Reaction Mass of (1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl formate and (1R,2S)-2,6,6-trimethylcycloheptyl formate

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

other: OECD TG 407

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Feb 2003 to 10 Apr 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is an OECD TG 407 under GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Feb 2003 to 10 Apr 2003

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)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health and Welfare Guidelines 1986 for a twenty-eight day repeat dose oral toxicity study as required by the Japanese Chemical Substances Control Law 1973 of the Ministry of International Trade and Industry amended 1986

Qualifier:

according to guideline

Guideline:

other: USA Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation: 28 day study: Males 140 to 190g, Females 122 to 159g; 14 day range-finding study: Males 127 to 162 g, Females 119 to 135 g.
- Housing: groups of five (28-day study) or three (14 day range-finding study) by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper. Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd., Hull, UK).
- Diet: A pelleted diet (Rodent 5LF2 (Certified) Diet, International Product Supplies Ltd., Wellingborough, Northants, UK) Ad libitum
- Water: Mains drinking water from polycarbonate bottles attached to the cage. Ad libitum
- Acclimation period: 28 day study: 8 days. 14 day range-finding study: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least fifteen
- Photoperiod (hrs dark / hrs light): 12/ 12

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

- 28-day repeated dose study: The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day of Arachis oil BP. Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
- 14-day range-finding study: The test material was administered daily, for fourteen consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day of Arachis oil BP. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at Days 4, 8 and 11.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken of each test material formulation and were analysed for concentration of the substance at Safepharm Analytical Services. The concentration of the substance in the test material formulations was determined by gas chromatography (GC) using an external standard technique. The results indicate that the prepared formulations were within ± 9% of the nominal concentration.

Duration of treatment / exposure:

28 days / 14 days (range-finding study)

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

14-day range finding study

No. of animals per sex per dose:

5 (28-day repeated dose study)
3 (14-day range finding study)

Control animals:

yes, concurrent vehicle

Details on study design:

- 28-day repeated dose study: The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.
- 14-day range finding study: Following single dose sighting work treating one male and one female with 1000 mg/kg, two groups, each of six rats (three males and three females) were dosed.

Observations and examinations performed and frequency:

28-DAY REPEATED DOSE STUDY:
- All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends). All observations were recorded.
- Prior to the start of treatment and on Days 7, 14, 21 and 26, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.
- Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait, Tremors, Twitches, Convulsions, Bizarre/ Abnormal/Stereotypic behavior, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin colour, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation.
- Motor Activity: Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was sixteen hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall sixteen hour period and also during the final 20% of the period (considered to be the asymptotic period).
- Forelimb/hindlimb Grip Strength: An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.
- Sensory Reactivity: Each animal was individually assessed for sensory reacttvtty to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, Vocalisation, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Startle reflex, Blink reflex.
- Bodyweight: Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28 and, in the case of recovery group animals, on Days 35 and 42. Bodyweights were also recorded at terminal kill.
- Food Consumption: Food consumption was recorded for each cage group at weekly intervals throughout the study.
- Water Consumption: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.
- Laboratory Investigations: Haematological and blood chemical investigations were performed on all non-recovery test and control group animals at the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 29 and 43. Animals were not fasted prior to sampling. Urinalytical investigations were performed on all non-recovery test and control group animals during Week 4 and on all recovery group animals during Week 6. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.
- Haematology: The following parameters were measured on blood collected into tubes containing potassium EDT A anti-coagulant: Haemoglobin, Erythrocyte count, Haematocrit, Erythrocyte indices (mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration), Total leucocyte count, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Platelet count, Reticulocyte count, Prothrombin time.
- Blood Chemistry: The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea, Glucose, Total protein, Albumin, Albumin/Globulin (A/G) ratio, Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Gamma glutamyltranspeptidase, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Creatinine, Triglycerides, Total cholesterol, Total bilirubin.
- Urinalysis: The following parameters were measured on collected urine: Volume, Specific Gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Reducing Substances, Blood.

