Acetic acid, anhydride, reaction products with 1,5,10-trimethyl-1,5,9-cyclododecatriene

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996, and OECD 407 (1995) and METI (2003)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

482-330-9

EC Name:

-

Cas Number:

144020-22-4

Molecular formula:

C17H26O

IUPAC Name:

1-[(1E,5Z,9Z)-2,5,10-trimethylcyclododeca-1,5,9-trien-1-yl]ethan-1-one; 1-[(1R)-2,5,10-trimethylcyclododeca-2,5,9-trien-1-yl]ethan-1-one; 1-[(1R)-4,9-dimethyl-12-methylidenecyclododeca-4,8-dien-1-yl]ethan-1-one; 1-[(1S)-2,5,10-trimethylcyclododeca-2,5,9-trien-1-yl]ethan-1-one; 1-[(1S)-4,9-dimethyl-12-methylidenecyclododeca-4,8-dien-1-yl]ethan-1-one

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

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: 29 to 35 days
- Weight at study initiation: 118-145 g for males and 112-134 g for females
- Fasting period before study: no
- Housing: The animals were housed five of one sex per cage. The cages were made of a polycarbonate body with a stainless steel mesh lid. The cages had wood shavings as bedding (Lignocel 3/4 woodflakes).
- Diet (ad libitum): standard rodent diet (Rat and Mouse No. 1 Maintenance Diet), except when urine was being collected and before routine blood sampling.
- Water (ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes, except when urine was being collected. All animals were allowed access to water for 1 hour before routine blood sampling.
- Acclimation period: 12 days before treatment commenced

DETAILS OF FOOD AND WATER QUALITY: The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Each batch of diet was analysed routinely by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinised and approved before any batch of diet was released for use. The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. Certificates of analysis were received routinely from the water supplier.

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

IN-LIFE DATES: From: 26 April 2007 (animal arrival) To: 5-19 June 2007 (necropsy main study - recovery study)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

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

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was prepared for administration as a series of formulations at the required concentrations prepared by dilution of individual weighings of the test substance. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

DIET PREPARATION
- Rate of preparation of diet (frequency): All formulations were prepared weekly.
- Mixing appropriate amounts with (Type of food): standard rodent diet (Rat and Mouse No. 1 Maintenance Diet)

VEHICLE
- Concentration in vehicle: 0, 3, 30, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Remarks:

The test formulations prepared for Week 1 of treatment were analysed for achieved concentration of the test substance.

Details on analytical verification of doses or concentrations:

Four samples (nominally 1 mL accurately weighed) were taken from each group; 2 samples were assayed from each formulation. The remaining
samples from each formulation were retained frozen (nominally -20°C) as contingency for analysis if any result required confirmation.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 at 0 and 1000 mg/kg bw/day
5 at 15 and 150 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE 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 (before dosing during the treatment period), by an observer unaware of the experimental group to which the animal belonged.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily observations at the following times in relation to dose administration. Week 1: Predose, 1 to 2 hours after completion of dosing all groups, as late as possible in the working day. Day 8 to Day 15: predose, 1 to 2 hours after completion of dosing all groups, an observation was performed as late as possible in the working day if considered necessary to better define the end point of any continuing signs. Day 16 to termination: Predose, as each animal was returned to its home cage, at the end of dosing each group, between 1 and 2 hours after completion of dosing of all groups or, when the duration of dosing was protracted, 1 to 2 hours after completion of each group. as late as possible in the working day. During the acclimatisation and recovery periods, observations of the animals and their cages were recorded once per day.

