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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 January 2009 to 27 June 2009
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 relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA: OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 19 August 2008; Date of signature: 04 March 2009
Limit test:
no

Test material

Constituent 1
Details on test material:
Sponsor's identification : H-MI ammonium salt
Description : dark red solid
Purity : 90.3%
Lot number : MB-1
Date received : 04 December 2008
Storage conditions : room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Blackthorn, Bicester, Oxon.
- Age at study initiation: Six to eight weeks old.
- Weight at study initiation: Males: 212 to 269 g; females: 166 to 192 g.
- Fasting period before study: Not stated.
- Housing: The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK).
- Diet: The animals were allowed free access to food, a pelleted diet (Rodent 2014C Teklad Global Certified Diet Harlan UK, Oxon, UK) was used.
- Water: The animals were allowed free access to water, mains drinking water was supplied from polycarbonate bottles attached to the cage.
The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: Nine days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): Twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: From: 0 To: Termination 28 days later for both male and female groups in all groups bar recovery group. 42 days later for males and females in the recovery groups.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0, 6, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of H-MI ammonium salt in the test material formulations was determined spectrophotometrically.

Samples
The test material formulations were diluted with water to give a final, theoretical test material concentration of approximately 0.01 mg/ml.

Procedure
The standard and sample solutions were analysed spectrophotometrically using the following conditions:

Spectrophotometer : Camspec M550
Wavelength : λ max at ~ 306 nm
Cell path length : 1 cm
Reference medium : Water

Homogeneity Determinations
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.

Stability Determinations♦
The test material formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for fourteen days.

Verification of Test Material Formulation Concentrations
The test material formulations were sampled and analysed within two days of preparation.


♦ The analytical method is considered to be non stability indicating. However the absorbance would likely change due to any decomposition of the test material and therefore the formulations are considered stable.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
Dose levels of 30, 300 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Male and Female: 5 animals per sex at 0 mg/kg/day
Male and Female: 5 animals per sex at 30 mg/kg/day
Male and Female: 5 animals per sex at 300 mg/kg/day
Male and Female: 5 animals per sex at 1000 mg/kg/day
Male and Female: 5 animals per sex at 1000 mg/kg/day (Recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of a range-finding study.
- Rationale for animal assignment (if not random): Random.
- Rationale for selecting satellite groups: Not stated.
- Post-exposure recovery period in satellite groups: 14 days.
- Section schedule rationale (if not random): Not stated.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule: Pre, post, 1 hour and 5 hours after dosing for all groups other than the recovery group. Recovery group: Pre, post, 1 hour, 5 hours and then daily (am and pm) until day 42.
- Cage side observations checked in table were included: yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pre, post, 1 hour and 5 hours after dosing for all groups other than the recovery group. Recovery group: Pre, post, 1 hour, 5 hours and then daily (am and pm) until day 42.
Functional observations were made prior to the start of treatment and on Days 7, 10, 21 and 24.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1, at weekly intervals and prior to terminal kill. Recovery: Day 1, at weekly intervals, day 36, day 43 and prior to terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 30 (15 male, 15 female) Non-recovery and 10 (5 male and 5 female) control animals (10 (5 male and 5 female) Recovery obtained at the end of the treatment free period).
- The following parameters were examined: Haemaglobin, Erythrocyte count, Haematocrit, Erythrocyte indices, Total leucocyte count (WB), Differential leucocyte count, Platelet count (PLT), Reticulocyte count and Prothrombin time

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period
- Animals fasted: No
- How many animals: All non-recovery and control animals (recovery animals assessed at end of treatment-free period).
- The following parameters were examined: Urea, Glucose, Total protein, Albumin, Albumin/Globulin ratio, Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Gamma glutamyltranspeptidase, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Creatinine, Triglycerides, Total cholesterol and Total Bilirubin.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected overnight during week 4 for non recovery animals and week 6 for recovery animals.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- The following parameters were examined: Volume, Ketones, Specific gravity, Bilirubin, pH, Urobilinogen, Protein, Reducing Substances, Glucose and Blood.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and on days 7, 10, 21 and 24 at the same time each day.
- Dose groups that were examined: All dose groups
- Battery of functions tested: Sensory activity, grip strength and motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free period, all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the plasma from each animal was stored frozen at approximately -20°C. As no treatment-related effect on the pituitary-thyroid axis was identified, these samples were discarded.