14-DAY RANGE FINDING STUDY:
- All animals were examined for overt signs of toxicity, ill health or behavioural change immediately before dosing and one hour after dosing. All observations were recorded.-
Bodyweight: Individual bodyweights were recorded on Days 1, 4, 8, 11 and 14.

Sacrifice and pathology:

28-DAY REPEATED DOSE STUDY:
- Pathology: On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free period, all surviving animals were killed by intravenous overdose of sodium pentobarbitone (Rhone Merieux, Dagenham, Essex, UK) followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
- Organ Weights: The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus.
- Histopathology: Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin: Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Epididymides, Eyes, Gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Muscle (skeletal), Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles, Skin (hind limb), Spinal cord (cervical), Spleen, Stomach, Testes, Thymus, Thyroid/parathyroid, Trachea, Urinary bladder, Uterus. The tissues (Adrenals, Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Epididymides, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Rectum, Seminal vesicles, Spleen, Stomach, Testes, Thymus, Thyroid/parathyroid, Trachea, Urinary bladder, Uterus) from all non-recovery and 1000 mg/kg/day dose group animals were prepared as paraffin wax blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. The liver and spleen from all 15 and 150 mg/kg/day dose group animals were also processed. Since there were indications of treatment-related changes in the liver, kidneys and thyroid glands, examination was subsequently extended to include similarly prepared sections of these tissues from all animals in the other treatment groups, including recovery groups.

14-DAY RANGE FINDING STUDY: On completion of the dosing period, all animals were killed by cervical dislocation and immediately subjected to an internal and external macroscopic examination. No tissues were retained.

Statistics:

Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative urinalytical data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOV A) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. In the case of recovery group data, the analysis performed was a two-tailed t-test incorporating Levene's test for homogeneity of variance. Where Levene's test showed unequal variances among either nonrecovery or recovery group data, the affected parameters were analysed using non-parametric methods: Kruskal-Wallis ANOV A and mann-Whitney 'U' test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 1000 mg/kg/day developed transient increased salivation around the time of dosing from Day 2 onwards together with associated signs of increased salivation approximately one hour after dosing and instances of noisy respiration and stained fur or fur loss. Hunched posture was seen in 1000 mg/kg/day animals of either sex from Day 21 and episodes of tiptoe gait were observed in two females treated with 1000 mg/kg/day on Day 26 only. All findings regressed in recovery 1000 mg/kg/day animals on cessation of treatment. Increased salivation of short duration is often reported following the oral administration of a test material and the daily occurrence of this finding around the time of dosing is usually considered attributable to an unpleasant tasting or locally irritant formulation rather than an indication of systemic toxicity. No treatment-related clinical signs were detected in animals treated with 150 or 15 mg/kg/day. The remaining incidental findings detected were considered to be of no toxicological importance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment-related deaths during the study although one recovery control male was killed in extremis on Day 42 when it sustained injuries while escaping the container in which it was held prior to blood sampling.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related effects in bodyweight. Test animals showed weight gains similar to those of the controls throughout the study period. Statistical analysis of weekly bodyweight gain showed no significant intergroup differences.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no adverse effect on food consumption throughout the study. Food efficiency in test animals was similar to that of controls.

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles throughout the study period revealed no intergroup differences.

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects were detected in the haematological parameters measured.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in the blood chemistry parameters measured.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No abnormalities were detected in any of the urinalysis parameters measured. Statistical analysis of the quantitative data revealed no significant intergroup differences.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Behavioural Assessments:
Hunched posture was noted in a number of animals of either sex treated with 1000 mg/kg/day during open field assessments at Week 3 and Week 4, which correlated with the daily clinical observations. Noisy respiration was observed for one 1000 mg/kg/day female at Week 3 and two females showed tiptoe gait during the Week 4 assessment. These isolated findings most likely resulted from a response to the dosing procedure and were considered not to be indicative of neurotoxicity. No such observations were reported for 150 or 15 mg/kg/day animals. All remaining inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used and were, therefore, of no toxicological importance.