BODY WEIGHT: Yes
- Time schedule for examinations: on the day that treatment commenced (Day 1), on Days 8, 15, 22 and 28 of treatment, on Days 1, 8, and 14 of the recovery period and before each 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 each week throughout the treatment and recovery periods. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On Day 29, at the end of the treatment period (Main study animals) and on Day 15 of recovery (Recovery animals)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined: Haematocrit (Hct), Haemoglobin (Hb), Erythrocyte count (RBC), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Platelet count (Plt), Total white cell count (WBC), Differential WBC count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)), Prothrombin time (PT) and Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On Day 29, at the end of the treatment period (Main study animals) and on Day 15 of recovery (Recovery animals)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined: Alkaline phosphatase (ALP), Alanine amino-transferase (ALT), Aspartate amino-transferase (AST), Total bilirubin (Bili), Urea, 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) and Albumin/globulin ratio (A/G Ratio)

PLASMA/SERUM HORMONES/LIPIDS: Yes

URINALYSIS: Yes
- Time schedule for collection of urine: On Day 29 (Main study animals) and Day 15 of recovery (Recovery animals), overnight urine samples were collected from all animals. Animals were placed in an individual metabolism cage without food or water at approximately 16.00 hours; urine was collected until approximately 07:00 hours (Day 29) or 08:00 hours (Day 15 of recovery) the following day.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Appearance (APP), Volume (Vol), pH, Specific gravity (SG), Protein (Prot), Glucose (Gluc), ketones (Keto), bile pigments (Bili), blood pigments (UBld), Deposit examination (Epithelial cells (Epi), Polymorphonuclear leucocytes (Leuc), Erythrocytes (RBC), Crystals (Cryst), Spermatozoa and precursors (Sperm), Casts and Other abnormal components (A))

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Sensory reactivity, grip strength and motor activity assessments were performed (before dosing) during Week 4 of treatment. Sensory reactivity assessments were repeated for all Recovery phase animals during Week 2 of the Recovery period.
- Dose groups that were examined: all recovery phase animals and all main study animals in 15 and 150 mg/kg bw/day dosing groups.
- Battery of functions tested: sensory activity, grip strength and motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- The following organs, taken from each animal killed after 4 weeks of treatment or 2 weeks of recovery, were dissected free of adjacent fat and other contiguous tissue and the weights recorded: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus
- Samples or the whole of the following tissues are preserved: Adrenals, Aorta - thoracic, Brain, Caecum, Colon, Duodenum, Epididymides, Eyes, Femurs, Harderian glands, Head, Heart, Ileum (including Peyer’s patch), Jejunum, Kidneys, Lachrymal glands, Liver, Lungs, Lymph nodes (mandibular, mesenteric and left axillary), Mammary area – caudal, Oesophagus, Optic nerves, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submandibular and sublingual), Sciatic nerves, Seminal vesicles, Skeletal muscle, Skin, Spinal cord, Spleen, Sternum, Stomach, Testes, Thymus, Thyroid with parathyroids, Tongue, Trachea, Urinary bladder, Uterus and cervix, Vagina and any abnormal tissue.

HISTOPATHOLOGY: Yes
- Tissues subject to histological processing: Adrenals - cortex and medulla, Brain - cerebellum, cerebrum and midbrain, Femur with joint - longitudinal section including articular surface, epiphysial plate and bone marrow, Heart - included auricular and ventricular regions, Kidneys - included cortex, medulla and papilla regions, Liver - section from all main lobes, Lungs - section from two major lobes, to include bronchi, Spinal cord - transverse and longitudinal section at the cervical, lumbar, and thoracic levels, Sternum - included bone marrow, Stomach - included keratinised, glandular and antrum in sections, Thyroid - included parathyroids in section where possible, Uterus - uterus section separate from cervix section

Statistics:

The following sequence of statistical tests was used for grip strength and motor activity, bodyweight, organ weight and clinical pathology data: If 75% of the data (across all groups) were the same value, for example c, then a frequency analysis was applied. Treatment groups were compared using pairwise Fisher's Exact tests for each dose group against the control both for i) values =c, and for ii) values <=c versus values >c, as applicable. If Bartlett's test for variance homogeneity was not significant at the 1% level, then parametric analysis was applied. For comparisons involving two groups only, a t-test was used. For all other comparisons the F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose-response was not monotone, Dunnett's test was performed instead. If Bartlett's test was significant at the 1% level, then logarithmic and square-root transformations were tried. If Bartlett's test was still significant, then non-parametric tests were applied. For comparisons involving two groups only a Wilcoxon rank sumtest was used. For all other comparisons the H1 approximate test,the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.
For organ weight data, analysis of covariance was initially performed using terminal bodyweight as covariate. Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All animals receiving 1000 mg/kg/day showed post-dose salivation. This sign was first apparent for some animals on Day 13. With the exception of one female on Day 23, from Day 16 all animals were showing this sign daily. The salivation was present either immediately after dosing or at the check performed after completion of dosing the group and it had generally resolved by the last check of the day (approx 3-4 hours post-dose). On Day 23 only, all males at this treatment level showed pre-dose salivation.
Post-dose salivation was also evident in a total of 3 animals of each sex receiving 150 mg/kg/day. In this group the sign was first recorded from Day 21 and was present on one to three occasions for each of the affected animals. Onset was at the end of group check, with resolution apparent by the 1-2 hour post-dose check.
Post-dose chin rubbing was evident in all males at 1000 mg/kg/day and 9/10 females. This sign was first recorded on Day 18 and was evident on an intermittent basis thereafter. The chin rubbing was generally evident from immediately after dosing but had usually ceased by the 1-2 hour post-dose check.
The same females and one male at 150 mg/kg/day which demonstrated post-dose salivation, plus a further two males at this dose level also showed a low incidence (one to three occasions) of post-dose chin rubbing. The sign was present immediately after dosing but had resolved by the 1-2 post-dose check.
In addition, repetitive movements was recorded in 3/10 males and 4/10 females receiving 1000 mg/kg/day. This sign was only recorded on Day 18 of Week 3 and was only noted immediately post dose. Due to the isolated occurrence of this sign, it is of doubtful relationship to treatment and not of toxicological importance.
No post dose signs were observed in animals receiving 15 mg/kg/day.

For more information see Appendix 2 of the attached report.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males at 150 or 1000 mg/kg/day showed treatment-related but not toxicologically important lower than control mean bodyweight gains.
The overall (Day 1 to 28) mean bodyweight gains for males receiving 150 or 1000 mg/kg/day were lower than control, showing statistical significance and an apparent dose-relationship. Review of the individual bodyweight gains for these males however revealed the majority of animals had bodyweight gain values which were within or just below the low end of the respective control ranges, although tending to congregating towards the low end of the concurrent control range.
Females receiving 1000 mg/kg/day also showed a lower overall mean bodyweight gain compared with controls. The spread of the individual bodyweight gains for these females was however similar to controls and other treated groups.
Bodyweight gains for males treated at 15 mg/kg/day and females treated at 15 or 150 mg/kg/day were generally similar to respective controls.
During the recovery period, both sexes previously treated 1000 mg/kg/day recorded bodyweight gains similar to those of respective controls, indicating recovery, in males, from the previous effects of treatment.

For more information see Figure 2, Table 3, and Appendix 5 of the attached report.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment.
During the treatment period, food consumption values for all treated groups from both sexes were generally similar to controls.
During the recovery period, both males and females previously treated at 1000 mg/kg/day, showed mean cage food consumption similar to those of respective controls.

For more information see Table 4, and Appendix 6 of the attached report.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related changes were recorded in red blood cell and clotting function, with general recovery seen after a period without treatment.

At the end of the treatment period:
- Statistically significant lower than control group mean haematocrit (-4.9%) and haemoglobin (-5.2%) were recorded for females receiving 1000 mg/kg/day. Female red blood cell counts were low to a similar degree, but not achieving statistical significance. With the exception of one individual, Hb values for these females were within the expected range. There was no effect on red blood cells parameters in treated males.
- Both sexes receiving 1000 mg/kg/day showed higher group mean platelet counts (19.1 and 19.3% for male and female, respectively), statistical significance attained and some individual values being outside the expected background range. Males receiving 1000 mg/kg/day also demonstrated a higher than control mean activated partial thromboplastin (23.8%); the three males which showed the highest activated partial thromboplastin times also showed the highest platelet values. There was no similar effect on activated partial thromboplastin times for treated females and all individual values were within the expected background range. Males receiving 1000 mg/kg/day exhibited a longer mean prothrombin time when compared with controls (4.4%); statistical significance was attained but all values were within the expected background range. In contrast, females at this dose level showed a statistically significant lower (-12.5%) than control mean prothrombin time, but all individual values for this group were within the expected background range.

