2,5-Furandione, reaction products with 2-ethyl-1-hexanamine and triethanolamine

Endpoint summary

Description of key information

NOAEL (systemic) = 1000 mg/kg bw/day

BASF SE, 2019, according to OECD 408, GLP compliant, no treatment-related, adverse effects observed in any test group (100, 300, 1000 mg/kg bw/day)

BASF SE, 2013, according to OECD 422, GLP compliant, no signs of systemic toxicity up to a dose level of 1000 mg/kg bw/d in animals of both sexes

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-08 until 2018-11-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

12 Sep 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Kaiser-Friedrich-Stra ße 7, 55116 Mainz, Germany

Limit test:

no

Specific details on test material used for the study:

Please note: This kind of substance was in the past handle under the CAS nr 85204-21-3
After analytical characterisation the substance was renamed and received a new CAS nr. Now the substance is characterised by the CAS 1471311-93-9 EC Number: 939-488-3

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF and 14671736W0
- Expiration date of the lot/batch: 01 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the vehicle: stability in drinking water with 5 mg/100 mL Cremophor EL over a period of 7 days at room temperature verified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: prepared once weekly, specific amount of test substance was weighed and filled up with deionized water containing Cremophor EL
- Final dilution of a dissolved liquid: suspension in deionized water with 5 mg/100 mL Cremophor EL, respectively

FORM AS APPLIED IN THE TEST: suspension; preparations were kept homogeneous with a magnetic stirrer

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation (mean): 174 g male, 123 g female; weight variation of the animals used did not exceed 20 percent of the mean weight of each sex
- Housing: group, 5 animals per cage, polysulfonate cages (TECNIPLAST, Hohenpeißenberg, Germany)
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP”, meal (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum, drinking water
- Acclimation period: up to 6 days

DETAILS OF FOOD AND WATER QUALITY:
- diet: assayed for chemical and microbiological contaminants by the supplier
- drinking water: regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (06.00-18.00 h / 18.00-06.00 h)

IN-LIFE DATES: From: 2018-01-16 To: 2018-04-24 / 2018-04-25

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized water containing 5 mg/100 mL Cremophor EL

Analytical verification of doses or concentrations:

yes

Remarks:

performed in all concentrations at the beginning and towards the end of the administration period

Details on analytical verification of doses or concentrations:

- samples stored in a freezer
- HPLC, with reversed-phase (RP-18e) stationary phase and mobile phase based on Acetonitrile/(NH4) H2PO4 Buffer
- approx. 1.3 min retention time

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

1 g/ 100 mL

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

3 g/ 100 mL

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

10 g/ 100 mL

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on available study information e.g. 85R0586/11S125
- Rationale for animal assignment: list of randomization instructions was compiled with a computer
- Fasting period before blood sampling for clinical biochemistry: at least 16 hours

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, before the administration as well as within 2 hours and within 5 hours after the administration
- Parameters: any abnormal clinically signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Parameters: Abnormal behavior when handled, 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: before the start of the administration period (randomization), during the administration period on day 0 and thereafter at weekly intervals

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly
- Parameter: mean food consumption in grams per animal and day

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily by visual inspection
- Parameter: any overt changes in volume

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of the administration period on day 0 and on study day 91
- Dose groups that were examined: control and high-dose animals
- Parameters: eyes examined for any changes using an ophthalmoscope after administration of a mydriatic agent

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning of day of study day 92 (male) and 93 (female)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Anticoagulant used: EDTA-K3
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- all details given for heamatology apply
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: in the afternoon of the study day 81
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (no food or drinking water provided during the unrinalysis)
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observational battery (FOB)
- Time schedule for examinations: at the end of the administration period at about 10:00 h
- Dose groups that were examined: all dose groups, all animals
- Battery of functions tested: passive observations, open field observations, sensory motor activity, reflex test
- Parameters:
- Home cage observations: Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings
- Open field observations: Behavior on removal from the cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/ pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/ stereotypes, Gait, Activity/ arousal level, Feces excreted within 2 minutes (appearance/ consistency), Urine excreted within 2 minutes (amount/ color), Rearing within 2 minutes
- Sensory motor tests/ reflexes: Reaction to an object being moved towards the face (approach response), Touch sensitivity (touch response), Vision (visual placing response), Pupillary reflex, Pinna reflex, Audition (auditory startle response), Coordination of movements (righting response), Behavior during handling, Vocalization, Pain perception (tail pinch), Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test

