Ethanamine, N-ethyl-, reaction products with polyethylene-polypropylene glycol ether with trimethylolpropane (3:1) acrylate (>1 <6.5 mol EO and >1 < 6.5 mol PO)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-05-2017 - 10-04-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

1

Reference substance name:

Ethanamine, N-ethyl-, reaction products with polyethylene-polypropylene glycol ether with trimethylolpropane (3:1) acrylate (>1 <6.5 mol EO and >1 < 6.5 mol PO)

EC Number:

605-659-3

Cas Number:

173046-61-2

Molecular formula:

Mixture of C6H11O3-[C3H6O]p-[C2H4O]q-[C3H3O]r-C7H14ON and C6H11O3-[C3H6O]p-[C2H4O]q-[C3H3O]s With: 0

IUPAC Name:

Ethanamine, N-ethyl-, reaction products with polyethylene-polypropylene glycol ether with trimethylolpropane (3:1) acrylate (>1 <6.5 mol EO and >1 < 6.5 mol PO)

Test material form:

liquid

Specific details on test material used for the study:

The analyses of the test item (= test substance) have been carried out at Competence Center Analytics, BASF SE, 67056 Ludwigshafen, Germany. The Sponsor is responsible for compliance for all test substance information and their storage, except for those test substance investigations commissioned by the test facility.

Name of test substance: Laromer LR 8889
Test substance No.: 16/0422-1

Batch No.: 160005P040
CAS No.: 173046-61-2
Purity: 98.2 area-% (HPLC, 201 nm)
98.4 area-% (HPLC, 231 nm)
Content: > 99 g/100 g

Homogeneity: given
Date of production: 21 Oct 2016
Storage stability: Expiry date: 16 Oct 2017
The stability of the test substance under storage conditions over the test period was guaranteed by the Sponsor, and the Sponsor holds this responsibility.
The test facility is organizationally independent from the BASF SE sponsor division

ADDITIONAL TEST SUBSTANCE INFORMATION
Physical state/Appearance: Liquid / yellowish, clear
Storage conditions: Room temperature

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The test guidelines require the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

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
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males 11 - 12 weeks old and females about 9 - 10 weeks old
- Fasting period before study: no
- Housing: During pretreatment, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Hohenpeißenberg, Germany.
During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop- Rauxel, Germany, with the following exceptions:
•During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
•Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
Pregnant females were provided with nesting material (cellulose wadding) towards the end of gestation.
For enrichment wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added. In addition, in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX B.V., Elst, Netherlands) were added.

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY:
The food used was ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study (from the day of supply to the day before necropsy). Drinking water was supplied from water bottles (ad libitum).

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

IN-LIFE DATES: From: To: 16-05-2017 - 16-08-2017

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.

Vehicle:

corn oil

Details on oral exposure:

TEST SUBSTANCE PREPARATIONS
The test substance preparations in corn oil were prepared in intervals, which took into account the analytical results of the stability verification. The test substance preparations were produced weekly, at least.

For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with corn oil and mixed with a magnetic stirrer.


TEST GROUPS AND DOSES F0 generation parental animals

Test group Dose Concentration Dose volume
(mg/kg body weight/day) (g/100ml) (ml/kg body weight/day)
0 0 0 4*
1 100 2.50 4**
2 300 7.50 4**
3 1000 25.00 4**

* = Corn oil
** = Test substance preparations in Corn oil

Justification for use and choice of vehicle: the test item is almost insoluble in water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses were carried out at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany.
Analytical verifications of the stability of the test substance in corn oil for a period of 7 days at room temperature were carried out prior to the start of the study.
At the beginning (during premating), twice during gestation and once during lactation of the study each 1 sample was taken from all concentrations for concentration control analyses. Of each sample, one additional reserve sample (described by the suffix “R”) was retained.
The samples collected at the beginning and during lactation period were analyzed in the Analytical Laboratory. All further samples were not analyzed and stored frozen (at -20 °C) in the Laboratory of the Mechanistic Toxicology until the finalization of the study.

