Fatty acids, tall-oil, ethoxylated

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:

2017-09-08 to 2017-11-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

dose range finder

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 Jul 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, ethoxylated

EC Number:

500-150-1

EC Name:

Fatty acids, tall-oil, ethoxylated

Cas Number:

61791-00-2

Molecular formula:

C(18-50)H(34-98)O(3-8)

IUPAC Name:

Fatty acids, tall-oil, ethoxylated

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0014857532
- Expiration date of the lot/batch: 2018-01-26
- Name of test substance: Emulgane 1729

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected. This Wistar rat strain (Crl:WI(Han)) was selected since extensive historical control data were available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks (female); 10-11 weeks (male)
- Weight at study initiation: 215 - 221 g (female); 377 - 379 g (male)
- Fasting period before study: No
- Housing:
During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm² (610 x 435 x 215 mm)
During pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III
For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III with wire covers.

- Diet: ad libitum, ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum, tap water from water bottles
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY:
The supplier assayed the food used in the study for chemical and microbiological contaminants. The drinking water is 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 (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionised

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, deionized water was filled up to the desired volume and subsequently released by a magnetic stirrer. The test substance preparations were produced twice a week, at least. During the first week of application deionized water containing 0.5% sodium carboxymethyl cellulose was used as vehicle. For practical reasons, the carrier was changed to deionized water without 0.5% sodium carboxymethyl cellulose.

VEHICLE
- Concentration in vehicle: 1, 3, 10 g/100 mL
- Amount of vehicle (if gavage): 10 mL/kg bw/d

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At the time points mentioned above, additionally one sample from the mid concentration was taken for concentration control analysis. The samples collected at the beginning of the administration period and during the lactation period were analyzed.

Duration of treatment / exposure:

All animals, with the exception of the controls, received the test substance daily by gavage according to the time schedule (exception: no administration to animals being in labor). All animals were daily observed for any clinical signs during the study period. The duration of exposure was at least 28 days, including 14 days of pre-mating.

Frequency of treatment:

Once daily for 7 days/week

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

yes, historical

Details on study design:

- Dose selection rationale: Doses were determined by performing a 14-day dose range finding study in which doses of 300 and 1000 mg/kg bw/day were tested, described in the supporting study record with report number "01 R0078/17R025" in section 'Repeated dose toxicity: oral" (BASF SE, 2017).

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Parameters observed: any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to administration period and at weekly intervals thereafter
- Parameters observed: Abnormal behavior in handling; Fur; Skin; Posture; Salivation; Respiration; Activity/arousal level; Tremors; Convulsions; Abnormal movements; Gait abnormalities; Lacrimation; Palpebral closure; Exophthalmus (Protruding eyeball); Assessment of the feces excreted during the examination (appearance, consistency); Assessment of the urine excreted during the examination; Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning)
- During the mating period, the 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), PND 4. PND 7 PND 10 and PND 13.
- Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of the administration volume, body weight data of these individuals were only reported in the individual tables).
- Females without litter and after weaning (PND 13) were weighed once a week (for the calculation of the administration volume, body weight data of these individuals were only reported in the individual tables).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption was determined once a week for the 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 parental animals).
- Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
- Food consumption of the females which gave birth to a litter was determined for PND 1-4, 4-7, 7-10 and 10-13.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily by visual inspection of the water bottles for any changes in volume

HAEMATOLOGY: Yes
- Time schedule: Prenatal day 14
- Anaesthetic used for blood collection: Yes, under isoflurane anesthesia
- Animals fasted: Yes
- How many animals: the first 5 surviving parental males per group at termination and the first 5 females with litters (in order of delivery) per group at PND 14.
- The following parameters were observed: leukocytes; erythrocytes; haemoglobin; haematocrit; mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin concentration (MCHC); platelets; differential blood count; reticulocytes; preparation of blood smears (only evaluated blood smears were archived); prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule: Prenatal day 14
- Anaesthetic used for blood collection: Yes, under isoflurane anesthesia
- Animals fasted: Yes
- How many animals: the first 5 surviving parental males per group at termination and the first 5 females with litters (in order of delivery) per group at PND 14.
- The following parameters were observed: alanine aminotransferase; aspartate aminotransferase; alkaline phosphatase; serum y-glutamyl transferase; sodium; potassium ; chloride; inorg. phosphate; calcium; urea; creatinine; glucose; total bilirubin; total protein; albumin; globulins; triglycerides; cholesterol; bile acids.

THYROID HORMONES: Yes
- Blood samples for T3, T4 and TSH measurement were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia.
- If not sufficient serum could be sampled from PND 4 pups, samples were pooled per sex and litter. If not at least 8 pools per sex were sufficient for the hormone measurements, samples were pooled regardless of sex per litter.
- Additionally, blood samples for the above mentioned hormones were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia from all dams at PND 14 and all adult males at termination. The adults were fastened before the blood sampling.
- Blood samples from the PND 13 pups and the adult males were assessed for serum levels for T4 and TSH.
All generated serum samples were frozen at -80° at least until finalization of the report.

