Cyclohexanemethanol, 4-(1-methylethyl)-

Repeated dose toxicity: oral

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

cis-4-(isopropyl)cyclohexanemethanol

EC Number:

237-539-8

EC Name:

cis-4-(isopropyl)cyclohexanemethanol

Cas Number:

13828-37-0

Molecular formula:

C10H20O

IUPAC Name:

cis-(4-propan-2-ylcyclohexyl)methanol

Constituent 2

Reference substance name:

trans-(4-propan-2-ylcyclohexyl)methanol

Cas Number:

13674-19-6

Molecular formula:

C10H20O

IUPAC Name:

trans-(4-propan-2-ylcyclohexyl)methanol

Test material form:

liquid

Details on test material:

- Physical state: colourless liquid
- Storage condition of test material: At ambient temperature and protected from light

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

Test animals

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:

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 ppm

Doses / concentrations 2

Dose / conc.:

1 000 ppm

Doses / concentrations 3

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

Examinations

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"

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

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.   

Applicant's summary and conclusion

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).