Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

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

Test material

Constituent 1
Reference substance name:
DMC
IUPAC Name:
DMC
Details on test material:
- Name of test material (as cited in study report): DMC
- Substance type: not stated
- Physical state: not stated
- Analytical purity: not stated
- Impurities (identity and concentrations): not stated
- Composition of test material, percentage of components: not stated
- Purity test date: September 1991
- Lot/batch No.: 9109/S
- Expiration date of the lot/batch: stated to be unlimited
- Stability under test conditions: not stated
- Storage condition of test material: room temperature

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 Italia S.p.A., via Indipendenza 11 - 22050 CALCO (Como)
- Age at study initiation: 29 d (at receipt), aclimatization period approx. 2 weeks
- Weight at study initiation: males 75 - 85 g; females 60 - 70 g
- Fasting period before study: not stated
- Housing: limited access barriered rodent facility
- Diet (e.g. ad libitum): pelleted diet coded 4 RF 21 GLP top certificate produced for Charel River Italia by Mucedola S.r.l., Settimo milanese offered ad libitum
- Water (e.g. ad libitum): municipal supply, filtered offered ad libtum
- Acclimation period: approx. two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2C
- Humidity (%): 55%=/- 10%
- Air changes (per hr): approx. 20/h filtered on HEPA
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 29 Oct 1991 To: 26 Feb 1992

Administration / exposure

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


DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:


VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0.1, 0.5, 5 and 50 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg b.w.
- Lot/batch no. (if required): -
- Purity: -
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No details provided
"The concentration checks were performed by the Sponsor on formulates prepared on November 4, 1991, December 9 , 1991 and January 23, 1992. The results reported in Sponsor Communlcation dated February 20, 1992, were in accordance with the expected values."
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once a day
Doses / concentrations
Remarks:
Doses / Concentrations:
1, 5, 50 and 500 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
75M + 75F (15M + 15F in each experimental group)
Control animals:
yes
Details on study design:
- Dose selection rationale: agreed with sponsor
(-> this is the original information as it can be found in the study report and as it has been submitted with the IUCLID dossier in 2010)

More information is provided about the dose selection rationale (in Dec 2016) due to a request of ECHA (31 Aug 2015) to specify this information.
The requested information has been attached to this endpoint record under "Overall remarks, attachments", "Attached background material".
Origin: The service provider of the lead registrant has been in contact with the data owner of the 90-d-study to have the rationale for the dose selection clarified and has received the attached explanations.

- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.


DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


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 / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data


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 / No / No data


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weeks 1, 7, 13 & 17
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 6, 13 & 18
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals/sex weeks 6 & 13. 5 animals/sex week 18
- Parameters checked in table [No.?] were examined. Erythrocytes, Hemoglobin, Leukocytes, Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Hematocr, Mean Corpuscular Volume, Mean Corpuscular HGB Concn., Mean Corpuscular HGB, Platelets & Prothrombin Time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 6, 13 & 18
- Animals fasted: No data
- How many animals: 10 animals/sex weeks 6 & 13. 5 animals/sex week 18
- Parameters checked in table [No.?] were examined. glucose, urea, creatinine, total bilurubin, alkaline phosphatase, serum glutamic oxaloacetic transanimase, serum glutamic pyruvic transanimase, total cholesterol, total protein, serum protein electrophoresis, A/G ratio, sodium, potassium, calcium, inorganic phosphorous, chloride


URINALYSIS: Yes
- Time schedule for collection of urine: weeks 6, 13 & 18
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined. diuresis, specific gravity, leukocytes, nitrites, pH, protein, glucose, ketone bodies, urobilinogen, bilirubin, blood. Sediment of each urine sample was examined microscopically for the presence of: epithelial cells, leukocytes, erythrocytes, crystals, casts, bacteria and other abnormal


NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. At the end of the treatment and recovery periods the body weight of each animal that had been fasted over night (16 hours ) was recorded before the animal was sacrificed by incision of the femoral arteries after having been completely anesthetized with an i .p . injection of an overdosage of sodium pentobarbital at the dose of 50 mg/kg . Each animal was subjected to a detailed gross necropsy.
The following organs were removed. Individual organ weight/fasted body weight ratios were calculated
Skin and mammary gland, urinary bladder, prostate, testes, epididymides, (seminal vesicles), (vagina), uterus, ovaries, spleen, stomach, intestine : duodenum, jejunum, ileum, cecum, colon, rectum, mesenteric lymph nodes, pancreas, liver, kidneys, adrenals, submandibular salivary glands (and lymph nodes), sternum with bone marrow, heart, thymus, lungs, aorta, trachea, esophagus, thyroid with parathyroids (if present in the thyroid section), eyes(exorbital lacrimal glands), (tongue), Brain - coronal sections at three levels, pituitary, (skeletal muscle : biceps femoris), peripheral nerve : sciatic nerve, spinal cord : 'thoracic , (cervical and lumbar), (vertebrae), (femur, including articular surface), gross lesions


HISTOPATHOLOGY: Yes. All or part of each of the above organs were fixed in 10% buffered neutral formalin .Histology was carried out on the above-listed (unbracketed) organs and tissuesof all animals that died before the end of the study and of animals sacrificed at the end of treatment in the control and high dosage groups. Lungs and gross lesions were also examined in the intermediate and low dosage groups sacrificed at the end of the treatment period and in the animals that underwent recovery .The organs to be histologically examined were post-fixed for about half an hour in Carnoy's fluid, embedded in paraffin blocks , sectioned and stained with hematoxylin and eosin. Bone tissues were decalcified before being embedded .
Other examinations:
none
Statistics:
The parameters statistically examined were:body weight, body weight gain, food intake, hematology, blood chemistry, urinalysis and organ weights (absolute and relative to body weight).Data processing and evaluation information is included as an attachment (see below)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
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:
no effects observed
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:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: Three rats died during the study. No morphological compound related modifications were seen in any of these rats. No compound-related clinical signs or behavioral disorders were sen either during the dosing or recovery period in any group.


BODY WEIGHT AND WEIGHT GAIN: No effects on body weight that could be attributed to DMC oral administration for 13 consecutive weeks. were observed at any of the dosage levels applied . No body weight changes were seen in males during the withdrawal period , while a mean body weight gain (incidentally lower than controls) was seen in females from the two lower dosage groups during the firstt week of the recovery period but comparable afterwards to that of the control group


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): During the dosing and recovery periods, no changes in food consumption thatcould be attributed to DMC administration were seen at any dose


FOOD EFFICIENCY: not examined


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not examined


OPHTHALMOSCOPIC EXAMINATION: No treatment-related changes were seen at any dose


HAEMATOLOGY: No changes in the hematological profile that could be attributed to DMC administration were seen either at the mid-term (week 6) or end-of-dosing (week 13) examination at any dose.


CLINICAL CHEMISTRY: No blood chemistry changes that could be related to DMC administration were seen at any dose in animals of either sex .


URINALYSIS: For the urine parameters statistically analyzed (diuresis. specific gravity and pH) no changes that could be related to DMC administration were seen at any time at any dose. Assessment o f the semiquantitative analysis and sediment microscopy did not reveal major changes from controls in any of the DMC-treated groups at an y time ; frequ e n c y and degree of the positive findings fell within the range of normality for rats of this strain and age . No urinary changes were seen after discontinuation of treatment for 4 weeks .


NEUROBEHAVIOUR: -


ORGAN WEIGHTS: No changes attributable to treatment were seen at the final or recovery killings, and all mean absolute and relative values were comparable in treated and control groups .


GROSS PATHOLOGY: No test-compound related changes were observed at either the final or recovery killings.


HISTOPATHOLOGY: NON-NEOPLASTIC: No morphological changes related with DMC administration were seen at any dosage.


HISTOPATHOLOGY: NEOPLASTIC (if applicable) -


HISTORICAL CONTROL DATA (if applicable) -


OTHER FINDINGS -

Effect levels

Dose descriptor:
NOEL
Effect level:
> 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No DMC related deaths were seen . Clinical observations (symptomatology, body weight , food intake and ophthalmoscopic examination). laboratory investigations (hematology, blood chemistry and urinalysis) and post-mortem examinations (organ weight, gross pathology and histology) did not show any changes attributable to the test article in rats of any group.

In conclusion, on the basis of the overall results obtained in this study, oral administration of the test article DMC daily for 13 consecutive weeks to Sprague Dawley rats was well tolerated up to and including the highest dosage of 500 mg/kg/day and therefore this dose level is considered the NEL both in male and female rats .