At the end of the recovery period:
- Four out of five haematocrit values for females previously treated at 1000 mg/kg/day were higher (3.6%) than concurrent control values, but all values were with the expected range. Hb and red blood cell values for previously treated males were similar to controls. As a result of the elevated Hct value females previously treated at 1000 mg/kg/day also showed a lower mean MCHC value (-1.7%) compared with control.
Females previously treated at 1000 mg/kg/day continued to show higher than control platelet counts but the magnitude of difference from the controls was less than seen at the end of the treatment period (X1.12 compared to X1.19 during treatment). Platelet values for previously treated males were considered similar to controls. The mean activated partial thromboplastin and prothrombin times for previously treated males was still marginally slower in previously treated males, when compared with controls, but this was mainly attributable to single animals (No.10 for APTT and No.8 for PT), with values for the remaining animals were generally similar to controls. The mean activated partial thromboplastin and prothrombin times for previously treated females were similar to controls.

For more information see Table 5 and Appendix 7 of the attached report.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related changes were recorded for bilirubin, alkaline phosphatase, alanine transferase, aspartate amino-transferase, glucose, cholesterol, calcium, phosphorus, chloride and protein values.

At the end of the treatment period:
- Statistically significant higher than control group mean bilirubin concentrations were evident for in both sexes receiving 1000 mg/kg/day (2.5x for both sexes). Males at 150 mg/kg/day also tended to show higher than control bilirubin levels (1.5x), although statistical significance was not achieved.
- Both sexes receiving 1000 mg/kg/day had statistically significantly lower alkaline phosphatase (Males -35%, Females -28%) and aspartate amino-transferase concentrations (Males -38%, Females -30%) when compared with controls. Males receiving 1000 mg/kg/day showed statistically significant lower then control mean concentration (-24%); however female alanine transferase concentrations at this dose level were similar to control. Individual values for these parameters in the aforementioned groups were below or towards the low end of the concurrent control range, with some values also being below the expected background range.
- Lower than control group mean glucose was recorded for both sexes receiving 1000 mg/kg/day (Males -19%, Females -18%) and for males receiving 150 mg/kg/day (-11%), with 1000 mg/kg/day attaining statistical significance. These differences from controls arose due to values for the mentioned treated groups being below or towards the low end of the concurrent controls, although values were within the expected background range. Statistically significant higher than control cholesterol was recorded for both sexes treated at 1000 mg/kg/day (Males 1.43x, Females 1.68x). Individual values for males were higher than the highest concurrent controls and expected background value, whilst values for females at 1000 mg/kg/day tended to be within both ranges.
- Mean calcium (Males 1.06x, Females 1.08x) and phosphorus (Males 1.11x, Females 1.15x) concentrations for both sexes receiving 1000 mg/kg/day were higher when compared with control . Lower then control mean chloride concentrations were recorded for both sexes receiving 1000 mg/kg/day (Males -3%, Females -2%). Statistical significance was attained for all 3 parameters.
- Higher than control group mean total protein was apparent for both sexes at 1000 mg/kg/day (Males 1.13x, Females 1.15x). For males this was mainly due to higher globulin levels, resulting in a lower mean A/G ratio (-15%). Females at this dosage also showed a lower than control mean A/G ratio (-7.8%) although both the albumin (1.14x) and globulin levels were higher than controls. Statistical significance was attained for the total protein and A/G ratios for both sexes and albumin value for females.

