Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Laromer LR 8869
- Physical state: colorless, clear liquid
- Analytical purity: for details see analytical report no. 11L00263
- Purity test date: 2012-01-11
- Lot/batch No.: 110009P040
- Expiration date of the lot/batch: 2012-07-4
- Stability under test conditions: verified for at least 7 days
- Storage condition of test material: at room temperature, light protected, avoid temperatures above 25°C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 wks;
- Weight at study initiation: Males: 294g; Females: 197g, on average
- Fasting period before study: no
- Housing: individually (except during overnight mating and lactation) in Makrolon type M III cages
- Diet (e.g. ad libitum): Kliba maintenance diet mouse-rat “GLP” meal, ad lib.
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr):15

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:The test substance was applied as suspension, which was prepared at least once a week. An appropriate amount of the test substance was weighed, and corn oil was added up to the desired volume. The suspension was kept homogenous during administration by stirring with a magnetic stirrer.

The administration volume was 4ml/kg b.w.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance is poorly soluble in water.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in corn oil for a period of 7 days at room temperature was proven before the start of the study (BASF Project No. 11L00386).
For homogeneity and concentration control analyses, each 6 samples of all concentrations was drawn at the start of the study and towards the end of the administration period. Homogeneity was confirmed, all concentrations measured were within 90-110% of the target concentration.
Duration of treatment / exposure:
males: 36 days
females: 51 days
Frequency of treatment:
daily, except to animals being in labor
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000mg/kg (high dose was reduced to 600mg/kg on day 19)
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of a range-finding study - no severe signs of toxicity were observed in animals treated with 1000mg/kg of the test substance for 14 days.
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, hematocrit, 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

URINALYSIS: Yes
- Time schedule for collection of urine: day 30 (males) and day 50 (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 32 males, day 49 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 following tissues were examined histotechnically in at least 5 animals per sex of the control and high dose group unless noted otherwise
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, forestomach (all animals from all dose groups), glandular stomach, 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

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 (study days 0 to 18) and 600 mg/kg bw/d (from study day 19 onwards):
- One female animal was found dead on study day 16 (mating day 3) and another one was sacrificed moribund on study day 18 (gestation day 2).
- One male animal was found dead on study day 35 (post-mating day 7).
- Piloerection was observed in 3 of 10 female animals during DCO, in 1 female animal during premating and post mating as well as in 2 females in mating and gestation.
- Smeared fur was observed in 1 of 10 female animals during gestation.
- Respiratory sounds were observed in 1 of 10 females during mating, in 2 of 10 females during gestation and in 1 of 10 females during lactation, gasping was observed in 1 of 10 female animals during gestation and lactation.
- Poor general condition was observed in 2 of 10 female animals during gestation.
- Hunched posture was observed in 1 of 10 female animals during gestation.
- Semiclosed eyelids were observed in 1 of 10 female animals during gestation.
Mortality:
mortality observed, treatment-related
Description (incidence):
due to local effects: ersoion/ulcer in the forstomach,
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
In test group 3 (1000 and 600 mg/kg bw/d) female animal No. 80 was found dead on study day 17 (Mating Day 3), female animal No. 74 was sacrificed moribund on study day 19 (GD 2), and male animal No. 32 was found dead on study day 35. These premature deaths were assessed as being related to the test substance. One female animal of test group 2 (300 mg/kg bw/d) was also found dead on study day 44. The animal died because of a gavage error. Therefore, a relation to treatment was excluded.

Almost all animals of both sexes of the mid and high dose group showed mostly slight, sometimes moderate salivation within 2 hours after the test substance administration on several days of the study. The same was true for individual low dose animals. From the temporary, short appearance immediately after dosing it could be assumed that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was related to the test substance but assessed as being non-adverse.

Animal No. 74 of test group 3 (1000 and 600 mg/kg bw/d) showed poor general condition, piloerection and respiration sounds on GD 1 and GD 2. Therefore, the animal was sacrificed in a moribund condition on GD 19. Animal No. 75 of the same group showed semiclosed eyelids, poor general condition, hunched posture, smeared fur, piloerection (up to GD 7) and gasping on GD 2 and respiration sounds on GD 3. The same animal showed respiration gasping and sounds on LD 1. These findings were assessed as being related to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No treatment-related changes in body weight data were observed in animals of all test groups.

FOOD CONSUMPTION
No treatment-related changes in food consumption were observed.

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

CLINICAL CHEMISTRY
No treatment-related changes were measured.

URINALYSIS
No test substance-related effects were observed.

NEUROBEHAVIOUR
No test substance-related effects were observed.

ORGAN WEIGHTS
Absolute thymus weights were significantly decreased in high dose male animals when compared to control group animals. This finding was not considered to be treatment related, since no relative organ weights were significantly altered.

GROSS PATHOLOGY
Erosions, ulcers, and in one case a thickened forstomach were observed in all high dose male and females (with the exception of one female) and 5 mid dose females and 4 mid dose males.

HISTOPATHOLOGY:
Five male and 6 female animals of test group 3 (1000 and 600 mg/kg bw/d) as well as each 2 males and females of test group 2 (300 mg/kg bw/d) revealed multifocal erosions/ulcers in the forestomach. Accompanying these erosions/ulcers a mild to extreme diffuse or focal squamous hyperplasia with hyperkeratosis and a mild to severe submucosal edema was present. When the hyperplasia of the squamous epithelium was focal, it was located at the cardia region (stomach entrance). Within the edema a higher number of inflammatory cells were observed. Not all animals with a macroscopically diagnosed erosion/ulcer showed this finding. But the consistency of the accompanying findings (hyperplasia with hyperkeratosis and edema) which were observed in animals with and without erosion/ulcer led to the conclusion that this all belongs to the same complex. As the erosions/ulcer that could be seen microscopically were very small it is possible that they were not on the level of section. But taken all these findings together they point towards an irritating effect of the substance on the squamous epithelium of the forestomach. Especially the cardia region (entrance of the stomach) was affected.
The three animals of test group 3 (1000 and 600 mg/kg bw/d) which were found dead or were sacrificed in a moribund (animal Nos. 32, 74 and 80) did not show any other findings that could explain the death beside the ones in the stomach. Two of them revealed a moderate to severe thymus atrophy which was regarded to be a consequence to the stress caused by the findings in the forestomach. But it is not clear whether these observed findings were the cause of the death although death was regarded to be a consequence to the treatment with the test substance.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: deaths and poor general state in high dose animals
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: irritation in the stomach

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion