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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012/2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted March 1996
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, adopted July 2000
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Laromer PO33F
- Physical state: yellowish liquid
- Analytical purity: The test item is a complex mixture of isomers and homologues components. Thus, no purity can be stated (see analytical report No: 11L00272 for details).
- Purity test date: 2011-12-19
- Lot/batch No.: 110007P040
- Expiration date of the lot/batch: 2012-11-30
- Stability under test conditions: confirmed for up to 7 days (see study report No. 11L00387)
- Storage condition of test material: room temperature under light exclusion

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-11 wks
- Weight at study initiation: on average: Males: 335g; Females: 197g
- Fasting period before study: no
- Housing: single (except during mating and during lactation) in Markrolon type M III cages
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse-rat “GLP”, meal ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: app. 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The desired amount of test substance was weighed, and corn oil was added up to the correct volume. To prepare a homogenous suspension, the mixture was stirred with a magnetic stirrer also during administration. The test substance preparations were produced at least once a week and were stored at room temperature. The administration volume was 4 mL/kg body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance is poorly soluble in water, but forms a homogenous suspension in corn oil, which is also non-toxic to rats.
- Concentration in vehicle: 1.25; 3.75; 12.5g/100mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and concentration control analyses of the test-substance preparations were performed in all concentrations at the start of the administration period. Additionally, samples from all concentrations as reverse samples for concentration control analysis were taken at the end of the study. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 90.0-110.0% of the nominal concentrations. Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that Laromer PO 33 F was distributed homogeneously in corn oil.
Duration of treatment / exposure:
Males: 35 days
Females: 56 days
Frequency of treatment:
daily (except to animals being in labor)
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 150, 500 mg/kg b.w.
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The selection of doses was based on the results of a test study in female Wistar rats (BASF project No. 10C0457/11S163) conducted at dose levels of 0, 600 and 1000 mg/kg bw/d. In this study, a NOAEL was not established due to erosions and ulcerations in the stomach of different animals at both dose levels of 600 and 1000 mg/kg bw/d as well as clinical findings like poor general condition, piloerection and body weight loss after 1 week of treatment.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on workdays, daily on weekends and public holidays
- Cage side observations: check for moribund animals, pertinent behavioral changes, signs of overt toxicity, parturation, littering and lactation behavior of the dams

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first administration, weekly thereafter
- The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, weekly thereafter with the following exceptions for females: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 4.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, except during mating

WATER CONSUMPTION: Monitored by daily visual inspection

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (16-20h)
- How many animals: 5 per sex and group
- The following parameters were examined: Leukocyte count, Erythrocyte count, hemoglobin, hemtocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differential blood count, reticulocytes, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Animals fasted: Yes (16-20h)
- How many animals: 5 per sex and group
- The following parameters were examined: ALT, AST, ALP, GGT, sodium, potassium, chlorid, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: day 31 (males), day 52 (females)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: yes, during collection
- How many animals: 5 per sex and group
- The following parameters were examined: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbity, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 3 days prior to necropsy (day 29 males, day 50 females)
- Dose groups that were examined: 5 animals per sex and group (only females with litter)
- Battery of functions tested: home cage and open field observation, motor activity, sensory motor test / reflexes (including: Reaction to an object being moved towards the face, touch sensitivity, vision (Visual placing response), pupillary reflex, pinna reflex, auditory startle response, righting response, behavior during handling, vocalization, pain perception (Tail pinch), grip strength of forelimbs and hindlimbs, landing foot-splay test)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
The following tissues were weighed
- in all animals: epididymides, testes
- in 5 males and females of each group: adrenal glands, brain, heart, kidneys, liver, spleen, thymus
The uteri of all cohabited female parental animals were examined for the presence and number of implantation sites. The uteri of apparently non-pregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations.
The following tissues were examined histotechnically in at least 5 animals per sex of the control and high dose group (reproductive organs were examined in all high dose and control animals):
adrenal glands, gross lesions (all affected animals in all dosage groups), bone marrow (femur), brain, cecum, cervix, coagulation glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (auxillary and mesenteric), ovaries, oviducts, prostate gland, peyer's patches, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, stomach (also all males from the low and mid dose groups), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina
Other examinations:
Reproductive performance: see entry in chapter 7.8.1

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Female animal No. 131 of test group 3 (500 mg/kg bw/d) was sacrificed in a moribund condition on study day 51 (GD 24). Vaginal discharge was observed on GDs 23-24 in this animal, which showed poor general state and was unable to deliver on GD 24. This animal was sacrificed moribund on the same day. According to the pathological results the findings were assessed as being incidental and not related to treatment.

Almost all male and most female animals of the high dose group showed salivation within 2 hours after the administration on several days of the study. Salivation within 2 hours after treatment was also seen in several mid dose male and female animals. From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract.

BODY WEIGHT AND WEIGHT GAIN
No test substance-related changes in body weight or body weight gain were observed for all test groups.

FOOD CONSUMPTION
No test substance-related, adverse findings were noted.

WATER CONSUMPTION
No test substance-related, adverse findings were noted.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.

NEUROBEHAVIOUR
All deviations from "zero" values were equally distribute between all groups including controls, the finding were assessed as being incidental and not treatment related.

ORGAN WEIGHTS
All mean weight parameters (absolute and relative) did not show significant differences when compared to the control groups.

GROSS PATHOLOGY
All gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
All findings were considered to be incidental or spontaneous in origin and without any relation to treatment.
The high dose female animal No. 131 that was sacrificed in a moribund state revealed a decidual reaction with consequent inflammation of the uterus. Decidual reaction is a proliferation of decidual cells (tissue of endometrial origin lining of the uterus, which is in contact with the fetal membranes and the placental plate). They often are assiocated with inflammation as observed in this animal. The local inflammation in the uterus can lead to disturbance of the general condition or, if the inflammation is spreading, sepsis. This was regarded to be the reason for the moribund state of this animal but was not regarded to be treatment-related.

Effect levels

Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion