Fatty acids, tall-oil, ethoxylated

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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

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

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 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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): in Polycarbonate cages type III

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

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

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

Parental animals: Observations and examinations:

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 h 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

Oestrous cyclicity (parental animals):

For all females of the pool estrous cycle normality was evaluated before the beginning of the administration. In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.

Sperm parameters (parental animals):

- Parameters examined in male parental generation: testes (histologically), weight of testes and of epididymides.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and their blood was sampled for determination of thyroid hormone concentrations. Furthermore they were all examined externally and eviscerated; their organs were assessed macroscopically.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities and the organs were assessed macroscopically; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

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.

Postmortem examinations (offspring):

- On PND 13, one selected male and one female pup per litter were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing.
- The remaining pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs will be assessed macroscopically.
- Pups showing clinical symptoms or gross-morphological findings may have been further examined using appropriate methods. Organs/tissues with gross-morphological findings may have been preserved in a suitable manner for potential histopathological examination. All pups without any notable findings were discarded after their macroscopic evaluation.

Statistics:

Statistics of clinical examinations and postmortem examinations of offspring:
- Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:
- Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days; anogenital distance, anogenital index: DUNNETT test (two-sided)
- Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups: FISHER'S EXACT test (one-sided)
- Mating days until day 0 pc, % post implantation loss, pups stillborn, % perinatal loss; nipple development: WILCOXON test (one-sided+) with BONFERRONI-HOLM
- Implantation sites, pups delivered, pups liveborn, life pups day x, viability index, survival index: WILCOXON test (one-sided-) with BONFERRONI-HOLM
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: KRUSKAL-WALLIS test and WILCOXON test (two-sided)
- % live male day x, % live female day x: WILCOXON test (two-sided)
- Number of cycles and cycle length: KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)

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

Reproductive indices:

- Male and female mating indices
- Male and female fertility indices
- Gestation index
- Anogenital index: anogenital distance [mm] / cubic root of pup weight [g]

Offspring viability indices:

- viability index
- survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

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):

Thyroid hormones
In parental males of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

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.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The stages of spermatogenesis in the testes of males of the high-dose test group were comparable to those of the controls.

Reproductive performance:

no effects observed

Description (incidence and severity):

Male mating index
The male mating index calculated after the mating period to produce F1 litter was 100% in all test groups.

Male fertility index
Fertility was proven in all of the F0 parental males within the scheduled mating interval to produce F1 litter.
Male animal No. 18 of test group 1 (100 mg/kg bw/d), which was mated with female No. 118, did not generate F1 pups but implants were found at necropsy. Male animal Nos. 31 and 33 of test group 3 (1000 mg/kg bw/d), which were mated with female animal Nos. 131 and 133, did not generate F1 pups and no implants were found at necropsy.
Thus, the male fertility index was 100% in test groups 0-2 and 80% in test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Female mating index
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) was 3.3 days for test group 0, 2.9 days for test group 1, 2.4 days for test group 2 and 2.5 days for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Female fertility index
Most sperm positive rats delivered pups with the exception of female animal No. 118 of test group 1 (100 mg/kg bw/d), which was mated with male No. 18, did not deliver pups but showed one implantation site. Female animal Nos. 131 and 133 of test group 3 (1000 mg/kg bw/d), which were mated with male animal Nos. 31 and 33, had sperm in vaginal smear but did not deliver pups and did not show any implants. Female animal No. 113 of test group 1 was not sperm positive but delivered pups. Thus, the female fertility index was 100% in test groups 0-2 and 80% in test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Gestation index
The gestation index was 100% in test groups 0, 2 and 3 (0, 300 and 1000 mg/kg bw/d) and reduced to 90% in test group 1 (100 mg/kg bw/d). The decreased value was assessed to be incidental and in the normal range of biological variation.