Oestrus Cycle Assessment
At termination, a vaginal smear was taken from all females and the stage of oestrus was 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: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin except where stated:

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)♦, Mammary gland♦, Muscle (skeletal), Oesophagus, Ovaries♦, Pancreas, Pituitary♦, Prostrate♦, Rectum♦, Salivary glands (submaxillary), Sciatic nerve♦, seminal vesicles (with coagulating glands and fluids)♦, skin (hind limb), Spinal cord (cervical, mid thoracic and lumbar)♦, Spleen♦, Stomach♦, Testes♦~, Thymus♦, Thyroid/parathyroid♦, Trachea♦, Urinary bladder♦, Uterus & Cervix♦, Vagina♦.

*: Eyes fixed in Davidson's fluid.
~: Preserved in Bouin's fluid then transferred to Industrial Methylated Spirits (IMS) approximately 48 hours later.
#: Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative.

All tissues were despatched to the Test Site Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford for processing. The tissues marked ♦ from all non-recovery control and 1000 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and spleen from all 30 and 300 mg/kg/day dose group animals.
Other examinations:
Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of stomachfrom all animals in the other 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.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Where appropriate, quantitative data were
analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test.
The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (nonparametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (P) are present as follows:

P < 0.01 **
P < 0.05 *
p ≥ 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Please see comments below.
Mortality:
mortality observed, treatment-related
Description (incidence):
Please see comments below.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment-related effect on bodyweight change was detected for treated animals, in comparison to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related effect on dietary intake or food efficiency was detected for treated animals, in comparison to controls.
Food efficiency:
no effects observed
Description (incidence and severity):
No treatment-related effect on dietary intake or food efficiency was detected for treated animals, in comparison to controls.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Please see comments below.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the haematological parameters measured.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the blood chemical parameters measured.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No adverse effect on uranalytical parameters was detected for treatment animals, in comparison to controls.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Please see comments below.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in organ weight measurement.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of the tissues did not reveal any toxicologically significant findings.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
However please see comments below.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Mortality. There were no deaths during the study.
Clinical Observations. An isolated episode of increased salivation was observed in one animal of either sex treated with 1000 mg/kg/day.
No such effect was detected in animals of either sex treated with 300 or 30 mg/kg/day.

WATER CONSUMPTION
An increase in water consumption was seen in non-recovery 1000 mg/kg/day males from Week 3 onwards and recovery 1000 mg/kg/day males up to and including Week 5. Females from this treatment group showed an increase in water consumption during Weeks 3 and 4.
No such effect was detected in animals of either sex treated with 300 and 30 mg/kg/day or recovery 1000 mg/kg/day females.

NEUROBEHAVIOUR
Behavioural Assessment. There were no treatment-related changes in the behavioural parameters measured.
Functional Performance Tests. There were no adverse changes in the functional performance parameters measured.
Sensory Reactivity Assessments. There were no treatment-related changes in sensory reactivity.

HISTOPATHOLOGY: NON-NEOPLASTIC
The following treatment-related changes were observed.

STOMACH: Agglomeration of secretion, mucous cell hypertrophy/hyperplasia, mucosal basophilia, and acanthosis/hyperkeratosis of the limiting ridge were seen in relation to treatment for animals of either sex treated with 1000 mg/kg/day or 300 mg/kg/day. One female treated with 30 mg/kg/day also demonstrated changes but these conditions are seen occasionally and spontaneously among control animals such that effects in one animal, even in the absence of such effects among concurrent controls, cannot be reliably regarded as an effect of treatment.

Conditions were considered to have generally regressed among recovery animals of either sex treated with 1000 mg/kg/day following an additional fourteen days without treatment, although two 1000 mg/kg/day males had residual changes. One recovery control animal demonstrated all conditions.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The oral administration of H-MI ammonium salt to rats for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg/day resulted in treatment related effects at 300 and 1000 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The oral administration of H-MI ammonium salt to rats for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg/day resulted in treatment related effects at 300 and 1000 mg/kg/day.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The oral administration of H-MI ammonium salt to rats by gavage for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg/day resulted in treatment related effects at 300 and 1000 mg/kg/day.