Functional Performance Tests:
There were no treatment-related changes detected in the functional performance parameters measured. Statistical analysis of the quantitative data revealed no significant intergroup differences.

Sensory Reactivity Assessments:
There were no treatment-related changes in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and was of no toxicological importance. Statistical analysis of the quantitative data revealed no significant intergroup differences.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in liver weight both absolute (p<0.01) and relative (p<0.001) to bodyweight was observed in females treated with 1000 mg/kg/day with an increase in relative (p<0.001) liver weight also detected in males from this dose group. An increase in relative (p<0.05) kidney weight was detected in males treated with 1000 mg/kg/day with an increase in the absolute, but not relative weight of this organ (p<0.01) observed in recovery males treated with 1000 mg/kg/day at the end of the fourteen day recovery period. No such changes in organ weights were detected for animals of either sex treated with 150 or 15 mg/kg/day. The remaining statistically significant intergroup differences detected were considered to be of no toxicological importance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Four males treated with 1000 mg/kg/day showed pale kidneys. No other treatment-related macroscopic findings were observed. Macroscopic examination of the recovery control male killed in extremis revealed gross organ damage consistent with the injury incurred. The other, incidental findings detected were of no toxicological importance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related changes were observed:
Liver: Centrilobular hepatocyte enlargement was observed in relation to treatment for rats of either sex dosed at 1000 mg/kg/day. A similar effect was also observed for one male rat dosed at 150 mg/kg/day but his condition is occasionally observed as a spontaneous entity in control rats and thus cannot be regarded convincingly as an effect of treatment at this dose level. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature, as evidenced by significant regression of the condition among recovery 1000 mg/kg/day animals following an additional fourteen days without treatment.
Kidneys: Globular accumulations of eosinophilic material, with associated renal tubular basophilia in some instances, were observed in the tubular epithelium of male rats dosed at 1000 mg/kg/day, 150 mg/kg/day, or at 15 mg/kg/day. There was partial regression of the condition among Recovery 1000 mg/kg/day animals after completion of the fourteen day recovery period. This finding is consistent with the presence of hydrocarbon nephropathy, which results from the excessive accumulation of alpha2-Microglobulin in renal proximal tubular epithelial cells. Alpha2-Microglobulin is found only in the proximal tubular epithelium of adult male rats.
Thyroids: A higher incidence of follicular cell hypertrophy was seen in relation to treatment for female rats dosed at 1000 mg/kg/day, but not at any other dose level. The condition was observed to have regressed among Recovery 1000 mg/kg/day animals following an additional fourteen days without treatment. The most plausible explanation for these changes is a secondary response to the changes occurring in the liver. In the rat, thyroxine is ultimately excreted via the bile, having first been conjugated by glucuronyltransferase in the liver. It is probable that this enzyme may have been induced in response to treatment, thereby increasing thyroxine excretion and stimulating a compensatory thyroid response. All remaining morphological changes seen in the heart, liver, spleen, kidneys and bone marrow were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

14-DAY RANGE FINDING STUDY:

- There were no deaths.

- Findings were limited to animals of either sex treated with 1000 mg/kg/day involving increased salivation detected up to ten minutes after dosing from Day 5 onwards. Excessive visible salivation is commonly reported following the oral administration of a test material, and in isolation considered to be attributable to the repeated administration of an unpalatable or locally irritant test material formulation by gavage rather than an indication of systemic toxicity. Generalised fur loss was noted in control females from Days 1 to 5.

- Bodyweight gain in test animals was comparable with that seen in controls.

- No macroscopic abnormalities were detected.

Conclusions:

Under the conditions of the test (28-days repeated dose toxicity study, in compliance with GLP, following OECD TG 407), the NOAEL was determined to be 1000 mg/kg bw/day.