Duration of treatment / exposure:

35 (males) - 70 (females) days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels are based on the results of a 2-week preliminary toxicity study in Han Wistar rats. In that study, exposure of four males and four females to 1000 mg/kg/day led to the following rather unspecific and/or randomly occuring observations:

• Poor, general condition in one male
• Salivation after administration in all females and one male
• Respiration sounds in 2/8 animals
• Food consumption and water consumption was decreased in males and increased in females on single occasions
• In agreement with the lower food consumption, there was a slight body weight loss until day 3 in males (-2.6g)
• Liver weights were increased in males by 17%
• In females, the red blood cell count and HCT were increased, while MCH and MCHC were decreased
• Due to the irritant properties of the test substance, foci in the forestomach and discoloration of the glandular stomach were observed in one male each.
• One male was found dead on day 4. Pathological examinations revealed discoloration of the lung, potential an indication of a gavage error. Since all other animals survived without relevant toxic effects, this death was not considered substance related.

300 mg/kg bw/day only led to salivation after administration in 7/8 animals and to an altered water consumption (decreased for males, increased for females, each on a single occasion only)

1000 mg/kg/day was therefore selected as the high dose for this study with a low dose of 100 mg/kg/day and an intermediate dose of 300 mg/kg/day.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data.


DETAILED CLINICAL OBSERVATIONS: Yes
The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.
For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed:
1.Abnormal behavior in “handling”
2.Fur
3.Skin
4.Posture
5.Salivation
6.Respiration
7.Activity/arousal level
8.Tremors
9.Convulsions
10.Abnormal movements
11.Gait abnormalities
12.Lacrimation
13.Palpebral closure
14.Exophthalmos
15.Assessment of the feces discharged during the examination (appearance /consistency)
16.Assessment of the urine discharged during the examination
17.Pupil size

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results. The following exceptions are notable for the female parental animals:
•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, 7, 10 and 13.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume;

FOOD CONSUMPTION AND COMPOUND INTAKE:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
•Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
•Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 7, 7 - 14, and 14 - 20.
•Food consumption of F0 females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10, 10 - 13.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.


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

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):
Parameters and methods:
Parameter Unit Method
Leukocyte count (WBC) giga/L cytochemistry coupled with flow cytometry
Erythrocyte count (RBC) tera/L flow cytometric laserlight scattering
Hemoglobin (HGB) mmol/L cyanmethemoglobin method; according to ICSH
Hematocrit (HCT) L/L calculation:MCV x erythrocytes
Mean corpuscular volume
(MCV) fL RBC/PLT method; mean of RBC volume distribution curve (histogram)
Mean corpuscular
hemoglobin (MCH) fmol calculation:hemoglobin/erythrocytes
Mean corpuscular hemoglobin
concentration (MCHC) mmol/L calculation:hemoglobin/hematocrit
Platelet count
(PLT) giga/L flow cytometric laserlight scattering
Differential blood count % and giga/L cytochemistry coupled with flow cytometry
Reticulocytes
(RETA) giga/L cytochemistry coupled with flow cytometry

Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Only evaluated blood smears were archived.

Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).


CLINICAL CHEMISTRY: Yes
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters

Parameters and methods:

Enzyme (systematic name and system number) Unit Method, wave- length and measuring
temperature (Detection limit)
Alanine aminotransferase (ALT)
(L-alanine: 2-oxoglutarate aminotransferase;
EC 2.6.1.2.) μkat/L kinetic UV test, 340 nm; 37°C, (0.08 μkat/L)
Aspartate aminotransferase (AST)
(L-aspartate: 2-oxoglutarate aminotransferase;
EC 2.6.1.1.) μkat/L kinetic UV test, 340 nm; 37°C, (0.08 μkat/L)
Alkaline phosphatase (ALP)
(orthophosphoric acid monoester phosphohydrolase;
EC 3.1.3.1.) μkat/L kinetic color test, 415 nm, 37°C, (0.084 μkat/L)
gamma-Glutamyltransferase (GGT)
(gamma-glutamyl) peptide: aminoacid-gamma- glutamyl-transferase;
EC 2.3.2.2.) nkat/L kinetic color test, 415 nm, 37°C, (25 nkat/L)
Sodium (NA) mmol/L
Potassium (K) mmol/L ion selective electrodes (ISE), (Na: 80, K: 1.5, Cl: 60 nmol/L)
Chloride (CL) mmol/L
Inorganic phosphate (INP) mmol/L molybdate reaction (0.1 mmol/L)
Calcium (CA) mmol/L mmol/L o-cresolphthalein complex without deproteinization (0.2 mmol/L)
Urea (UREA) mmol/L mmol/L enzymatic determination with the urease/ glutamate dehydro- genase method (0.5 mmol/L)
Creatinine (CREA) μmol/L enzymatic determination with the creatininase/ creatinase /sarcosinoxidase method (5 μmol/L)
Glucose (GLUC) mmol/L hexokinase/glucose-6-phosphate dehydrogenase method (0.11 mmol/L)
Total bilirubin (TBIL) μmol/L DPD method (0.56 μmol/L)
Total protein (TPROT) g/L biuret method (2 g/L)
Albumin (ALB) g/L bromocresol green method (3.2 g/L)
Globulins (GLOB) g/L difference between total protein and albumin
Triglycerides (TRIG) mmol/L enzymatic color test with lipase esterase/ glycerokinase/ glycerol- 3-phosphate oxidase/4-amino- phenazone (0.1 mmol/L)
Cholesterol (CHOL) mmol/L enzymatic determination with cholesterol esterase/ cholesterol oxidase/4-amino-phenazone (CHOD-PAP method) (0.1 mmol/L)
Bile acids (TBA) μmol/L enzymatic colorimetric determination with 3-hydroxy-steroid dehydrogenase and NAD (1 μmol/L)


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in the first 5 male and the first 5 female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations:
The animals were observed in their closed home cages (for a short period: about 10- 30 seconds); any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:

1.Posture
2.Tremors
3.Convulsions
4.Abnormal movements
5.Gait
6.Other findings

Open field observations:
The animals were transferred to a standard arena (50 × 50 × 25 cm) and observed. The following parameters were examined:

1.Behavior on removal from cage
2.Fur
3.Skin
4.Salivation
5.Nasal discharge
6.Lacrimation
7.Eyes/pupil size
8.Posture
9.Palpebral closure
10.Respiration
11.Tremors
12.Convulsions
13.Abnormal movements/stereotypy
14.Gait
15.Activity/arousal level
16.Feces (consistency/color) within 2 minutes
17.Urine (amount/color) within 2 minutes
18.Rearing within 2 minutes
19.Other findings

Sensory motor tests/Reflexes:
The animals were removed from the open field and subjected to following sensory motor or reflex tests:

1.Reaction to an object being moved towards the face (Approach response)
2.Touch sensitivity (Touch response)
3.Vision (Visual placing response)
4.Pupillary reflex
5.Pinna reflex
6.Audition (Startle response)
7.Coordination of movements (Righting response)
8.Behavior during handling
9.Vocalization
10.Pain perception (Tail pinch)
11.Other findings
12.Grip strength of forelimbs
13.Grip strength of hindlimbs
14.Landing foot-splay test


Motor activity measurement

The measurement of motor activity (MA) was measured at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.

IMMUNOLOGY: No

OTHER:
Thyroid Hormones
Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling.

Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH).

All generated serum samples were frozen at -80°C until measurement or at least until finalization of the report. This needs a separate approval of the study director. The documentation of the disposal of the samples will be archived retrospectively with the raw data.

The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Parameters and methods:
Hormone parameter Unit Method
Total thyroxine (T4) nmol/L ELISA
Thyroid stimulating hormone (TSH) μg/L direct, competitive radioimmunoassay

Sacrifice and pathology:

All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs. The animal no. 32 died intercurrently and was necropsied and assessed by gross pathology as soon as possible after their death.

Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below.

Following histotechnical processing, all histological slides, tables of individual macroscopic findings as well as organ weight tables and historical control data were sent to InSight Pathology BV, Chopinlaan 6, NL-5343EM Oss, The Netherlands. Light microscopical examination of the hematoxylin-eosin and special stained slides, and assessment of findings were performed under the responsibility of the Principal Investigator Eric van Esch at the test site InSight Pathology BV under the test site phase number ISP 17281.