At the end of the recovery period:
- Minor differences from control were evident in sodium and albumin levels. Full recovery was considered to be evident in the other affected parameters mentioned above. The mean alkaline phosphatase value for males previously treated at 1000 mg/kg/day continued to be slightly low (-11%) when compared with controls however this was due to a single male (No.9).
- A statistically significantly higher mean sodium level (1.01x) was recorded for females previously treated at 1000 mg/kg/day when compared with controls, however as the majority of individual values for previously treated females were within the control ranges recorded at the end of the treatment and recovery phase this was considered not to be an effect of previous treatment.
- A statistically higher than control albumin value was evident for males (1.06x) previously treated at 1000 mg/kg/day; however again there was overlap between values recorded for controls at both sets of investigations and thus this difference was considered to be a result of normal variation.
- There were no other differences from control, indicating good general recovery from the
previous effects of treatment.

For more information see Table 6 and Appendix 8 of the attached report.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant lower group mean urinary protein was recorded for both sexes receiving 1000 mg/kg/day (Males: -71%, Females: -100%) and in females receiving 150 (-78%) or 15 mg/g/day (-67%) when compared with control; dose-related in females. This effect was most marked in females receiving 1000 mg/kg/day, where every individual value was zero.
At the end of the recovery period, urinary protein values were considered similar to controls in both sexes indicating recovery.

For more information see Table 7 and Appendix 9 of the attached report.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Sensory reactivity observations and grip strength (Tables 1; Appendix 4 of attached report)
Sensory reactivity observations during Week 4 of treatment were unaffected by treatment.
Some males receiving 1000 mg/kg/day showed reduced sensory reactivity responses compared with controls; with no response to touch in two animals (Nos. 6 and 7) and a weak response to tail pinch in three animals (Nos. 6, 7 and 9). During Week 2 of the Recovery phase, when the sensory reactivity assessments were repeated, these individuals all showed a normal response. In the absence of other findings, the nature and incidence of these inter-group differences were considered to indicate natural variation rather an effect of treatment. Sensory reactivity responses of females in all treated groups were similar to controls.
Grip strength values during Week 4 of treatment were unaffected. As no treatment related changes were noted during treatment, this parameter was not monitored during the recovery phase.
Compared with controls, group mean forelimb grip strength values were slightly low for males in all treated groups and for females receiving 1000 mg/kg/day. There was, however, no dose-relationship between group mean values for treated males and none of the differences achieved statistical significance. These differences were therefore considered to be fortuitous. Mean forelimb strength for males at 15 or 150 mg/kg/day and hindlimb strength for females at 150 mg/kg/day were lower than controls, however values for higher treated groups were considered to be similar to controls indicating the differences were not due to treatment.

Motor activity (Figure 1; Table 2; Appendix 3 of attached report)
Motor activity scores for males and females during Week 4 of treatment were unaffected. As no treatment related changes were noted during treatment, this parameter was not monitored during the recovery phase.
Male activity - Overall mean low and high beam scores for all treated groups were similar to controls. Three males at 1000 mg/kg/day showed lower individual high beam overall scores than controls, but when comparing the individual time-points, the values were generally similar to those of controls except at 42 minutes, whereas controls seem to have a "burst" of activity, during which the low beam scores were up as well. Looking at the individual low beam scores for males at 1000 mg/kg/day for the various time points, they generally reflect the ranges seen in controls and thus the overall conclusion is there were no treatment-related effects on the male activity.
Female activity - With exception of one value (No. 49), overall values for individual animals within or very close to concurrent control range and during first part of the hour, values for females at 1000 show similar activity to controls. Group mean total high beam scores (rearing activity) showed a weak dose-related increase compared with controls (from about 48 minutes), but this was due to largely to the considerable inter and intra-group variation during the second half of the 1-hour test period, when individual animal variation tends to be higher anyway. Therefore this was considered not to be treatment-related.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Adjusted group mean liver weights were higher for female groups receiving 150 (1.22x) or 1000 (1.67x) mg/kg/day, exhibiting a dose related trend, and in males receiving 1000 mg/kg/day (1.32x) when compared with controls, attaining statistical significance.
Group mean adjusted kidney weights were higher for female groups receiving 150 (1.12x) or 1000 (1.14x) mg/kg/day, achieving statistical significance but not showing a dose related trend when compared with control. No similar effect was noted for males.
Absolute group mean brain weights for all male treated groups were slightly higher than control (1.06x, 1.04x, 1.06x for 50, 150, and 1000 mg/kg bw, respectively). This small, yet statistical significant difference was obtained in all male groups. There was no dose relationship, no similar effect observed in female treated groups and values were within the expected background range. Therefore these differences were considered not to be due to treatment.