Executive summary:

In a study performed according to OECD TG 407 and in compliance with GLP, the test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no treatment-related deaths during the study. No treatment related changes in functional performance parameters, sensory reactivity, bodyweight gain, food consumption, water consumption, haematological parameters, blood chemical parameters, and urinalysis parameters were observed. Animals treated with 1000 mg/kg/day developed transient increased salivation around the time of dosing and approximately one hour after dosing and instances of noisy respiration and stained fur or fur loss. Hunched posture was seen in 1000 mg/kg/day animals from Day 21 and episodes of tiptoe gait were observed in two females treated with 1000 mg/kg/day on Day 26 only. All findings regressed in recovery 1000 mg/kg/day animals. An increase in liver weight was detected in animals treated with 1000 mg/kg/day. Four males treated with 1000 mg/kg/day showed pale kidneys. Centrilobular hepatocyte enlargement was observed in relation to treatment for rats at 1000 mg/kg/day. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive. Globular accumulations of eosinophilic material, with associated renal tubular basophilia in some instances, were observed in the tubular epithelium male rats dosed at 1000 mg/kg/day, 150 mg/kg/day, or at 15 mg/kg/day. There was partial regression of the condition among recovery 1000 mg/kg/day animals after completion of the fourteen day recovery period. This finding is consistent with the presence of hydrocarbon nephropathy, which results from the excessive accumulation of alpha2 -microglobulin in renal proximal tubular epithelial cells. A higher incidence of follicular cell hypertrophy was seen in relation to treatment for female rats dosed at 1000 mg/kg/day, but not at any other dose level. The condition was observed to have regressed among Recovery 1000 mg/kg/day animals following an additional fourteen days without treatment.

In conclusion, oral administration of the test material, to rats, by gavage, for a period of twenty-eight consecutive days resulted in treatment-related but non-adverse changes in either sex at a dose level of 1000 mg/kg/day and in males treated with 150 or 15 mg/kg/day. There was no effect of treatment in females treated with 150 or 15 mg/kg/day. The treatment-related renal changes seen in male rats treated with 150 or 15 mg/kg/day were consistent with a well-documented condition known as hydrocarbon nephropathy, which only occurs in male rats and is not indicative of a hazard to human health. Furthermore, the effects seen in animals treated with 1000 mg/kg/day were confined to minimal clinical observations and adaptive, reversible liver and thyroid changes so the NOAEL was considered to be 1000 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 407

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

455-260-1

EC Name:

-

Cas Number:

478695-70-4

Molecular formula:

C15H26O4

IUPAC Name:

(3,3-DIMETHYLCYCLOHEXYL)ETHYL ETHYL PROPANE-1,3-DIOATE

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation: 28 day study: Males 140 to 190g, Females 122 to 159g; 14 day range-finding study: Males 127 to 162 g, Females 119 to 135 g.
- Housing: groups of five (28-day study) or three (14 day range-finding study) by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper. Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd., Hull, UK).
- Diet: A pelleted diet (Rodent 5LF2 (Certified) Diet, International Product Supplies Ltd., Wellingborough, Northants, UK) Ad libitum
- Water: Mains drinking water from polycarbonate bottles attached to the cage. Ad libitum
- Acclimation period: 28 day study: 8 days. 14 day range-finding study: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least fifteen
- Photoperiod (hrs dark / hrs light): 12/ 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

- 28-day repeated dose study: The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day of Arachis oil BP. Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
- 14-day range-finding study: The test material was administered daily, for fourteen consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day of Arachis oil BP. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at Days 4, 8 and 11.

Details on mating procedure:

no mating occurred in the OECD TG 407 study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken of each test material formulation and were analysed for concentration of the substance at Safepharm Analytical Services. The concentration of the substance in the test material formulations was determined by gas chromatography (GC) using an external standard technique. The results indicate that the prepared formulations were within ± 9% of the nominal concentration.