BEHAVIOUR (FUNCTIONAL FINDINGS): Yes
FUNCTIONAL OBSERVATIONAL BATTERY
- The functional observational battery (FOB) was carried out once, towards the end of the administration period, in the first 5 surviving parental males and the first 5 surviving parental females with litter per group (in order of delivery).
- The examinations were generally started in the morning at about 10:00 h. The FOB was carried out in a randomized sequence. The animals were not transferred to new cages before the test, nor were food or drinking water withdrawn. The FOB was started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable.
- Home cage observation: besides other abnormalities, posture; tremors; convulsions; abnormal movements; gait were observed.
- Open field observation: besides other abnormalities, 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), rearings within 2 minutes and other findings were observed.
- Sensory motor tests/reflex tests: the animals were removed from the open field and were subjected to the sensory motor and reflex tests; reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), other findings, grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test were performed.

MEASUREMENT OF MOTOR ACTIVITY
- The measurement of motor activity (MA) was carried out once, towards the end of the administration period in the first 5 surviving parental males per group and the first 5 surviving parental females with litter per group (in order of delivery).
- For this purpose, the animals were placed in clean polycarbonate cages with a small amount of bedding for the duration of the measurement. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. Eighteen beams will be allocated per cage.
- The number of beam interrupts were counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the polycarbonate cages was selected at random. Motor activity measurements were carried out from 14.00 h onwards.
- On account of the measuring variant "staggered", the starting time varied by the time which was needed to place the animals in the cages. For each animal, measurement started individually when the 1st beam was interrupted and ended exactly 1 hour later.
- The animals were given no food or water during the measurements. During measurement the pups were placed in a different room, separated from the dams. After the transfer of the last animal in each case, the room of measurement was darkened.

Sacrifice and pathology:

SACRIFICE
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention was given to the reproductive organs. Animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.

GROSS NECROPSY
Gross necropsy consisted of external and internal examinations including the cervical and thoracic caves.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following weights were determined in all animals sacrificed on schedule: Anesthetized animals; Epididymides; Ovaries; Prostate; Seminal vesicles with coagulating glands; Testes; Thyroid glands; 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): Adrenal glands; Brain; Heart; Kidneys; Liver; Spleen; Thymus
- The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson's solution: All gross lesions; Adrenal glands; Aorta; Bone marrow (femur); Brain; Cecum; Cervix; Coagulating glands; Colon; Duodenum; Eyes with optic nerve; Esophagus; Extraorbital lacrimal glands; Epididymides (modified Davidson's solution); Femur with knee joint; Heart; Ileum; Jejunum (with Peyer's patches); Kidneys; Larynx; Liver; Lungs; Lymph nodes (axillary and mesenteric); Mammary gland (male and female); Nose (nasal cavity); Ovaries (modified Davidson's solution); Oviducts; Pancreas; Parathyroid glands; Pharynx; Pituitary gland; Prostate gland; Rectum; Salivary glands (mandibular and sublingual); Sciatic nerve; Seminal vesicles; Skeletal muscle; Spinal cord (cervical, thoracic and lumbar cord); Spleen; Sternum with marrow; Stomach (forestomach and glandular stomach); Testes (modified Davidson's solution); Thymus; Thyroid glands; Trachea; Urinary bladder; Uterus; Vagina.

Statistics:

Statistics of clinical examinations:
- Means and standard deviations were calculated. In addition, the following statistical analyses were carried out for Food consumption (parental animals), body weight and body weight change (parental animals): DUNNETT test (two-sided)


Statistics of clinical pathology
- Means, medians and standard deviations were calculated
- Clinical pathology parameters: KRUSKAL-WALLIS and WILCOXON