Live birth indices
The rate live birth indices were 100% in test groups 1-3 (100, 300, 1000 mg/kg bw/d) and 99.2% in test group 0 (control). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Postimplantation loss
The postimplantation loss was 0.9% in control group 0, 15.8% in test group 1 (100 mg/kg bw/d), 0.5% in test group 2 (300 mg/kg bw/d) and 1.8% in test group 3 (1000 mg/kg bw/d). Although the postimplantation loss was rather high in test group 1, the value was still within the normal range of biological variation inherent in the strain of rats used for this study. In addition, a dose-response relationship was not observed.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

2

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse ffects observed.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Each one pup of both sexes of control group (0 mg/kg bw/d), 2 male pups of test group 1 (100 mg/kg bw/d), 1 female pup of test group 2 (300 mg/kg bw/d), each one pup of both sexes of test group 3 (1000 mg/kg bw/d) died prematurely. Female pup No. 127-15 of test group 2 (300 mg/kg bw/d) showed thread-like tail between PND 1 and 4. These changes were assessed to be incidental.
All other F1 pups in all test groups (0-3) did not show clinical signs up to scheduled sacrifice on PND 4 and PND 13.

Mortality / viability:

no mortality observed

Description (incidence and severity):

The viability index indicating pup mortality between PND 0 and 4 was 99.3% in test groups 0 (control) and 2 (300 mg/kg bw/d), 97.5% in test group 1 (100 mg/kg bw/d), and 98.4% in test group 3 (1000 mg/kg bw/d). The rate live birth indices were 100% in test groups 1-3 (100, 300, 1000 mg/kg bw/d) and 99.2% in test group 0 (control group). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group with the following exceptions:
Mean pup body weight of female pups in test group 1 (100 mg/kg bw/d) was significantly higher on lactation day 13 (+7.7%) when compared to control. The mean pup body weight change values of female pups in test group 1 between lactation days 1-13 and in female pups of test group 2 between lactation days 7-13 and 1-13 were significantly higher. These deviations to the control were assessed to be incidental and not related to the test item.
One male runt was found in one litter of test group 0 (control), 3 male runts and 2 female runts were found in three different litters of test group 2 (100 mg/kg bw/d) and 1 female runt was found in one litter of test group 3 (1000 mg/kg bw/d). A relation to dosing was not observed, test substance-related effects did not occur.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

One male and 1 female pup of test group 0 (control group), 2 male pups of test group 1 (100 mg/kg bw/d) and 1 female pup of test group 2 (300 mg/kg bw/d) showed post mortem autolysis. These findings were observed as the pups were found dead and the autolysis was in progress before the daily clinical observations started. One male pup of test group 1 (100 mg/kg bw/d) and 1 female pup of test group 3 (1000 mg/kg bw/d) were cannibalized. Thread-like tail was observed in one female pup (No. 127-15) of test group 2 (300 mg/kg bw/d). In addition to the finding the stained skeleton showed fused sacral centrum with arches (3rd, bilateral), absent sacral vertebra (4th) and absent caudal vertebrae (all).
These findings were assessed to be incidental and not related to the test substance.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Pup number and status at delivery
The mean number of delivered F1 pups per dam was equally distributed among test groups 0, 1, 2 and 3. No significant deviations occurred.

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Anogenital distance
No test substance-related effects were observed.

Nipple/areola anlagen
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13. The number of nipples in male animals of test group 3 (1000 mg/kg bw/d) was significantly increased. In addition, the percentage of male pups having areolae was significantly increased. However, both values were clearly within the historical control data. A relation to treatment was not considered.

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive 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

Table 1 Reproductive parameter indices in percent

Test group (mg/kg bw/d)	0 (0)	1 (100)	2 (300)	3 (1000)
P0 Males
Mating Index	100	100	100	100
Fertility Index	100	100	100	80
P0 Females
Mating Index	100	100	100	100
Fertility Index	100	100	100	80
Gestation Index	100	90	100	100
Live birth Index	99.2	100	100	100
F1 Pups
Viability Index	99.3	97.5	99.3	98.4
Survival Index	99.1	100	100	89.5

 

Table 2 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