Duration of treatment / exposure:

28 days / 14 days (range-finding study)

Frequency of treatment:

Daily

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)

Doses / concentrations 4

Dose / conc.:

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

Remarks:

14-day range finding study

No. of animals per sex per dose:

5 (28-day repeated dose study)
3 (14-day range finding study)

Control animals:

yes, concurrent vehicle

Details on study design:

- 28-day repeated dose study: The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.
- 14-day range finding study: Following single dose sighting work treating one male and one female with 1000 mg/kg, two groups, each of six rats (three males and three females) were dosed.

Examinations

Parental animals: Observations and examinations:

28-DAY REPEATED DOSE STUDY:
- All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends). All observations were recorded.
- Prior to the start of treatment and on Days 7, 14, 21 and 26, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.
- Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait, Tremors, Twitches, Convulsions, Bizarre/ Abnormal/Stereotypic behavior, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin colour, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation.
- Motor Activity: Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was sixteen hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall sixteen hour period and also during the final 20% of the period (considered to be the asymptotic period).
- Forelimb/hindlimb Grip Strength: An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.
- Sensory Reactivity: Each animal was individually assessed for sensory reacttvtty to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, Vocalisation, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Startle reflex, Blink reflex.
- Bodyweight: Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28 and, in the case of recovery group animals, on Days 35 and 42. Bodyweights were also recorded at terminal kill.
- Food Consumption: Food consumption was recorded for each cage group at weekly intervals throughout the study.
- Water Consumption: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.
- Laboratory Investigations: Haematological and blood chemical investigations were performed on all non-recovery test and control group animals at the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 29 and 43. Animals were not fasted prior to sampling. Urinalytical investigations were performed on all non-recovery test and control group animals during Week 4 and on all recovery group animals during Week 6. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.
- Haematology: The following parameters were measured on blood collected into tubes containing potassium EDT A anti-coagulant: Haemoglobin, Erythrocyte count, Haematocrit, Erythrocyte indices (mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration), Total leucocyte count, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Platelet count, Reticulocyte count, Prothrombin time.
- Blood Chemistry: The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea, Glucose, Total protein, Albumin, Albumin/Globulin (A/G) ratio, Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Gamma glutamyltranspeptidase, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Creatinine, Triglycerides, Total cholesterol, Total bilirubin.
- Urinalysis: The following parameters were measured on collected urine: Volume, Specific Gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Reducing Substances, Blood.

14-DAY RANGE FINDING STUDY:
- All animals were examined for overt signs of toxicity, ill health or behavioural change immediately before dosing and one hour after dosing. All observations were recorded.-
Bodyweight: Individual bodyweights were recorded on Days 1, 4, 8, 11 and 14.

Oestrous cyclicity (parental animals):

No

Sperm parameters (parental animals):

No

Litter observations:

No

Postmortem examinations (parental animals):

28-DAY REPEATED DOSE STUDY:
- Pathology: On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free period, all surviving animals were killed by intravenous overdose of sodium pentobarbitone (Rhone Merieux, Dagenham, Essex, UK) followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
- Organ Weights: The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus.
- Histopathology: Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin: Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Epididymides, Eyes, Gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Muscle (skeletal), Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles, Skin (hind limb), Spinal cord (cervical), Spleen, Stomach, Testes, Thymus, Thyroid/parathyroid, Trachea, Urinary bladder, Uterus. The tissues (Adrenals, Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Epididymides, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Rectum, Seminal vesicles, Spleen, Stomach, Testes, Thymus, Thyroid/parathyroid, Trachea, Urinary bladder, Uterus) from all non-recovery and 1000 mg/kg/day dose group animals were prepared as paraffin wax blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. The liver and spleen from all 15 and 150 mg/kg/day dose group animals were also processed. Since there were indications of treatment-related changes in the liver, kidneys and thyroid glands, examination was subsequently extended to include similarly prepared sections of these tissues from all animals in the other treatment groups, including recovery groups.

14-DAY RANGE FINDING STUDY: On completion of the dosing period, all animals were killed by cervical dislocation and immediately subjected to an internal and external macroscopic examination. No tissues were retained.