A review of the organ weights of animals killed after two weeks of recovery indicated that higher than control group mean adjust liver and kidney weights were still evident in females previously treated at 1000 mg/kg/day (Liver: 1.27x at recovery compared with 1.67x at treatment; Kidney - 1.10x at recovery compared with 1.14x at treatment), indicating a degree of recovery in females from the effects of previous treatment. No appreciable difference from control was observed in absolute liver weight for previously treated males, indicating recovery from treatment.

In addition, recovery animals exhibited slightly lower than control group mean absolute brain weights in both sexes (Males: -2%, Females: -5%), attaining statistical significance in previously treated females, however, the recorded values were well within the expected background control range and are thus considered not to be an effect of previous treatment.

For more information see Table 8 and Appendix 10 of the attached report.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Enlargement liver was seen in one male rat and all female rats treated at 1000 mg/kg/day, but not in any control rats. The incidence and distribution of all the other findings were considered to fall within the background range of macroscopic changes.

Animals killed after 2 weeks of recovery: The macroscopic examination performed at the recovery kill revealed no changes attributable to treatment with the substance. The incidence and distribution of all the findings were considered to fall within the background range of macroscopic changes.

For more information see Table 9, Appendix 11, and Annex 3 of the attached report.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Animals killed after four weeks of treatment: Treatment-related findings
- Liver: Minimal or slight centrilobular hepatocyte hypertrophy was seen in three males and three females given 1000 mg/kg/day, and minimal or slight generalised hepatocyte hypertrophy was seen in the remaining two males and two females of this group. Two females given 150 mg/kg/day showed minimal generalised hepatocyte hypertrophy, and minimal centrilobular hepatocyte hypertrophy was seen in one other female of this group.
- Thyroids: Minimal or, in one case, slight follicular cell hypertrophy was seen in three males and four females given 1000 mg/kg/day.
- Spleen: A slightly increased severity of haemosiderosis and incidence of extramedullary haemopoiesis were seen in females given 1000 mg/kg/day, compared with controls. Neither increase was present in males given 1000 mg/kg/day.
- Incidental findings: All other microscopic findings were considered to be incidental and unrelated to the test substance.

Animals killed after two weeks of recovery: Treatment-related findings
- Liver: Minimal centrilobular hepatocyte hypertrophy was seen in a single female previously given 1000 mg/kg/day. It was considered that almost complete recovery from the hypertrophy seen after 4 weeks of treatment had occurred.
- Thyroids: Minimal follicular cell hypertrophy was seen in one control male and one male previously given 1000 mg/kg/day. It was therefore considered that recovery had occurred.
- Spleen: A marginally increased severity of haemosiderosis was still seen in females previously given 1000 mg/kg/day, compared with controls. However, no increase in incidence of extramedullary haemopoiesis was present. It was considered that partial recovery from the changes seen after 4 weeks of treatment had occurred.
- Incidental findings: All other microscopic findings were considered to be incidental and unrelated to the test substance.

Testes: Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Histopathology conclusion: Treatment-related findings were seen in the liver and thyroids of both sexes given 1000 mg/kg/day and in the spleen of females of this dose group. They comprised centrilobular or generalised hepatocyte hypertrophy in the liver, follicular cell hypertrophy in the thyroids, and an increased severity of haemosiderosis and increased incidence of extramedullary haemopoiesis in the spleen. Centrilobular or generalised hepatocyte hypertrophy was also seen in several females given 150 mg/kg/day. Following a 2-week recovery period, there was evidence of partial or complete recovery from all changes.

For more information see Table 10, Appendix 11, and Annex 3 of the attached report.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: Treatment-related findings were also evident at 150 mg/kg/day, but the degree of difference and number of changes seen at this level was less than at 1000 mg/kg/day. Good evidence of recovery was seen at 1000 mg/kg/day.

