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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 February 2004 to 19 August 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
Annex V
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 2 December 2002 Date of Signature: 13 February 2003
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TIPX
- Substance type: Colourless liquid.
- Physical state: Liquid.
- Lot/batch No.: 042028.
- Storage condition of test material: Approximately 4ºC in the dark.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, UK.
- Age at study initiation: 5 to 7 weeks old.
- Weight at study initiation: 149 - 180 g (male) and 128 - 166 g (female).
- Fasting period before study: Overnight fasting immediately before dosing and for approximately 2 hours after dosing.
- Housing: The animals were housed in groups of 5 by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet: Rodent 5LF2 and 5LF3 (Certified) Diets BCM IPS Limited, London, UK. Ad libitum.
- Water: Mains drinking water ad libitum.
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC.
- Humidity (%): 55 ± 15%. On 2 occasions the humidity deviated from the respective target but this was considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): ≥ 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness.

IN-LIFE DATES: Day 0 (the day of dosing) up to Day 29.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was prepared at 3.75, 37.5 and 250 mg/ml, as a solution in Arachis oil BP. The stability and homogeneity of the test material formulations were determined at the test laboratory. The formulations to be stable for at least 14 days. Formulations were therefore prepared weekly and stored at approximately 4ºC in the dark.

DIET PREPARATION
Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The stability and homogeneity of the test material formulations were determined at the test laboratory. The samples used were test material extracted with methanol with final theoretical concentration of 0.1 mg/ml. The formulations were found to be stable for at least 14 days.
- Amount of vehicle (if gavage): 4 ml/kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Summary; The concentration of TIPX in the test material formulations was determined by gas chromatography (GC) using an external standard technique.

Samples; The test material formulations were extracted with methanol with final theoretical concentration of 0.1 mg/ml.

Standards; Standard solutions of test material were prepared in methanol at a nominal concentration of 0.1 mg/ml.

Procedure; The sample and standard solutions were analysed by GC.

Homogeneity determinations; The test material formulations were mixed throughly and samples were taken from the top, middle and bottom of the container, shaking between sampling in triplicate.

Stability determinations; The test material formulations were sampled and analysed initially and then after storage at 4ºC in the dark for 14 days.

Verification of test material formulation concentrations; The test material formulations were sampled and analysed within 2 days of preparation.

Results; Mean concentration found was in the range of 90 – 102% of nominal concentrations. The results showed the formulation to be stable for at least 14 days.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
15 mg/kg/day
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
150 mg/kg/day
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
other: nominal in vehicle
No. of animals per sex per dose:
5 animals per sex per dose and the control group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of the range-finding study performed. See the summary of the range-finding study in 'any other information on materials and methods incl. tables'.
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, and 1 and 5 hours after dosing during the working week. Animals were observed immediately before dosing, and 1 hour after dosing at weekends and public holidays.
- Cage side observations checked: Overt signs of toxicity, ill-health and behavioural change.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 0 (the day before the sart of treatment) and on Day 7, 14, 21 and 28. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION: Yes
Weekly food consumption was recorded for each cage group.

FOOD EFFICIENCY: Yes
- Food efficiency (the ratio of bodyweight gain / food consumption) was calculated.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily for each cage group by visual inspection of the water bottles for any overt changes.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Haematological and blood chemical investigations were performed on Day 28. Blood samples were obtained from the lateral tail vein.
- Anaesthetic used for blood collection: Not reported.
- Animals fasted: No.
- How many animals: All animals.
- Parameters checked in tables 17 - 20 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Behavioural assessment: Prior to the start of treatment and on Days 2, 9, 16 and 23. Observation was carried out from approximately 2 hours after dosing on each occasion.
Functional Performance Tests: During Week 4. Observation was carried out from approximately 2 hours after dosing on each occasion.
Sensory Reactivity: During Week 4. Observation was carried out from approximately 2 hours after dosing on each occasion.

- Dose groups that were examined:
All animals.

- Parameters examined:
Behavioural assessment: Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed:
Gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation.

Functional Performance Tests: Motor Activity. 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 16 hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall 16 hour-period and also during the final 20% of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grip Strength. An automated 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 reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex, tail pinch, blink reflex and finger approach.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were killed by intravenous overdose of sodium pentobarbitone, followed by exsanguination on Day 29. 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 at the end of the study, were dissected free from fat and weighed before fixation.
Adrenals, liver, brain, ovaries, epididymides, spleen, heart, testes, kidneys and thymus.

HISTOPATHOLOGY: Yes
Samples of the following tissues were preserved 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. Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), 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 and uterus.

All tissues were despatched to Precision Histology International, One Eyed Lane, Weybread, Diss, Norfolk, UK. The tissues (except aorta, bone & bone marrow, eyes, muscle, oesophagus, pancreas, pituitary, salivary glands and skin) from all control and 1000 mg/kg/day group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. Theliver and slpeen from all 150 and 15 mg/kg/day were also processed.
Since there were indications of treatment-related changes in the liver, kidneys, throid gland, duodenum, stomach, mesenteric lymph nodes and bone marrow, examination was subsequently extended to include ssimilarly prepared sections of these tissues from all animals in the treatment groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.
Other examinations:
No.
Statistics:
Haematology, blood chemistry and absolute and bodyweight relative organ weights, weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.