Postmortem examinations (offspring):

No

Statistics:

Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative urinalytical data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOV A) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. In the case of recovery group data, the analysis performed was a two-tailed t-test incorporating Levene's test for homogeneity of variance. Where Levene's test showed unequal variances among either nonrecovery or recovery group data, the affected parameters were analysed using non-parametric methods: Kruskal-Wallis ANOV A and mann-Whitney 'U' test.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals of either sex treated with 1000 mg/kg/day developed transient increased salivation around the time of dosing from Day 2 onwards together with associated signs of increased salivation approximately one hour after dosing and instances of noisy respiration and stained fur or fur loss. Hunched posture was seen in 1000 mg/kg/day animals of either sex from Day 21 and episodes of tiptoe gait were observed in two females treated with 1000 mg/kg/day on Day 26 only. All findings regressed in recovery 1000 mg/kg/day animals on cessation of treatment. Increased salivation of short duration is often reported following the oral administration of a test material and the daily occurrence of this finding around the time of dosing is usually considered attributable to an unpleasant tasting or locally irritant formulation rather than an indication of systemic toxicity. No treatment-related clinical signs were detected in animals treated with 150 or 15 mg/kg/day. The remaining incidental findings detected were considered to be of no toxicological importance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment-related deaths during the study although one recovery control male was killed in extremis on Day 42 when it sustained injuries while escaping the container in which it was held prior to blood sampling.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related effects in bodyweight. Test animals showed weight gains similar to those of the controls throughout the study period. Statistical analysis of weekly bodyweight gain showed no significant intergroup differences.

Description (incidence and severity):

There was no adverse effect on food consumption throughout the study. Food efficiency in test animals was similar to that of controls.

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles throughout the study period revealed no intergroup differences.

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects were detected in the haematological parameters measured.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in the blood chemistry parameters measured.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No abnormalities were detected in any of the urinalysis parameters measured. Statistical analysis of the quantitative data revealed no significant intergroup differences.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Behavioural Assessments:
Hunched posture was noted in a number of animals of either sex treated with 1000 mg/kg/day during open field assessments at Week 3 and Week 4, which correlated with the daily clinical observations. Noisy respiration was observed for one 1000 mg/kg/day female at Week 3 and two females showed tiptoe gait during the Week 4 assessment. These isolated findings most likely resulted from a response to the dosing procedure and were considered not to be indicative of neurotoxicity. No such observations were reported for 150 or 15 mg/kg/day animals. All remaining inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used and were, therefore, of no toxicological importance.

Functional Performance Tests:
There were no treatment-related changes detected in the functional performance parameters measured. Statistical analysis of the quantitative data revealed no significant intergroup differences.

Sensory Reactivity Assessments:
There were no treatment-related changes in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and was of no toxicological importance. Statistical analysis of the quantitative data revealed no significant intergroup differences.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related changes were observed:
Liver: Centrilobular hepatocyte enlargement was observed in relation to treatment for rats of either sex dosed at 1000 mg/kg/day. A similar effect was also observed for one male rat dosed at 150 mg/kg/day but his condition is occasionally observed as a spontaneous entity in control rats and thus cannot be regarded convincingly as an effect of treatment at this dose level. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature, as evidenced by significant regression of the condition among recovery 1000 mg/kg/day animals following an additional fourteen days without treatment.
Kidneys: Globular accumulations of eosinophilic material, with associated renal tubular basophilia in some instances, were observed in the tubular epithelium of male rats dosed at 1000 mg/kg/day, 150 mg/kg/day, or at 15 mg/kg/day. There was partial regression of the condition among Recovery 1000 mg/kg/day animals after completion of the fourteen day recovery period. This finding is consistent with the presence of hydrocarbon nephropathy, which results from the excessive accumulation of alpha2-Microglobulin in renal proximal tubular epithelial cells. Alpha2-Microglobulin is found only in the proximal tubular epithelium of adult male rats.
Thyroids: A higher incidence of follicular cell hypertrophy was seen in relation to treatment for female rats dosed at 1000 mg/kg/day, but not at any other dose level. The condition was observed to have regressed among Recovery 1000 mg/kg/day animals following an additional fourteen days without treatment. The most plausible explanation for these changes is a secondary response to the changes occurring in the liver. In the rat, thyroxine is ultimately excreted via the bile, having first been conjugated by glucuronyltransferase in the liver. It is probable that this enzyme may have been induced in response to treatment, thereby increasing thyroxine excretion and stimulating a compensatory thyroid response. All remaining morphological changes seen in the heart, liver, spleen, kidneys and bone marrow were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Absence of effects on gonads