Motor activity:
- Time schedule for examinations: the same day as the FOB from 14:00 h, each measurement lasted exactly 1 hour
- Dose groups that were examined: all dose groups, all animals
- Parameters: animals individually house in cages, 18 beams were allocated per cage, number of beam interrupts was counted over 12 intervals for 5 minutes per interval, rats were fasted during the measurement

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Parameters: Organ weights and incidence of gross lesions for the following organs:
- Organs examined: Anesthetized animals (final body weight), Adrenal glands (fixed), Brain, Epididymides, Heart, Kidneys, Liver, Ovaries (fixed), Spleen, Testes, Thymus (fixed), Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix

HISTOPATHOLOGY: Yes (see table)

Statistics:

CLINICAL EXAMINATIONS
- Body weight, body weight change: DUNNETT's test (two-sided) for the hypothesis of equal means
- Rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided), if p<=0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians

CLINICAL PATHOLOGY
- blood paramters:
- parameters with bidirectional changes, Non-parametric one-way analysis using KRUSKAL-WALLIS test; if p<=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
- 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
- Urinalysis, (apart from urine volume, specific gravity, color and turbidity): Pairwise comparison with the WILCOXON-test (one-sided) for the hypothesis of equal medians (if exactly the same numbers of the dose group and the control, no statistical test)
- Urine volume and specific gravity: Non-parametric one-way analysis using KRUSKAL-WALLIS test, if p<=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
- Urine color and turbidity: not evaluated statistically

PATHOLOGY
- Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided), if p<=0.05, a pairwise comparison of each test group with the control group was performed using WILCOXON-test (two-sided) for the equal medians

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related, adverse effects were obtained in any test group (100, 300 and 1000 mg/kg bw/d).
- salivation shortly after treatment was observed in all animals of the highest test group on several days
From the temporary, short appearance immediately after dosing it was concluded that the finding was induced by a bad taste of the test substance or local affection of the upper digestive tract.

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- no test substance-related, adverse effects on body weight development were obtained
- mean body weights and body weight change values of male and female animals did not show any significant deviations to the control

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- no test substance-related, adverse changes observed

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

no effects observed

Description (incidence and severity):

- no test substance-related, adverse changes observed with regard to water consumption

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- no treatment-related findings observed
- other apparent findings were assessed as being incidental in nature since they occurred in control as well as in treated animals without a dose-response relationship

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- no treatment-related changes among hematological parameters observed
- observed effects were within historical control range or statistically significant but not dose-dependent and therefore 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
- observed effects were within historical control range or statistically significant but not dose-dependent and therefore regarded as incidental and not treatment-related

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- in males of the highest test group there were significant increased ketone bodies found, considered an isolated finding, which was not accompanied by any changed metabolism parameter
- this alteration was regarded as potentially treatment-related, but non-adverse
- no treatment-related, adverse changes among urinalysis parameters were observed
- potential in kidneys observed for concentrating the urine, regarded as an adaptive but not as an adverse effect
- observed effects were within historical control range or statistically significant but not dose-dependent and therefore regarded as incidental and not treatment-related

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- deviations from "zero values" obtained
- findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only
- observations were considered to have been incidental
- no test substance-related deviations to the control animals were noted for male and female animals

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- there were significantly increases in absolute and relative organ weights in some test groups, results given in table 5 and table 6, respectively
- significant weight increases of the kidneys in males (relative: 0.70%) and females (absolute/ relative: 1.77 g/ 0.78%) of the highest test group, above the historical control range (males, relative: 0.544-0.654%; females, absolute: 1.388-1.628 g; relative: 0.652-0.746%)
- no histopathological correlate was noted, these changes were considered treatment-related but not adverse
- weak significant absolute weight increase of the kidneys in females of test group 2 above the historical range (1.69 g)
- no significant relative weight deviations were noted, the absolute weight increase was regarded as possibly treatment-related and not adverse

- significant mean absolute liver weight increase in females of test group 3 (5.87 g) was beyond the historical control range (4.883-5.577 g)
- relative weight was not statistically significant changed and no histopathological correlate was observed, this change was considered to be possibly treatment-related but not adverse

- significant decrease of the heart in males of the highest test group (1.01 g) within the historical control values (0.905-1.134 g)
- no histopathological correlate observed, regarded as incidental and not treatment related

- incidentally significant spleen weight increase in males, not related to treatment

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- findings occurred individually and were considered to be incidental or spontaneous in origin and without any relation to treatment

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- findings occurred either individually or were biologically equally distributed over control and treatment groups
- findings considered to be incidental or spontaneous in origin and without any relation to treatment

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: there were no adverse signs of toxicity even at a dose level of 1000 mg/kg bw/d

Critical effects observed:

no

Table 5: Absolute organ weights

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Kidneys				+5.04%	+11.80%*	+17.43%**
Liver				+5.26%	+1.84%	+12.16%**
Heart	-0.93%	-2.60%	-5.86%*
Spleen	+14.38%*	+11.64%	+5.99%

* : p ≤ 0.05, **: p ≤ 0.01

Table 6: Relative organ weights

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Kidneys	+3.54%	-0.06%	+12.61%**	+5.54%	+9.87%	+13.68%*

* : p ≤ 0.05, **: p ≤ 0.01

Conclusions:

The test substance did not reveal adverse signs of toxicity even at a dose level of 1000 mg/kg bw/d by gavage to male and female Wistar rats over a period of 3 months .
Thus, the no observed adverse effect level (NOAEL) was set to 1000 mg/kg bw/d for male and female Wistar rats.

Executive summary:

The test substance was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 100 (test group 1), 300 (test group 2) and 1000 mg/kg body weight/day (mg/kg bw/d; test group 3) over a period of 3 months according to OECD 408.

Deionized water containing 5mg/100mL Cremophor EL served as vehicle, control animals were dosed daily with the vehicle only.

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and at the end of the administration period. A functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Clinico-chemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period, all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations.

The various analyses confirmed the stability of the test-substance preparations for a period of 7 days at room temperature and the correctness of the prepared concentrations.

There were no treatment-related, adverse effects observed after the examination of clinical parameters, clinical pathology and pathology in any of the three dose groups.

The findings let to the conclusion that the oral administration of the test item by gavage to male and female Wistar rats over a period of 3 months did not reveal adverse signs of toxicity even at a dose level of 1000 mg/kg bw/d.

Thus, the no observed adverse effect level (NOAEL) was set to 1000 mg/kg bw/d for male and female Wistar rats.

Reference 2

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 11-12 weeks (upon receipt)
- Weight at study initiation: the animals were of comparable size and weight
- Housing: individually with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
• Pregnant animals and their litters were housed together until PND 4 (end of lactation).
• For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

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

Route of administration:

oral: gavage

Vehicle:

other: water containing 5 mg/100 mL Cremophor EL

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Korantin MAT was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water containing 5 mg/100 mL Cremophor EL was filled up to the desired volume, subsequently released with a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week and were stored at room temperature. The administration volume was 10 mL/kg body weight.


VEHICLE
- Justification for use and choice of vehicle (if other than water): the vehicle was suitable for this type of test
- Concentration in vehicle: 1, 3 and 10 g/100 mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
The stability of the test substance in drinking water containing 5 mg/100 mL Cremophor EL for a period of 7 days at room temperature was proven during the study.
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.

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice.

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: on request of the sponsor
- Rationale for animal assignment (if not random): random, the list of randomization instructions was compiled with a computer.

Positive control:

N/A

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.

The littering 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 public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

The day of littering 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 prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high).

The following parameters were examined:

1. abnormal behavior when handled
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results.

The following exceptions are notable for the female animals:

- During the mating period the parental fe¬males 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.

- Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.


FOOD CONSUMPTION: Yes
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:

- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14, 14-20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 1-4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

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

CLINICAL PATHOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.
For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence.
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in International System (SI) units.
The following examinations were carried out in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group:

HAEMATOLOGY: Yes
The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes.
Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
Prothrombin time (Hepato Quick’s test) (HQT)
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).

CLINICAL CHEMISTRY: Yes
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters :
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), gamma-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)

URINALYSIS: Yes
The dry chemical reactions on test strips (Combur 10 test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany).
Urine-Parameters examined: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observational battery
The functional observational battery (FOB) will be carried out in the first 5 parental males and the first 5 females with litter per group once at the end of the administration period. The examinations will generally start in the morning. The FOB will be carried out in a randomized sequence. The findings will, as far as possible, be graded according to their intensity on the basis of an index of findings attached to the raw data or will be described in detail. The animals will not be transferred to new cages, and food or drinking water will not be withdrawn before the tests.

Home cage observations:

The animals were observed in their closed home cages; 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 abnormalities

Open field observations:

The animals were transferred to a standard arena (50 × 50 cm with side heights of 25 cm) and observed for at least 2 minutes. The following parameters were examined:

1. behavior when removed from cage
2. fur
3. skin
4. salivation
5. nose discharge
6. lacrimation
7. eyes/pupil size
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements
14. impairment of gait
15. activity/arousal level
16. feces (number of fecal pellets/appearance/consistency) within two minutes
17. urine (appearance/quantity) within two minutes
18. number of rearings within two minutes

Sensorimotor tests/reflexes:

The animals were removed from the open field and subjected to following sensorimotor or reflex tests:

1. approach response
2. 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. grip strength of forelimbs
12. grip strength of hindlimbs
13. landing foot-splay test
14. other findings


Motor activity assessment
The measurement of motor activity (MA) was carried out in the first 5 parental males and the first 5 parental females with litter per group at the end of the administration period. The MA was carried out in a randomized sequence on the respective day of the study. 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 for the time of measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes. The sequence at which the animals were placed in the polycarbonate cages was selected at random. Motor activity measurements were carried out starting at 14.00 h. On account of the measuring variant "staggered", the starting time varied according to the time needed to place the animals in the cages. For each animal, measurement started individually when the 1st beam was interrupted and was finished exactly 1 hour later. The animals did not receive any food or water during the measurements. After the transfer of the last animal the room of measurement was darkened. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.

Sacrifice and pathology:

GROSS PATHOLOGY / HISTOPATHOLOGY: Yes
Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights
Weight assessment was carried out on all animals. The following weights were determined:

1. Anesthetized animals
2. Epididymides
3. Testes


The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations):

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

Organ/ tissue fixation
The following organs / tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:

1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal gland
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
48. Vagina

Paraffin embedding, sectioning and staining
After the organs were fixed, processing, examination by light microscopy and evaluation of findings were performed, staining with Hematoxylin-eosin (H&E):
all gross lesions (all affected animals per group),
control and high dose animals only:
Adrenal glands (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Bone marrow (femur) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Brain (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Cecum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Cervix (all animals per group)
Coagulation glands (all animals per group)
Colon (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Duodenum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Epididymides (all animals per group)
Heart (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Ileum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Jejunum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Kidneys (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Liver (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Lung (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Lymph nodes (mesenteric and axillary lymph nodes) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Ovaries (all animals per group)
Oviducts (all animals per group)
Peyer’s patches (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Prostate (all animals per group)
Rectum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Sciatic nerve (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Seminal vesicles (all animals per group)
Spinal cord (cervical, thoracic and lumbar cords) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Spleen (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Stomach (forestomach and glandular stomach) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Testes (all animals per group)
Thymus (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Thyroid glands (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Trachea (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Urinary bladder (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Uterus (all animals per group)
Vagina (all animals per group)

Special attention was given on stages of spermatogenesis in the male gonads.

A correlation between gross lesions and histopathological findings was attempted.

Other examinations:

N/A

Statistics:

Detailed statistical analyses were conducted

Details on results:

CLINICAL SIGNS AND MORTALITY
No animal died prematurely in the present study.

All animals of both sexes in test group 3 (1000 mg/kg bw/d) showed salivation within 2 hours after the administration on several days of the study. In test group 2 (300 mg/kg bw/d) male animal Nos. 25, 27 and 30 showed salivation within 2 hours after the administration on several days during the pre-mating period as well on mating day 0 (male animal No. 27, only). Female animal Nos. 122, 123, 126, 128 and 130 of test group 2 (300 mg/kg bw/d) showed salivation within 2 hours after the administration on several days during the pre-mating period. 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.

During the mating phase male animal No. 6 of test group 0 (control group) showed respirations sounds on mating days 5-6. This finding was assessed as being incidental and not related to treatment.

The Detailed Clinical Observations on study days 0, 7, 14, 21, 28 in male animals and on study days 0, 7, 14, 21, 28, 35, 42, 49, 56 in female animals of test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d) did not reveal any abnormalities.

BODY WEIGHT AND WEIGHT GAIN
No test substance-related changes in mean body weights or body weight change values were observed in any test group.
On lactation day 4, mean body weights of female animals in test groups 2 and 3 (300 and 1000 mg/kg bw/d) were significantly lower. In addition, mean body weight change value during lactation was significantly lower in female animals of test group 2 (300 mg/kg bw/d) between study days 0-4. However, no significant changes were observed in on the other time points during the lactation period.