Organs Test group
0 1 2 3
1.All gross lesions A2 A2 A2 A2
2.Adrenal glands A4 A4
3.Brain A4 A4
4.Cecum A4 A4
5.Cervix A1 A3 A3 A1
6.Coagulating glands A1 A3 A3 A1
7.Colon A4 A4
8.Duodenum A4 A4
9.Epididymides A1 A3 A3 A1
10.Eyes with optic nerve A4 A4
11.Heart A4 A4
12.Ileum A4 A4
13.Jejunum A4 A4
14.Kidneys A4 A4
15.Liver A1 A1 A1 A1
16.Lungs A4 A4
17.Lymph nodes
(axillary and mesenteric) A4 A4
18.Ovaries A1 A3 A3 A1
19.Oviducts A1 A3 A3 A1
20.Prostate gland A1 A3 A3 A1
21.Peyer’s patches A4 A4
22.Rectum A4 A4
23.Sciatic nerve A4 A4
24.Seminal vesicles A1 A3 A3 A1
25.Skeletal muscle A4 A4
26.Spinal cord
(cervical, thoracic, lumbar)A4 A4
27.Spleen A4 A4
28.Sternum with marrow A4 A4
29.Stomach
(forestomach and
glandular stomach) A1 A1 A1 A1
30.Testes A1 A3 A3 A1
31.Thymus A4 A4
32.Thyroid glands A4 A4
33.Trachea A4 A4
34.Urinary bladder A4 A4
35.Uterus A1 A3 A3 A1
36.Vagina A1 A3 A3 A1
A=Hematoxylin and Eosin stain
1=all animals/test group
2=all animals affected/test group
3=mating pairs suspected of reduced fertility
4=5 animals per sex/test group, females with litters only, same animals as used for clinical pathological examinations

The animal that died (no.32) was processed histotechnically and assessed like control animals

Special stains (Oil-Red-O and Sudan-Black) were performed exemplarily in the liver of selected animals (no. 1 and 101 of control and no. 35 and 133 of test group 3).
Special attention was given to the stages of spermatogenesis in the testes and histopathology of interstitial testicular cell structure. Whenever in the ovary the diagnosis: „no abnormalities detected” was used, that implies that all different stages of functional bodies (especially corpora lutea) were present and normal.
The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
A correlation between gross lesions and histopathological findings was attempted.

The following weights will be determined in all animals sacrificed on schedule:

1.Anesthetized animals (terminal body weight)
2.Epididymides
3.Ovaries
4.Prostate
5.Seminal vesicles with coagulating glands
6.Testes
7.Thyroid glands (fixed)
8.Uterus (with cervix)

The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):

1.Adrenal glands
2.Brain
3.Heart
4.Kidneys
5.Liver
6.Spleen
7.Thymus

Organ/tissue fixation

Parental animals
The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde or in modified Davidson’s solution:

1.All gross lesions
2.Adrenal glands
3.Aorta
4.Bone marrow (femur)
5.Brain
6.Cecum
7.Cervix
8.Coagulating glands
9.Colon
10.Duodenum
11.Eyes with optic nerve (modified Davidson’s solution)
12.Esophagus
13.Extraorbital lacrimal glands
14.Epididymides (modified Davidson’s solution)
15.Femur with knee joint
16.Heart
17.Ileum
18.Jejunum (with Peyer’s patches)
19.Kidneys
20.Larynx
21.Liver
22.Lungs
23.Lymph nodes (axillary and mesenteric)
24.Mammary gland (male and female)
25.Nose (nasal cavity)
26.Ovaries (modified Davidson’s solution)
27.Oviducts
28.Pancreas
29.Parathyroid glands
30.Pharynx
31.Pituitary gland
32.Prostate gland
33.Rectum
34.Salivary glands (mandibular and sublingual)
35.Sciatic nerve
36.Seminal vesicles
37.Skeletal muscle
38.Spinal cord (cervical, thoracic and lumbar cord)
39.Spleen
40.Sternum with marrow
41.Stomach (forestomach and glandular stomach)
42.Testes (modified Davidson’s solution)
43.Thymus
44.Thyroid glands
45.Trachea
46.Urinary bladder
47.Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E, 1964)
48.Vagina

The testes and epididymides of animal no. 32, that died intercurrently were fixed in 4% neutral-buffered formaldehyde solution.

