxicol Pathol. 2010;38(suppl):5S-81S.

Justification for selection of 90 -day repeated dose toxicity via oral route - systemic effects endpoint:

Klimisch 1 study conducted to testing guideline with no deviations.  This is the longest study available that includes evaluation of systemic toxicities.  

Justification for classification or non-classification

No adverse effects were observed in the Key study, a 90 -day repeat dose oral study via the diet, consequently no target organs were identified.

The results of the 28-day study showed non-adverse morphological changes in the liver, and changes in sperm motility. However, it should be noted that the effects on sperm motility observed in this study are considered to fall within the category of data contributing to an understanding of the classification of the substance for reproductive toxicity and is not further addressed here, other than to note that the effects on sperm motility are considered not to be a primary toxicity of the substance itself. All findings related to the sperm effects are discussed in IUCLID Section 7.8.

 

Therefore, the discussion of possible primary target organs for the substance should consider the findings in the liver. Based on the results and the discussion above, and taking into consideration the “slight” nature of some of the changes (e.g., the clinical chemistry findings were values that were within the Historical Control Data range), that the liver finding of vacuolation may represent an adaptive change, and the lack of any degenerative changes in the liver, it is considered that the liver findings at 300 mg/kg/day do not represent an adverse toxic effect.

 

The liver morphology changes together with the minor clinical chemistry changes are not considered to meet the criteria for classification, based on the following considerations:

--there were no degenerative changes observed in the liver; and

--the changes observed (hepatocyte vacuolation) were not indicative of significant functional changes in the liver, nor significant organ damage, nor necrosis, fibrosis or granuloma, nor a morphological change that provides evidence of marked organ dysfunction, nor was there evidence of cell death (see criteria for classification Category 2, in Annex 1, Section 3.9.2.7.3). 

As an additional note, morbidity or death resulting from long-term exposure to the substance is not expected, based on in silico predictions that it would be rapidly metabolized and would not bioaccumulate (see IUCLID Section 7.2). In this study, there were no indications of systemic toxicities due to the adaptive changes in the liver. The liver-related clinical chemistry findings themselves are not considered to support classification for specific target organ toxicity following repeated exposure (STOT-RE) as these were “small changes in clinical biochemistry” that may be of “doubtful or minimal toxicological importance” (see Annex 1, Section 3.9.2.8).

 

In summary, with regard to classification, it is considered that the liver findings observed following 28 days of oral dosing with the substance do not indicate adverse toxicity, and consequently the substance does not meet the criteria for classification of STOT-RE according to Regulation (EC) No 1272/2008, Annex I section 3.9.