FOOD CONSUMPTION
In female animals of test group 3 (1000 mg/kg bw/d) food consumption during gestation was significantly lower between study days 7-14 and 14-20. This finding was assumed to be incidental because the mean value was still within the normal range for pregnant animals at the end of gestation. In addition, no significant impacts on body weight data were observed.

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

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

In males of test group 1 (100 mg/kg bw/d), mean corpuscular hemoglobin content (MCH) and mean corpuscular volume (MCV) were decreased, but both parameters were not dose-dependently changed. Therefore, these alterations were regarded as incidental and not treatment-related.

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

In females of test group 3 (1000 mg/kg bw/d), triglyceride levels were increased, but the mean was within the historical control range (0.29-0.71 mmol/L, PART III, Supplement). Therefore, this change was regarded as incidental and not treatment-related.


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

In male animals of test group 3 (1000 mg/kg bw/d) higher incidences of phosphate crystals were found in the urine sediment. This was related to higher pH values in these rats compared to controls (not significantly increased), which led to a precipitation of these crystals during the urine collection. However, neither any other urine parameter nor any blood parameters were changed. Therefore, these alterations were regarded as maybe treatment-related but not as adverse.

NEUROBEHAVIOUR
Functional observational battery
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.
The following examinations were performed during FOB and have to be assessed individually:

Home cage observations
No test substance-related effects were observed.

Open field observations
No test substance-related effects were observed.

Sensorimotor tests/reflexes
No test substance-related effects were observed.

Quantitative Parameters
No test substance-related effects were observed.

Motor activity measurement
There were no significant deviations concerning the overall motor activity (summation of all intervals) and regarding the single intervals in male and female animals of all test groups in comparison to the concurrent control group.

ORGAN WEIGHTS
Absolute weights
When compared with control group 0 (set to 100%), the mean absolute weights of the kidneys were significantly increased in males and females of test group 3 (1000 mg/kg bw/d).

Relative weights
When compared with control group 0 (set to 100%), the mean relative weights of livers (males only) and kidneys (both sexes) were significantly increased in test group 3 (1000 mg/kg bw/d).

The increase in absolute kidney weights of males and females of test group 3 (1000 mg/kg bw/d) was most likely treatment-related due to a dose response-relationship and also altered relative kidney weights. However, as no histopathologic correlate was present the finding was regarded to be not adverse.
The increase of relative liver weights in males of test group 3 (1000 mg/kg bw/d) was also regarded to be most likely treatment-related although no histopathologic finding could explain the weight increase. But as no other parameter with regard to liver cell metabolism was altered it was regarded to be not adverse in nature.

All other 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.
The female that was not pregnant and its male mating partner did not show gross lesions in reproduction relevant organs which could explain the lack of offspring.

HISTOPATHOLOGY:
All findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility:
The investigated not pregnant female as well as its male mating partner of test group 3 (1000 mg/kg bw/d) did not show relevant histopathological findings that could explain infertility.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test substance-related effects were observed.

Critical effects observed:

not specified

Korantin MATwas administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2)and 1000 mg/kg bw/d (test group 3).

 

Regarding clinical examinations,signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) during the entire study period.

Salivation after treatment was seen in all animals of test group 3 (1000 mg/kg bw/d) and sporadically in a few animals of test group 2 (300 mg/kg bw/d). From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

 

Fertility indices for male and female animals were not impaired by test-substance administration even at a dose level of 1000 mg/kg bw/d. In addition, live birth indices of pups in all test groups were not influenced.

 

The viability index as indicator for pup mortality was not altered dose-dependently.

 

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d.

 

Regarding pathology, the treatment of male and female Wistar rats with up to 1000 mg/kg bw/d of the test substance led to treatment-related increase in male and female absolute and relative kidney weights in test group 3 (1000 mg/kg bw/d). In addition, the relative liver weight in males of test group 3 (1000 mg/kg bw/d) was also significantly and treatment-related increased. As there were no histopathologic findings in both organs these weight increases were not regarded to be adverse in nature but an adaptive effect.

No findings on the reproduction tract were observed.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

CONCLUSION

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage of KORANTIN MAT to Wistar rats revealed no signs of systemic toxicity up to a dose level of 1000 mg/kg bw/d in animals of both sexes. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000mg/kg bw/d in male and in female Wistar rats.

The NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d in male and female Wistar rats.

TheNOAEL for developmental toxicity was effective 1000 mg/kg bw/d.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reaction product of Maleic anhydride, 2-Ethylhexylamine and Triethanolamine was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). Drinking watercontaining 5 mg/100 mL Cremophor ELserved as vehicle. The study was conducted according to OECD 422 guideline and GLP (BASF SE, 2013) 

The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice.