Statistics:

Means, medians, standard deviations and deviation vs control of each test group were calculated for several parameters.

The following statistical analyses for blood parameters were used in this report:

For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two- sided) for the hypothesis of equal medians.
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Markers in the tables:
* for p < 0.05
** for p < 0.01

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the entire study period salivation from slight to severe was seen in all animals of test group 3 (1000 mg/kg bw/d) 0 to 2 hours after treatment. Salivation was first seen on pre- mating day 0 in one male and 4 female animals and afterwards gradually in all males and females of test group 3. Salivation was also observed in males and females of test group 2 (300 mg/kg bw/d). Due to the short time interval and its relation directly to the dosing procedure, this finding is attributed to the bad taste and/or irritant properties of the test substance and not considered adverse.

Furthermore respiration sounds were observed in one male (no. 34, test group 3) on pre- mating day 1 and in one female (no. 133, test group 3) on gestation day 5. Piloerection was seen in one male (no. 33, test group 3) on two consequent days (mating day 14 and post- mating day 0). These findings were considered as treatment related but based on their isolated and short time occurrence as not adverse.

A swelling of the mammary line was observed in one female of test group 1 (no. 119) and in one female of test group 3 (no. 139) on lactation days 14 to 15. This finding showed no significant difference to control. It was considered as incidental and likely to be a side effect of weaning the litter on PND 13.

Inadvertently, clinical signs are missing for all animals on pre-mating day 6 for the interval 2 to 5 hours after treatment. In the clinical observation before and thereafter, the salivation of all grades observed in the interval 0 to 2 hours after treatment was not observed in the interval 2 to 5 hours after treatment. Furthermore, no clinical signs were observed in the interval 2 to 5 hours that have not be determined in the interval 0 to 2 hours before. Therefore, it was assumed that on gestational day 6 this was also the case. Thus, the missing data were considered as not affecting the outcome and validity of this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male animal (no. 32) of test group 3 (1000 mg/kg bw/d) was found dead on mating day 7. The days before this animal merely showed moderate salivation (mating days 3 to 6) and no graver clinical signs. Histopathology revealed necrotic alterations in the trachea and bronchi, which point to trauma during the dosing procedure and have likely contributed to the death of this animal. No comparable changes were seen in the remaining animals, nor any clear signs of toxicity. Therefore, this death is considered to be unrelated to substance exposure.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and body weight change of all male and female F0 generation parental animals in all test substance-treated groups were comparable to the concurrent control values during the entire study period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of the F0 animals in all test groups was not influenced by the treatment throughout the entire study period.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.

At the end of the administration period in females of test groups 1 and 2 (100 and 300 mg/kg bw/d) mean corpuscular volume (MCV) values were significantly higher compared to controls. However, the change of this calculated parameter was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

One high dose male rat showed increased ALT, AST, and ALP activities. Histopathologically, a foci (4mm) containing necrotic cells was found in this animal. This isolated finding was not considered treatment related.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observational battery (FOB)

Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

Open field observations:
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.

Sensorimotor tests/reflexes:
There were no test substance-related findings in male and female animals of all test groups.

Quantitative Parameters:
There were no test substance-related findings in male and female animals of all test groups.

Motor activity measurement
No treatment-related changes on motor activity data (summation of all intervals) were observed in all male and female animals of all dose groups in comparison to the concurrent control group.