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related, adverse signs of toxicity were observed.
Salivation shortly after treatment was observed in test group 3 (1000 mg/kg bw/d), only. Male animal Nos. 33 and 34 as well as in female animal No. 138 showed salivation at the end of the premating period. During mating, salivation after treatment was observed in male animal Nos. 31, 32, 33, 34, 36, 37, 39 and 40 as well as in female animal No. 138 on several mating days. Male animal Nos. 35 and 40 showed salivation after treatment during postmating. From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that both kind of findings were induced by a bad taste of the test substance or local affection of the upper digestive tract.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related changes in mean body weights and mean body weight change values were observed in male and female animals during the entire study. Mean body weight change values were significantly increased in female animals of test group 3 (1000 mg/kg bw/d) between pre-mating days 0-7 and 0-13. Furthermore, in male animals of test group 3 (1000 mg/kg bw/d) the mean body weight change value between mating days 7-14 was significantly decreased. These changes were assessed to be incidental.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related changes in food consumption were observed in male and female animals during the entire study.
Food consumption was significantly decreased in male animals of test group 3 (1000 mg/kg bw/d) between pre-mating days 7-13. However, the value was still within the normal range typical for rats of this strain and age. In addition, no change in mean body weight was observable. Thus, the change was assessed to be incidental.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related, adverse changes among hematological parameters were observed.
At the end of the administration period in males and females of test group 3 (1000 mg/kg bw/d), absolute reticulocyte counts were significantly decreased. The reticulocyte mean in males was within, that one of females marginally below the historical control range (absolute reticulocytes: males 99.5 - 174.4 Giga/L; females 182.1 - 235.8 Giga/L). No other red blood cell parameters (i.e. red blood cell (RBC) counts, hemoglobin and hematocrit values) were altered. No histopathologic finding in the spleen indicating a red blood cell synthesis dysregulation was observed. Therefore, the decreases of absolute reticulocyte counts in rats of both sexes of test group 3 were regarded as maybe treatment-related, but non-adverse (ECETOC Technical Report No 85, 2002).
In males of test group 1 (100 mg/kg bw/d) relative monocyte counts were significantly increased whereas in males of test group 3 (1000 mg/kg bw/d) the same parameter was significantly decreased. All values were within the historical control range (males, relative monocytes 1.3 - 2.6 %). Therefore, this alteration was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.
In females of test groups 2 and 3 (300 and 1000 mg/kg bw/d) potassium values were significantly decreased. The mean of test group 2 was within, that one of test group 3 marginally below the historical control range (females, potassium 4.23 - 4.90 mmol/L). No other clinical chemistry parameter was changed. Therefore, the decrease of potassium values in females of test groups 2 and 3 were regarded as incidental and not treatment related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observational battery
Deviations from "zero values" were obtained in quantitative parameters in male and female animals. Without a dose-response relationship or occurred in single animals only, these observations were considered as incidental. No test substance-related effects were observed for homecage or open field observations, as well as sensorimotor tests/reflexes and quantative parameters.
Grip strength of forelimbs was significantly decreased in male animals of test group 1 (100 mg/kg bw/d). Since no dose-response relationship occurred and grip strength of hindlimbs was not affected, the change was assessed to be incidental. In female animals of test group 3 (1000 mg/kg bw/d), grip strength of hindlimbs was significantly lower. Again, the finding was assessed to be incidental as this was the only changed parameter and grip strength of forelimbs was not affected.

Motor activity measurement
Regarding the overall motor activity and single intervals, no test substance-related deviations were noted for male and female animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

When compared to control group 0 (set to 100%), the mean absolute weight of the heart was significantly increased in test group 2 females (111%). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
When compared to control group 0 (set to 100%), the mean relative weight of the heart was significantly decreased in test group 3 females (92%). All other mean relative weight parameters did not show significant differences when compared to the control group 0.
The significantly increased absolute heart weights in test group 2 females and the significantly decreased relative heart weights in test group 3 females were regarded as incidental, since there was no dose-response relationship and no correlating histopathologic change.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All findings occurred individually without a relation to the dose level. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility:
The female animal Nos. 131 and 133, which were not pregnant as well as the male mating partners Nos. 31 and 33, did not show relevant gross lesions. Female No. 118, which showed implantations on GD 0 and the male mating partner No. 18 did not show any gross internal lesions.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All 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. The stages of spermatogenesis in the testes of males of the high-dose test group were comparable to those of the controls. In high-dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.

Fertility
The female animal Nos. 131 and 133, which were not pregnant as well as the male mating partners Nos. 31 and 33, did not show relevant histopathological findings. Female 118 and mating partner No. 18 did not show any histophatological findings during examination.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycle
Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. The mean estrous cycle duration in the different test groups (0-3) was 3.9 to 4.0 days.

Thyroid hormones
In parental males of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) and in male and female pups of test groups 11, 12 and 13 (100, 300 and 1000 mg/kg bw/d) at PND 13, no treatment-related alterations of T4 and TSH levels were observed.

Effect levels

Key result

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: general systemic toxicity

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Analyses

Stability

The stability of the test substance in deionized water over a period of 4 days is given. As the mixtures were stored no longer than this time period, the stability was guaranteed.

Homogeneity

Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test substance was distributed homogeneously in deionized water.

Concentration

The concentrations of the test substance in cornoil were found to be in the range of 90-110% of the nominal concentration. The results demonstrated the correctness of the concentrations of the test substance in deionized water.

Food

On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 1*10E5/g food.

Drinking water

On the basis of the analytical findings the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

Bedding and enrichment

On the basis of the analytical findings, the bedding and the enrichment are found to be suitable. Levels given in Lab. Animal, Nov-Dec 1979, pp. 24-34, served as a guideline for maximum tolerable contaminants.

Relevant result tables

Absolute and relative organ weights parental animals

	Males				Females
Test group (mg/kg bw/d)	0 (0)	1 (100)	2 (300)	3 (1000)	0 (0)	1 (100)	2 (300)	3 (1000)
Relative organ weight
Heart	-	-	-	-	-	103	106	92*
Absolute organ weight
Heart	-	-	-	-	-	106	111*	96

*p ≤ 0.05; **p ≤ 0.01, X = group excluded from statistics, n = DUNNETT