 

Observations

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear.

A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4.

Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, after the day of parturition (postnatal day [PND] 0) and on PND 4.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO₂and examined macroscopically for external and visceral findings.

Clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period.

 

Towards the end of the administration period a functional observational battery was performed and motor activity was measured in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.

All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Results

Analytics

The various analyses confirmed

-     the stability of the test substance in drinking water at room temperature over a period of 7 days,

-     the homogeneous distribution of the test substancein drinking water,

-     the correctness of the prepared concentrations of the test-substance preparations in drinking water.

 

Findings

The following test substance-related, relevant findings were noted:

 

Test group 3: 1000 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations, Reproductive Performance, Clinical Pathology and Pathology

- No testsubstance-related, adverse findings were noted.

 

F1 PUPS

 

Clinical Examinations/ Gross Findings

- Notestsubstance-related, adverse findings were noted.

Test group 2: 300 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations, Reproductive Performance, Clinical Pathology and Pathology

- Notestsubstance-related, adverse findings were noted.

 

F1 PUPS

 

Clinical Examinations/ Gross Findings

- Notestsubstance-related, adverse findings were noted.

Test group 1: 100 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations, Reproductive Performance, Clinical Pathology and Pathology

- No testsubstance-related, adverse findings were noted.

 

F1 PUPS

 

Clinical Examinations/ Gross Findings

- Notestsubstance-related, adverse findings were noted.

 

 Conclusion

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage of Reaction product of Maleic anhydride, 2-Ethylhexylamine and Triethanolamine to Wistar rats revealed no signs of systemic toxicity up to a dose level of 1000 mg/kg bw/d in animals of both sexes. Thus,the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000mg/kg bw/d in male and in female Wistar rats.

The NOAEL for reproductive performance and fertility was set to 1000mg/kg bw/d in male and female Wistar rats.

The NOAEL for developmental toxicity was effective 1000 mg/kg bw/d.

Sub-chronic repeated dose testin, according to OECD 408, 2019

The test substance was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 100 (test group 1), 300 (test group 2) and 1000 mg/kg body weight/day (mg/kg bw/d; test group 3) over a period of 3 months according to OECD 408.

Deionized water containing 5mg/100mL Cremophor EL served as vehicle, control animals were dosed daily with the vehicle only.

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and at the end of the administration period. A functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Clinico-chemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period, all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations.

The various analyses confirmed the stability of the test-substance preparations for a period of 7 days at room temperature and the correctness of the prepared concentrations.

There were no treatment-related, adverse effects observed after the examination of clinical parameters, clinical pathology and pathology in any of the three dose groups.

The findings let to the conclusion that the oral administration of Reaction product of Maleic anhydride, 2-Ethylhexylamine and Triethanolamine by gavage to male and female Wistar rats over a period of 3 months did not reveal adverse signs of toxicity even at a dose level of 1000 mg/kg bw/d.

Thus, the no observed adverse effect level (NOAEL) was set to 1000 mg/kg bw/d for male and female Wistar rats.

Further

Reaction product of Maleic anhydride, 2-Ethylhexylamine and Triethanolamine was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 100 (test group 1), 300 (test group 2) and 1000 mg/kg body weight/day (mg/kg bw/d; test group 3) over a period of 3 months according to OECD 408 (BASF SE, 2019).

Deionized water containing 5mg/100mL Cremophor EL served as vehicle, control animals were dosed daily with the vehicle only.

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning and at the end of the administration period. A functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Clinico-chemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period, all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations.

The various analyses confirmed the stability of the test-substance preparations for a period of 7 days at room temperature and the correctness of the prepared concentrations.

There were no treatment-related, adverse effects observed after the examination of clinical parameters, clinical pathology and pathology in any of the three dose groups.

The findings let to the conclusion that the oral administration of

Reaction product of Maleic anhydride, 2-Ethylhexylamine and Triethanolamine

by gavage to male and female Wistar rats over a period of 3 months did not reveal adverse signs of toxicity even at a dose level of 1000 mg/kg bw/d.

Thus, the no observed adverse effect level (NOAEL) was set to 1000 mg/kg bw/d for male and female Wistar rats.

Justification for classification or non-classification

Based on the available data, the test substance is not classified with regard to repeated dose toxicity according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP), respectively.