An episode of increased salivation pre-dosing was observed in one male whilst increased salivation to thirty minutes post dosing was observed in one female treated with 1000 mg/kg/day. Increased salivation is often recorded following the oral administration of an unpleasant tasting and or irritant test material formulation. Although such isolated episodes would not be indicative of toxicity, laboratory investigations revealed an increase in water intake for non-recovery animals of either sex treated with 1000 mg/kg/day from Week 3 onwards. This also extended into Week 5 for the recovery 1000 mg/kg/day males. Histopathological evaluation of the tissue showed associated gastric changes comprising of agglomeration of secretion, mucous cell hypertrophy/ hyperplasia, mucosal basophilia and acanthosis/hyperkeratosis of the limiting ridge were seen in animals of either sex treated with 1000 mg/kg/day. These changes also extended to animals of either sex treated with 300 mg/kg/day. Following cessation of treatment the histopathological findings identified in the non-recovery animals were considered to have regressed in the recovery 1000 mg/kg/day animals.

Applicant's summary and conclusion

Conclusions:
The oral administration of H-MI ammonium salt to rats for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg/day resulted in treatment-related effects at 300 and 1000 mg/kg/day.

The changes seen at treatment levels up to 1000 mg/kg/day were considered to be adaptive and not represent an adverse health effect. The ‘No Observed Adverse Effect Level’ (NOAEL) should, therefore, be regarded as 1000 mg/kg/day.

No treatment-related effects were identified at 30 mg/kg/day the “No Observable Effect Level” (NOEL) for animals of either sex was therefore considered to be 30 mg/kg/day.
Executive summary:

Introduction. The study was designed to investigate the systemic toxicity of the test material and complies with the following regulatory guidelines:

i) Commission Directive 96/54/EC (Method B7).

ii) The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973 of Ministry of International Trade and Industry (MITI) amended 2004.

iii) The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

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

Methods. The test material was administered by gavage to three groups, each of five male and five female Wistar Han™:HsdRccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 300 and 1000 mg/kg/day (incorporating a correction factor for 90.3% purity). A control group of five males and five females was dosed with vehicle alone (Distilled water). 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, 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.

Results.

Mortality. There were no deaths during the study.

Clinical Observations. An isolated episode of increased salivation was observed in one animal of either sex treated with 1000 mg/kg/day.

No such effect was detected in animals of either sex treated with 300 or 30 mg/kg/day.

Functional Observations.

Behavioural Assessment. There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests. There were no adverse changes in the functional performance parameters measured.

Sensory Reactivity Assessments. There were no treatment-related changes in sensory reactivity.

Bodyweight. No treatment-related effect on bodyweight change was detected for treated animals, in comparison to controls.

Food Consumption. No treatment-related effect on dietry intake of food efficiency was detected for treated animals, in comparison to controls.

Water Consumption. An increase in water consumption was seen in non-recovery 1000 mg/kg/day males from Week 3 onwards and recovery 1000 mg/kg/day males up to and including Week 5. Females from this treatment group showed an increase in water consumption during Weeks 3 and 4.

No such effect was detected in animals of either sex treated with 300 and 30 mg/kg/day or recovery 1000 mg/kg/day females.

Haematology. There were no toxicologically significant changes in the haematological parameters measured.

Blood Chemistry. There were no toxicologically significant changes in the blood chemical parameters measured.

Urinalysis: No adverse effect on uranalytical parameters was detected for treatment animals, in comparison to controls.

Organ Weights. There were no toxicologically significant changes in organ weight measurement.

Necropsy. Macroscopic examination of the tissues did not reveal any toxicologically significant findings.

Histopathology. The following treatment-related changes were observed.

STOMACH: Agglomeration of secretion, mucous cell hypertrophy/hyperplasia, mucosal basophilia, and acanthosis/hyperkeratosis of the limiting ridge were seen in relation to treatment for animals of either sex treated with 1000 mg/kg/day or 300 mg/kg/day. One female treated with 30 mg/kg/day also demonstrated changes but these conditions are seen occasionally and spontaneously among control animals such that effects in one animal, even in the absence of such effects among concurrent controls, cannot be reliably regarded as an effect of treatment.

Conditions were considered to have generally regressed among recovery animals of either sex treated with 1000 mg/kg/day following an additional fourteen days without treatment, although two 1000 mg/kg/day males had residual changes. One recovery control animal demonstrated all conditions.

Conclusion. The oral administration of H-MI ammonium salt to rats for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg/day resulted in treatment-related effects at 300 and 1000 mg/kg/day.

The changes seen at treatment levels up to 1000 mg/kg/day were considered to be adaptive and not represent an adverse health effect. The ‘No Observed Adverse Effect Level’ (NOAEL) should, therefore, be regarded as 1000 mg/kg/day.

No treatment-related effects were identified at 30 mg/kg/day the “No Observable Effect Level” (NOEL) for animals of either sex was therefore considered to be 30 mg/kg/day.