In female animals of test group 1 (100 mg/kg bw/d), 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) a significantly increase of beam interruptions was recorded for interval 6. In this interval the number of beam interruptions of the control group was relative low, breaking isolated the continuity of the habituation curve. Therefore, these isolated single findings of interval 6 in test group 1-3 without any effect on the respective sum of all intervals, were assessed as incidental and spontaneous in nature.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver
Slightly, but statistically significant increased relative mean liver weight (115% from control) present in males treated with 1000 mg/kg bw/d Laromer LR 8889. Liver weight in females of this group were slightly increased to 110% of the control value, but were still within the historical control data.
The weight change is considered to be adaptive.

Seminal vesicles
Slightly, but statistically significant increased absolute and relative mean seminal vesicles weight (120 and 121% from control, respectively), above historical control values, present in males treated with 300 mg/kg bw/d Laromer LR 8889.
The weight change was not dose dependent an is therefore considered not to be induced by the test substance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Forestomach
Focus/foci (organ-like color or white) noted in a single male (animal 39) and single female (139) of the 1000 mg/kg bw/d Laromer LR 8889 treated group.
The test substance is irritating to the eye. Foci in the forestomach of 2 out of 20 animals are most probably a result of the irritating test substance nature.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Forestomach
Morphologic changes were encountered within the forestomach of rats treated with Laromer LR 8889 and comprised:
Hyperplasia, squamous cell, in some affected animals accompanied by hyperkeratosis was observed in 2 out of 10 males and 4 out of 10 females of the group treated with 1000 mg/kg bw/d Laromer LR 8889.
Few small ulcers found within the area of hyperplasia, squamous cell in a single male and female of the group treated with 1000 mg/kg bw/d Laromer LR 8889.
The test substance is irritating to the eye. Hyperplasia and ulcers are most probably a result of the irritating test substance nature.

A small erosion noted at the limiting ridge of a single male (22) treated with 300 mg/kg bw/d, while male 31 treated with 1000 mg/kg bw/d Laromer LR 8889 showed degeneration of the squamous epithelium at the limiting ridge. Meaning this was not dose dependent and therefore most probably coincidential.


Liver
An increased incidence and severity of fine hepatocellular cytoplasmic vacuolation in the periportal/midzonal areas noted in males treated with 1000 mg/kg bw/d Laromer LR 8889. Using both the Oil-red-O and Sudan Back staining method, the hepatocellular intracytoplasmic vacuoles were proven to contain slightly increased amount of lipids (neutral fat) in the selected male animal from the group treated with 1000 mg/kg bw/d, compared to the control male. The increase incidence and severity of lipid accumulation - to the extent observed - is not considered an adverse finding. In the selected female from the high dose group, the amount of lipids was similar to that observed in the control female.


Testes/Epididymides
In the testes of male 33 of the group treated with 1000 mg/kg bw/d Laromer LR 8889, minimal vacuolation and degeneration of (elongated) spermatocytes and minimal loss of elongated spermatids in mainly stage XII-XIII tubules was present. The change resulted in minimal increased cellular debris within the epididymal ducts. The changes observed did not cause significant functional changes, since the affected male was able to produce normal offspring.
Thorough microscopic examination of all other males and all female reproductive organs (especial those from the two couples that had no offspring) did not reveal any other relevant histopathologic change. The morphology and distribution of the different successive stages during spermatogenesis (Russell et al., 1990) was normal for all other males examined. Therefore the degeneration of spermatocytes in male 33 is considered to be conicidencial and not adverse.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No alterations in T4 and TSH levels were observed.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

2

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

The concentrations of Laromer LR 8889 in corn oil were found to be in the range of 102 to 105% at the beginning of the premating period and 102 to 113% of the nominal concentration during lactation period. The 113% was observed in the sample of the low dose.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of Laromer LR 8889 to Wistar rats revealed no treatment-related adverse systemic findings in parental animals (F0) and pups (F1). Only signs of local toxicity were observed in local irritations to the mucosa of the forestomach at 1000 mg/kg bw/d in both sexes.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity as well as for reproductive performance and fertility was the dose 1000 mg/kg bw/d in both sexes of parental animals. The NOAEL for local toxicity was the dose 300 mg/kg bw/d in both sexes of parental animals.

The NOAEL for developmental toxicity in the F1 progeny was the dose 1000 mg/kg bw/d.