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

DISCUSSION

Treatment at these doses was well tolerated, with no toxicologically important clinical signs or effects on bodyweight gain and food consumption. There were however a number of treatment related changes which although reversible in nature and considered not adverse individually, when considered together within the context of the study, indicate a degree of underlying systemic toxicity at the high dose level (1000 mg/kg/day).

Despite exhibiting a dose related trend and a delayed onset, the transient post dose salivation and chin rubbing seen in this study are commonly encountered findings on oral gavage rat studies at this facility and are often associated with the dosing procedure and/or the palatability of the test formulation and not toxicity.

Liver: The centrilobular and generalised hepatocyte hypertrophy seen in all animals given 1000 mg/kg/day correlates with the statistically significantly increased adjusted group mean liver weights and macroscopic enlargement recorded in this dose group. Centrilobular hepatocyte hypertrophy is a common effect of treatment with xenobiotics. Typically, this is due to proliferation of smooth endoplasmic reticulum with increased activity of drug-metabolising enzymes, and as such represents an adaptive response. Increased metabolic activity in the liver may also correlate well with the observed increases in albumin and cholesterol levels which may be due to increased liver activity and consequently decreased glucose levels due to increased glucagon production during the treatment period. Lower than control liver enzyme levels recorded in animals receiving 1000 mg/kg/day also suggest disruption of normal liver activity, although how this correlates with the other findings is unclear. There were however no histopathological findings which indicated the lower enzyme levels were due to an adverse event in the liver or other tissue.

Thyroid: The thyroid follicular cell hypertrophy seen in both sexes receiving 1000 mg/kg/day is often concomitant with centrilobular hepatocyte hypertrophy in the rat. Hepatic microsomal enzymes are important in the deiodinisation and biliary excretion of thyroid hormones, and increased metabolism of these hormones will affect the feedback control of thyroid secretion, resulting in stimulation of the thyroid gland. This is considered to be a rodent specific effect and not of relevance to human health. Plasma calcium levels can rise as a result of thyroid hypertrophy and this may at least be part of the cause for the raised levels seen at 1000 mg/kg/day on this study. In this study there was no indication that the elevated calcium levels were associated with an adverse event. This is further supported by the fact that values for individual males were generally within the expected background range and there was overlap with the background for females also.

Spleen: The increased severity of haemosiderosis and slightly increased incidence of extramedullary haemopoiesis seen in the spleen of females given 1000 mg/kg/day, compared with controls, were considered to be associated with the lower than control red blood cell parameters recorded in females of this dose group. These findings are considered to indicate a slight increase in red cell destruction, with evidence of a regenerative response. Increased red blood cell turnover is further supported by the higher bilirubin levels and possibly by the increased phosphorus level recorded in females receiving 1000 mg/kg/day. Males treated at his dosage also showed elevated bilirubin and phosphorus levels although there were no corresponding effects on red blood cells nor the spleen, thus the aetiology of these findings in males at 1000 mg/kg/day is unclear. Similarly the reason for the lower chloride values for both sexes at 1000 mg/kg/day is not known.

Both sexes at 1000 mg/kg/day showed a clear treatment-related effect on platelet numbers, although within this study this was not clearly linked with an adverse event. Females did not show full resolution of this finding at the end of the two period without treatment. The extended clotting times which indicate a disturbance in the common clotting pathway are contrary to the elevated platelet levels and may be in some way associated with the microscopic changes in the liver. As individual values for the clotting parameters were within the expected background range, in isolation these changes are considered not to be of toxicological importance.

Kidney: Females at 1000 mg/kg/day showed higher kidneys weights but the urinary protein output was lower. Lower urinary protein output in isolation is not usually an indication of toxicity. There were no microscopic histopathology findings which could be associated with the higher kidney weights.

