Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11th February 1993 to 7th December 1993
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:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: 97.3%
Batch No.: 9236-23
Appearance: white powder

Test animals

Species:
rat
Strain:
other: Sprague-Dawley: Ico:OFA.SD. (IOPS Caw)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: IFFA CREDO, BP 0109, 69592 L'Arbresle, Cedex, France
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 218 to 265 g; Females: 181 to 219 g
- Fasting period before study: No - powdered diet was offered from 7 days before the start of treatment
- Housing: Animals were housed in groups of 5 of the same sex and dose group in stainless steel mesh cages (555 x 350 x 200 mm).
- Diet (e.g. ad libitum): Rat and mouse complete diet available ad libitum.
- Water (e.g. ad libitum): Filtered (0.2 µm) main drinking water available ad libitum from an automatic watering system.
- Acclimation period: 13-14 days between animal arrival and start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C (target range). On three occasions during the study the temperature was above the target range (with a maximum of 25.5°C)
- Humidity (%): 35 to 75% (target range). Relative humidity was outside the target range on 3 occasions (between 20 to 80%).
- Air changes (per hr): Minimum 15 air changes per hour
- Photoperiod: 12 hours light (artificial)/ 12 hours dark

IN-LIFE DATES: Not specified

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test article was prepared in a form suitable for admixture in basic powdered diet. For each concentration a weighed amount of test article was mixed into a small amount of diet in a mixer to produce a pre-mixture of high test article concentration. The pre-mixture was then diluted to the final desired concentration with the necessary quantity of basic diet and mixed in a blender for 12 minutes.

DIET PREPARATION
- Rate of preparation of diet (frequency): Test article/ diet mixes were prepared at least weekly.
- Mixing appropriate amounts with (Type of food): Basic powdered diet (Diet A04C-10, Usine d'Alimentation Rationnelle, Villemoisson, 91360 Epinay-sur-Orge, France)
- Storage temperature of food: Mixes were prepared up to 3 days before distribution and stored at room temperature until filling of the food hoppers.

VEHICLE
Not applicable - the test material was mixed directly into the diet.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At weeks 1, 4, 8 and 13, the preapred diets for each sex/dose group were analysed for achieved concentration of the bulk diets on the day of preparation.
Similar analysis was performed at week 1, on the last day of the treatment week for the diet placed in extra food hoppers in an empty cage for one week.
Duration of treatment / exposure:
Males: 92 days
Females: 93 days
Reversibility animals: 91 days
Frequency of treatment:
Dosing regime: 7 days/week, continuous
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2000, 6500 and 20000 mg/kg powdered diet
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
123.1, 406.5 and 1261.3 mg/kg bw/day for males
Basis:
actual ingested
Remarks:
Doses / Concentrations:
146.9, 478.5 and 1479.2 mg/kg bw/day for females
Basis:
actual ingested
No. of animals per sex per dose:
Male: 15 animals at 0 mg/kg bw/day
Male: 10 animals at 123.1 mg/kg bw/day
Male: 10 animals at 406.4 mg/kg bw/day
Male: 15 animals at 1261.3 mg/kg bw/day
Female: 15 animals at 0 mg/kg bw/day
Female: 10 animals at 135.7 mg/kg bw/day
Female: 10 animals at 478.5 mg/kg bw/day
Female: 15 animals at 1479.2 mg/kg bw/day
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The concentrations were specified by the study sponsor.
- Rationale for animal assignment: Random

After week 13, 10 animals/group/sex were killed the day after the last day of treatment. 5 animals/group/sex in the control and high dose groups (the remaining animals) were then sacrificed in after week 17 i.e. following a 4 week treatment free period.
Positive control:
Not used

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily, at the beginning and end of each working day for mortality/morbidity.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily to detect any clinical signs or reaction to treatment. A full clinical examination was performed weekly.


BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly during the treatment period and the treatment free period.


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 - food consumption was measured weekly for each cage of animals during the treatment period and the treatment-free period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes - the mean achieved intake of test article expressed in mg/kg bw/day was calculated weekly for each group and sex by the computer according to a formula based on test article concentration in the diet, food consumption over the week, body weight at the beginning of the week and body weight gain for the week.


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: No data


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable


OPHTHALMOSCOPIC EXAMINATION: Yes - a mydriatic agent (tropicamide) was instilled into the eyes before examination
- Time schedule for examinations: Week 13
- Dose groups that were examined: All animals were examined pretest and all animals in groups 1 (control) and 4 (high dose) at week 13.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: 10 animals/sex for groups 1 to 4 after week 13 and and all recovery animals after week 17.
- Anaesthetic used for blood collection: Yes - light ether
- Animals fasted: Yes - for at least 16 hours
- How many animals: 10 animals/sex for groups 1 to 4 and and all recovery animals
- Parameters examined: Haemoglobin, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, packed cell volume, red blood cell count, mean corpuscular volume, total white blood cell count, differential white blood cell count, platelet count.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 10 animals/sex for groups 1 to 4 after week 13 and and all recovery animals after week 17.
- Animals fasted: Yes - for at least 16 hours
- How many animals: 10 animals/sex for groups 1 to 4 and all recovery animals (only for blood urea nitrogen)
- Parameters examined: Sodium, potassium, chloride, calcium inorganic phosphorus, glucose, blood urea nitrogen, total cholesterol, total bilirubin, total protein, albumin, globulin (calculated), albumin/ globulin ratio, creatinine, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After weeks 13 and 17, all animals were necropsied in random order after fasting. Animals were killed by carbon dioxide inhalation and exsanguination. All animals were submitted to full necropsy procedures including examination of:
- the external surface
- all orifices
- the cranial cavity
- the carcass
- the external surface of the brain and of samples of the spinal cord
- the thoracic, abdominal and pelvic cavities and viscera
- the cervical tissues and organs.
The following organs were weighed at scheduled necropsy for all animals:
- adrenals
- brain
- kidneys
- liver
- testes

HISTOPATHOLOGY: Yes - histopatholgical examinations were performed for all organs/ tissues, with the exception of those marked with a *, for all animals in groups 1 (control) and 4 (high dose) killed after week 13. The liver, kidney and lungs were examined for all animals in all group killed after week 13. In each group, histopathological examination was performed for all gross lesions, ecpet those for which diagnosis is judged unnecessary for the outcome of the study by the pathologist. The following organs/ tissues were sampled for all animals:
- adrenals
- aorta
- bone (femur)* and articulation*
- bone (sternum) with bone marrow
- bone marrow smears
- bronchi (mainstem)
- brain
- caecum
- colon
- duodenum
- epididymides
- eyes
- heart
- ileum
- jejunum
- kidneys
- lacrimal gland* (exorbitol)
- liver
- lungs
- lymph node (submaxillary)
- lymph node (mesenteric)
- mammary gland*
- oesophagus
- optic nerves*
- ovaries
- pancreas
- parathyroids
- pituitary
- prostate*
- rectum
- salivary gland (submaxillary)*
- sciatic nerve
- seminal vessels
- skeletal muscle* (quadriceps femoris)
- skin
- spinal cord (cervical, thoracic, lumbar)
- spleen
- stomach (fundus, pylorus)
- testes
- thymus
- thyroids
- trachea
- urinary bladder
- uterus (horn & cervix)
- vagina
- all gross lesions
Other examinations:
None reported
Statistics:
Data from concurrent controls and historical data from (gavage) control rats were used to evaluate the effect of the test article.
For body weight gains, food consumption, food efficiency, test article intake, haematology, blood clinical chemistry parameters and organ weights, the arithmetic mean and standard deviation were calculated for each group and sex.
For each parameter, Levene's test was used to test the equality of variance across groups. However, as Levene's test showed no significant difference in the group variances, data were analysed using parametric procedures. Such analysis consisted of a one way analysis of variance (ANOVA) allowing for a group effect, followed by pairwise comparisons using a protected t-test or Dunnett's t-test to assess the significance of intergroup differences.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality and no treatment-related clinical signs were noted during the study.

BODY WEIGHT AND WEIGHT GAIN
A very small decreased body weight gain was noted for group 3 and 4 males from the beginning of treatment and for group 4 females (not statistically significant)after 1 month of treatment. Evidence of reversibility was noted for the group 4 females.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A very slight decrease of food consumption (not statistically significant), without adverse effect, was noted for group 3 and 4 males. It was considered not to be treatment related.

OPHTHALMOSCOPIC EXAMINATION
No treatment related changes were noted.

HAEMATOLOGY
No treatment related changes were noted.

CLINICAL CHEMISTRY
A very slight increase of blood urea nitrogen was noted in group 4 males (not statistically significant when compared to controls). This effect was reversible after 4 weeks without treatment.; hence, this effect was considered to be without toxicological significance.
The slight increase in alkaline phosphatase activity noted in Group 4 males was not considered to be of toxicological significance since the mean value was within the background range.

ORGAN WEIGHTS
No treatment related changes were noted.

MACROSCOPIC and MICROSCOPIC PATHOLOGY FINDINGS
There were no findings or lesions considered to be related to treatment.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
406.5 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: decreased body weight gain
Dose descriptor:
NOAEL
Effect level:
478.5 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: decreased body weight gain
Dose descriptor:
NOEL
Effect level:
123.1 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: decreased body weight
Dose descriptor:
NOEL
Effect level:
146.9 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: decreased body weight gain

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Administration of the test article induced a decreased body weight gain in group 3 male and in group 4 of both sex, reversible in group 4 females. The concentration of 2000 ppm which corresponded to mean intakes of between 123.1 and 146.9 mg/kg bw/day in males and females, respectively is considered as a NOEL (NOAEL 406.5 mg/kg bw/day and 478.5 mg/kg bw/day in males and females, respectively).
Executive summary:

The oral administration (dietary admixture) of the test article Millad 3988 to Sprague-Dawley rats for 13 consecutive weeks of concentrations of 2000, 6500 and 20000 ppm induced a decreased body weight gain in group 3 male and in group 4 of both sex, reversible in group 4 females. The concentration of 2000 ppm which corresponded to mean intakes of between 123.1 and 146.9 mg/kg bw/day in males and females, respectively is considered as a NOEL (NOAEL 406.5 mg/kg bw/day and 478.5 mg/kg bw/day in males and females, respectively).