Probability values (p) were calculated as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no deaths. In top dose males, hunched posture was evident from Day 16, followed by incidents of abdominal distention, laboured respiration, increased respiratory rate, pilo-erection, tiptoe gait, tail elevation and wet fur during the final 2 weeks of treatment, more severely observed in males than females at 1000 mg/kg/day. Increased salivation was observed but this is not considered to be signs of systematic toxicity. See tables 1 and 2 as attached.

BODY WEIGHT AND WEIGHT GAIN
Males treated at 1000mg/kg/day showed a slight reduction in bodyweight gain compared to controls during the study, with statistical significance achieved during week 3. See tables 3 and 4 as attached.

FOOD CONSUMPTION / FOOD EFFICIENCY
A lower food intake and food efficiency was evident for this group during weeks 1 and 3 in males at 1000mg/kg/day. See tables 5 and 6 as attached.

WATER CONSUMPTION
Daily visual inspection of water bottles revealed no intergroup differences.

HAEMATOLOGY
Animals of either sex treated with 1000mg/kg/day showed elevated total leucocyte count, specifically in the lymphocyte and neutrophil fractions. No such effects were observed at 150 or 15mg/kg/day. See tables 7 and 8 as attached.

CLINICAL CHEMISTRY
Animals of either sex treated with 1000mg/kg/day showed a reduction in total plasma protein and albumin levels. This was statistically significant in males but not in females. Elevated cholesteral and alanine aminotransferase levels were also observed for males at this dose but not statistically significant. See tables 9 and 10 as attached.

NEUROBEHAVIOUR
Hunched posture, pilo-erection, increased salivation and decreased respiratory rate were observed in males during weeks 3 and 4, whilst hunched posture was confined to the females during Week 4 at 1000 mg/kg/day. See tables 11 and 12 as attached.

ORGAN WEIGHTS
Animals of either sex treated with 1000mg/kg/day showed a statistically significant increase in liver weight, both absolute and relative to terminal bodyweight. The mean increase over control absolute liver weight was 36% for males and 52% for females. Relative kidney and adrenal weight was also elevated for 1000mg/kg/day males. See tables 13 to 16 as attached.

GROSS PATHOLOGY
There were no treatment-related macroscopic abnormalities detected for animals treated with 1000 mg/kg/day. All males treated with 150mg/kg/day showed dark contents in the stomach and small intestine. No such effects were detected for 150mg/kg/day females or for animals of either sex treated with 15 mg/kg/day.

HISTOPATHOLOGY
See tables 17 and 18 as attached.
Liver: Higher grades of severity and/or a higher incidence of glycogen type hepatocyte vacuolation were observed in relation to treatment for animals of either sex treated with 1000mg/kg/day. Centrilobular hepatocyte enlargement was also seen for two males at this dose level.

Kidneys: Globular accumulations of eosinophilic material were observed in the tubular epithelium of males treated with 1000 and 150mg/kg/day and for one male treated with 15mg/kg/day. This finding is consistent with male rodent specific hydrocarbon nephropathy and does not represent an
adverse human effect.

Thyroid glands: Follicular cell hypertrophy was seen in relation to treatment for males treated with 1000 and 150mg/kg/day.

Duodenum: Treatment-related mucosal hypertrophy was seen for animals of either sex treated with 1000mg/kg/day and in one female treated with 150mg/kg/day.

Stomach: Acanthosis and hyperkeratosis were observed as an effect of treatment in the forestomach of animals of either sex treated with 1000mg/kg/day.

Mesenteric lymph nodes: Haemorrhage was seen in the sinusoids of the mesenteric lymph nodes of all males and of one female treated with 1000mg/kg/day.

Bone marrow: Higher grades of severity of adipose infiltration of the bone marrow, indicative of marrow hypoplasia, were seen as an effect of treatment for males treated with 1000mg/kg/day.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Treatment-related effects observed at 1000 mg/kg/day.
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Treatment-related effects observed at 1000 and 150 mg/kg/day.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of the test material, TIPX, to rats for a period of 28 consecutive days at a maximum dose level of 1000 mg/kg/day resulted in treatment-related changes at 1000 and 150 mg/kg/day. No such effects were demonstrated at 15 mg/kg/day and the ‘No Observed Effect Level’ (NOEL) is therefore considered to be 15 mg/kg/day.
The treatment-related changes detected at 150 mg/kg/day were minimal and, in isolation were considered not to represent an adverse health effect. The ‘No Observed Adverse Effect Level’ (NOAEL) was, therefore considered to be 150 mg/kg/day.
The test material does not meet the criteria for classification according to EU classification system (Council Directive 67/548/EEC and Regulation (EC) No 1272/2008).
Executive summary:

Introduction.The study was designed to investigate the systemic toxicity of the test material. It complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 407 “Repeated Dose 28-Day Oral Toxicity in Rodents” (adopted 27 July 1995).