As indicated above, although when taken in isolation none of the differences from controls seen at 1000 mg/kg/day are considered to be of toxicological importance. Taken together however provides evidence that there is a general disturbances in the physiology of the high level animals compared with controls, which is considered indicative of mild toxicity. Thus 1000 mg/kg/day has not been classed a No Observerable Adverse Effect level (NOAEL) on this study.

There were some treatment-related findings at 150 mg/kg/day (lower bodyweight gain, higher bilirubin levels and reduced glucose in males and higher liver and kidney weights and lower urinary protein levels in females). The magnitude of the difference from controls was however generally less than seen for animals at 1000 mg/kg/day and considering all affected parameters at 1000 mg/kg/day showed at least partial recovery the disturbances seen at 150 mg/kg/day were considered not to be of toxicological importance. Thus within the context of this study 150 mg/kg/day was classed as the highest NOAEL.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD TG 407, GLP), the NOAEL was determined to be 150 mg/kg bw/day for males and females, based on haematological, biochemical, organ weight and histopathological effects.

Executive summary:

In a 28-days repeated dose toxicity study (OECD TG 407, GLP) with a 2-week recovery period, the substance was administrated via oral gavage to Sprague-Dawley rats at dosages of 15, 150 and 1000 mg test item/kg bw/day. Control animals received the vehicle, corn oil, alone. All parameters measured from the OECD TG 407 have been recorded.

Clinical signs: No adverse effects considered to be associated with treatment were observed for mortality, clinical signs, neurobehaviour, bodyweight and food consumption.

Haematology: Treatment resulted in lower group mean haematocrit and haemoglobin for females at 1000 mg/kg/day, increased group mean platelet counts for both sexes and activated partial thromboplastin times for males at 1000 mg/kg/day. At the end of the recovery period, general recovery from the previous changes in red blood cell parameters was evident, however higher than control platelet counts were still evident in females previously treated at 1000 mg/kg/day, though magnitude of difference was much less than during treatment.

Urinalysis: No adverse effects considered to be associated with treatment were observed

Biochemical parameters: Higher group mean bilirubin and cholesterol and lower ALP, AST, glucose for both sexes and lower ALT in males, were recorded for animals receiving 1000 mg/kg/day. Males receiving 150 mg/kg/day also recorded lower AST and glucose compared with controls. In addition, higher than control group mean total protein, albumin, calcium and phosphorus and lower mean chloride and AG ratios were recorded for both sexes receiving 1000 mg/kg/day. No toxicologically important changes were recorded at the end of the recovery period, indicating general recovery from the previous effects of treatment.

Organ effects weight: Higher than control adjusted group mean liver weights were recorded for both sexes at 1000 mg/kg/day and for females at 150 mg/kg/day. Group mean adjusted kidney weights were higher for females receiving 150 or 1000 mg/kg/day. At the end of the recovery period, higher than control group mean adjust liver and kidney weights were still evident in females previously treated at 1000 mg/kg/day though recovery was evident. Enlargement of the liver was seen in one male rat and all female rats treated at 1000 mg/kg/day.

Macroscopy: No adverse effects considered to be associated with treatment were observed

Histopathology: Treatment-related histopathological findings were seen in the liver and thyroids of both sexes given 1000 mg/kg/day and in the spleen of females of this dose group. They comprised centrilobular or generalised hepatocyte hypertrophy in the liver, follicular cell hypertrophy in the thyroids, and an increased severity of haemosiderosis and increased incidence of extramedullary haemopoiesis in the spleen. Centrilobular or generalised hepatocyte hypertrophy was also seen in several females given 150 mg/kg/day. Following a 2-week recovery period, there was evidence of partial or complete recovery from these changes.

Conclusions: Under the conditions of this study, the NOAEL for repeated dose toxicity was established to be 150 mg/kg bw/day for males and females, based on haematological, biochemical, organ weight and histopathological effects. Treatment-related findings were also evident at 150 mg/kg/day, but the degree of difference and number of changes seen at this level was less than at 1000 mg/kg/day. Thus as good evidence of recovery was seen at 1000 mg/kg/day, 150 mg/kg/day was concluded to be the highest NOAEL on this study.