Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-05-02 to 2007-05-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
1996
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Analytical purity: 50.6%
- Physical state: liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation:
- Fasting period before study: no
- Housing: in groups of 5 per sex in Macrolon cages (M IV type, height 18 cm; during overnight activity monitoring individual housing in
Mlll type; height 15 cm) with sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (e.g. ad libitum): pelleted rodent diet (SM R/M-Z from SSNlFF Spezialdiaten GmbH, Soest, Germany), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 - 22.7°C
- Humidity (%): 30 - 74%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Main study: From 02 May 2007 To: 30 May 2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was administered undiluted.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
50 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)
No. of animals per sex per dose:
5
Control animals:
other: yes, from day 15 onwards Milli-Q water was used
Details on study design:
- Dose selection rationale: The dose levels were selected on the basis of a 5-day dose range finding study
- Rationale for animal assignment: By computer-generated random algorithm according to body weight

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/kg body weight/day

WATER CONSUMPTION: subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled postmortem examination
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes, overnight (with a maximum of 20 hours)
- How many animals: all
- Parameters examined: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled postmortem examination
- Animals fasted: Yes, overnight (with a maximum of 20 hours)
- How many animals: all
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, functional observations
- Time schedule for examinations: during week 4 of treatment
- Dose groups that were examined: all
- Battery of functions tested:
a. hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent).
b. motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals assigned to the study
ORGAN WEIGHTS: Yes, see table 1
HISTOPATHOLOGY: Yes, see table 1
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted over the 28-day observation period.
Salivation as observed among all animals at 1000 mg/kg bw/day and three males at 150 mg/kg bw/day was considered to be of no toxicological significance taking into account the nature and minor severity of this finding. Instead, salivation was considered to be a physiological response to taste and/or irritancy of the test substance upon oral administration.
Alopecia of the forelegs of one male at 50 mg/kg bw/day was a sign within the range of findings encountered in this type of study and was of no toxicological relevance. No clinical signs were noted among control animals, and females at 50 and 150 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically significant changes in body weights and body weight gain of treated animals were noted.
The slightly lower body weight and body weight gain of males at 1000 mg/kg bw/day in the first week of treatment (achieving a level of statistical significance for body weights) was considered to be of no toxicological significance. The change was of a temporary and slight nature (i.e. within the range considered normal for rats of this age and strain).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes occurred in haematological parameters of treated rats.
No toxicological significance was ascribed to the statistically significant lower haemoglobin levels in males at 150 and 1000 mg/kg bw/day and lower mean corpuscular haemoglobin concentration levels (MCHC) in males at 50 mg/kg bw/day, as these alterations were of a minor nature and occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment.
Any statistically significant changes noted among the dose groups were considered not to represent a sign of toxicity as these changes were of a minor nature and occurred in the absence of a dose-related trend. These alterations included lower alkaline phosphatase activity (ALP) and higher glucose levels in males at 50 mg/kg bw/day, lower cholesterol levels in males at 50 mg/kg bw/day and higher (a similar variation in this parameter was also noted among female dose groups), higher sodium and lower calcium levels in males at 150 mg/kg bw/day, and lower inorganic phosphate levels in females at 50 and 1000 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically significant changes were noted in organ weights and organ to body weight ratios.
The statistically significant lower brain weight of males at 1000 mg/kg bw/day was of a very minor nature (i.e. well within the range considered normal for rats of this age and strain). Also, brain weights of these males were similar to controls when corrected for terminal body weight. Therefore, this alteration was considered to be of no toxicological significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy did not reveal any toxicologically relevant alterations.
Incidental findings included a diaphragmatic hernia of the right lateral lobe of the liver in one male (no. 19) at 1000 mg/kg bw/day (this correlated to a high ASAT level in this male), fluid in the uterus in one female at 50 and 1000 mg/kg bw/day, and a yellowish focus on the left median lobe of the liver with enlargement of the mandibular lymph node in one female at 1000 mg/kg bw/day. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.
No macroscopic abnormalities were noted in control animals, in males at 50 and 150 mg/kg bw/day, and in females at 150 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no microscopic findings recorded which could be attributed to treatment with the test substance.
All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
No toxicologically significant changes were noted in any of the parameters examined/determined in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination.
Therefore a definitive No Observed Adverse Effect Level (NOAEL) for Amphopropionate C8 of 1000 mg/kg bw/day was established.
Executive summary:

In this Repeated Dose, 28-day Oral Toxicity Study performed according to OECD guideline 407 (1995) and EU Method B.7 (1996) Amphopropionate C8  (50.6 % a.i) was administered to groups of 5 male and 5 female Wistar rats by oral gavage at dose levels of 10, 150 and 1000 mg/kg bw/day for 28 days. The control group animals received MilliQ water from day 15 onwards.

All animals survived to study termination. No test substance-related findings were detected or observed in clinical examinations, body weights, food consumption values, neurobehaviour, haematology, clinical biochemistry and clinical pathology evaluations. During the anatomical pathology examinations, no test substance-related findings were observed or detected during macroscopic examinations, organ weight evaluations or microscopic examinations. In specific, no effects were noted on reproductive organs examined in this study.

There is no evidence for specific target organ toxicity in this study.

The NOAEL is ≥ 1000 mg/kg bw/day in this study.