Methods. The test material was administered by gavage to 3 groups, each of 5 male and 5 female Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, for 28 consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of 5 males and 5 females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural assessments, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results

Mortality.There were no deaths during the study.

Clinical Signs.Increased salivation was detected for animals for either sex treated with 1000 mg/kg/day from Day 6 onwards. Hunched posture was evident from Day 16, followed by incidents of abdominal distention, pilo-erection, respiration pattern changes, tiptoe gait and tail elevation. Findings in 1000 mg/kg/day females were less severe than those seen in the males. No clinically observable signs of toxicology were detected at 150 or 15 mg/kg/day.

Behavioural Assessment.Weekly open-field assessments confirmed the signs of hunched posture, pilo-erection, increased salivation and decreased respiratory rate reported for1000 mg/kg/day animals during weeks 3 and 4. No such effects were detected at 150 or 15 mg/kg/day.

 

Functional Performance Tests.No treatment-related effects were detected.

 

Sensory Reactivity Assessments.No treatment-related effects were detected.

 

Bodyweights.Males treated at 1000 mg/kg/day showed a slight reduction in bodyweight gain compared to controls during the study, with statistical significance achieved during week 3. No such effects were detected for females treated with 1000 mg/kg/day for animals of either sex treated with 150 or 15 mg/kg/day.

 

Food Consumption and Food Efficiency.A lower dietrary intake was evident for 1000 mg/kg/day males during weeks 1 and 3. Food efficiency was also slightly lower in the male 1000 mg/kg/day dose group.

 

Water Consumptions.No intergroup differences were noted.

 

Haematology.Animals of either sex treated with 1000 mg/kg/day showed elevated total leucocyte count, specifically in the lymphocyte and neutrophil fractions. No such observations were detected for animals of either sex treated with 150 or 15 mg/kg/day.

 

Blood Chemistry.Animals of either sex treated with 1000 mg/kg/day showed a statistically significant increase in liver weight, both absolute and relative to terminal bodyweight. Relative kidney and adrenal weight was also elevated for 1000 mg/kg/day males. No such organ weight changes were detected for animals of either sex treated with 150 or 15 mg/kg/day.

 

Necropsy.There were no treatment-related macroscopic abnormalities detected for animals treated with 1000 mg/kg/day. All males treated with 150 mg/kg/day however, showed

dark contents in the stomach and small intestine. No such effects were detected for 150 mg/kg/day females or for animals of either sex treated with 15 mg/kg/day.

Histopathology.Histopathological examination revealed the following treatment-related changes:

Liver:Higher grades of severity and/or a higher incidence of glycogen type hepatocyte vacuolation were observed in relation to treatment for animals of either sex treated with 1000mg/kg/day. Centrilobular hepatocyte enlargement was also seen for two males at this dose level, but this is of doubtful toxicological significance. Such changes are commonly observed in the rodent liver following the administration of xebobioticis and are generally regarded as adoptive in nature.

Kidneys:Globular accumulations of eosinophilic material were observed in the tubular epithelium of males treated with 1000 and 150 mg/kg/day and for one male treated with 15 mg/kg/day. This finding is consistent with male rodent specific hydrocarbon nephropathy and does not represent an adverse human effect.

Thyroid glands:Follicular cell hypertrophy was seen in relation to treatment for males treated with 1000 and 150 mg/kg/day.

Duodenum:Treatment-related mucosal hypertrophy was seen for animals of either sex treated with 1000mg/kg/day. One male treated with 150 mg/kg/day was also affected but cannot be reliably considered as a consequence of treatment at this treatment level.

Stomach:Acanthosis and hyperkeratosis were observed as an effect of treatment in the forestomach of animals of either sex treated with 1000 mg/kg/day and not at any other dose level.

 

Mesenteric lymph nodes:Haemorrhage was seen in the sinusoids of the mesenteric lymph nodes of all males and of one female treated with 1000 mg/kg/day. Animals from the remaining dose levels were not similarly affected.

Bone marrow:Higher grades of severity of adipose infiltration of the bone marrow, indicative of marrow hypoplasia, were seen as an effect of treatment for males treated with 1000 mg/kg/day, but not at any other treatment level.

 

Conclusion.The oral administration of the test material, TIPX, to rats for a period of 28 consecutive days at dose levels of up to 1000 mg/kg/day resulted in treatment-related changes at 1000 and 150 mg/kg/day. No such effects were demonstrated at 15 mg/kg/day and the ‘No Observed Effect Level’ (NOEL) is therefore considered to be 15 mg/kg/day.

The treatment-related changes detected at 150 mg/kg/day were minimal and in isolation were considered not to represent an advers health effect.

The ‘No Observed Adverse Effect Level’ (NOAEL) is therefore considered as 150 mg/kg/day.

The test material does not meet the criteria for classification according to EU classification system (Council Directive 67/548/EEC and Regulation (EC) No 1272/2008).