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

Effect levels (F1)

Remarks on result:

not measured/tested

Remarks:

No F1 generation was present in the OECD TG 407 study

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

14-DAY RANGE FINDING STUDY:

- There were no deaths.

- Findings were limited to animals of either sex treated with 1000 mg/kg/day involving increased salivation detected up to ten minutes after dosing from Day 5 onwards. Excessive visible salivation is commonly reported following the oral administration of a test material, and in isolation considered to be attributable to the repeated administration of an unpalatable or locally irritant test material formulation by gavage rather than an indication of systemic toxicity. Generalised fur loss was noted in control females from Days 1 to 5.

- Bodyweight gain in test animals was comparable with that seen in controls.

- No macroscopic abnormalities were detected.

Applicant's summary and conclusion

Conclusions:

No effects on male and female gonads and therefore no effect on fertility are expected.

Executive summary:

In a study performed according to OECD TG 407 and in compliance with GLP, the test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no treatment-related deaths during the study. No treatment related changes in functional performance parameters, sensory reactivity, bodyweight gain, food consumption, water consumption, haematological parameters, blood chemical parameters, and urinalysis parameters were observed. Animals treated with 1000 mg/kg/day developed transient increased salivation around the time of dosing and approximately one hour after dosing and instances of noisy respiration and stained fur or fur loss. Hunched posture was seen in 1000 mg/kg/day animals from Day 21 and episodes of tiptoe gait were observed in two females treated with 1000 mg/kg/day on Day 26 only. All findings regressed in recovery 1000 mg/kg/day animals. An increase in liver weight was detected in animals treated with 1000 mg/kg/day. Four males treated with 1000 mg/kg/day showed pale kidneys. Centrilobular hepatocyte enlargement was observed in relation to treatment for rats at 1000 mg/kg/day. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive. Globular accumulations of eosinophilic material, with associated renal tubular basophilia in some instances, were observed in the tubular epithelium male rats dosed at 1000 mg/kg/day, 150 mg/kg/day, or at 15 mg/kg/day. There was partial regression of the condition among recovery 1000 mg/kg/day animals after completion of the fourteen day recovery period. This finding is consistent with the presence of hydrocarbon nephropathy, which results from the excessive accumulation of alpha2 -microglobulin in renal proximal tubular epithelial cells. A higher incidence of follicular cell hypertrophy was seen in relation to treatment for female rats dosed at 1000 mg/kg/day, but not at any other dose level. The condition was observed to have regressed among Recovery 1000 mg/kg/day animals following an additional fourteen days without treatment.

In conclusion, oral administration of the test material, to rats, by gavage, for a period of twenty-eight consecutive days resulted in treatment-related but non-adverse changes in either sex at a dose level of 1000 mg/kg/day and in males treated with 150 or 15 mg/kg/day. There was no effect of treatment in females treated with 150 or 15 mg/kg/day. The treatment-related renal changes seen in male rats treated with 150 or 15 mg/kg/day were consistent with a well-documented condition known as hydrocarbon nephropathy, which only occurs in male rats and is not indicative of a hazard to human health. Furthermore, the effects seen in animals treated with 1000 mg/kg/day were confined to minimal clinical observations and adaptive, reversible liver and thyroid changes so the NOAEL was considered to be 1000 mg/kg/day.