Fatty acids, tall-oil, ethoxylated

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

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:

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

Version / remarks:

29 July 2016

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 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

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.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- 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

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

Duration of test:

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.

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).

Examinations

Maternal 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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

Pup status and litter size after birth: the status (sex, liveborn or stillborn) and number of all pups delivered from the parents were determined as soon as possible after birth. At the same time, the pups were examined for gross-morphological changes.
- Pup viability/mortality: in general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on weekends and public holidays. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.
- Clinical signs: all live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.
- Nipple/areola anlagen: all surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen were counted.
- Body weights: the pups were weighed on the day after birth (PND 1) as well as on PNDs 4, 7 and 13. The body weight determined on PND 1 was also used to determine runts. Those pups whose body weight was ≥ 25% below the mean body weight of the control group (separately according to male and female pups) were defined as runts.
- Anogenital distance: anogenital distance (AGD; defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were done blind to treatment in a randomized order, using a measuring ocular, on all live male, female and uncertain pups on day 1 after birth.

THYROID HORMONES: Yes
- Blood samples for T3, T4 and TSH measurement were taken from all surplus pups at PND 4 as well 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.

POSTMORTOM EXAMINATIONS
- On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and their organs will be assessed macroscopically.
- 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 that die or were sacrificed in a moribund state were eviscerated and examined for possible defects and/or the cause of death, paying particular attention to potential pericardial blood vessel effects.
- 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

Indices:

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

Offspring indices
- viability index
- survival index

Historical control data:

Extensive historical control data is available for Wistar rats strain (Crl:WI(Han))

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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. During mating, salivation was observed in female animal No. 138 on several mating days. 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 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. 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 during the entire study.

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 test group 3 (1000 mg/kg bw/d), absolute reticulocyte counts were significantly decreased. The reticulocyte mean in one female was marginally below the historical control range (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 animals of test group 3 were regarded as maybe treatment-related, but non-adverse (ECETOC Technical Report No 85, 2002).

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 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.
In female animals of test group 3 (1000 mg/kg bw/d), grip strength of hindlimbs was significantly lower. 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 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 did not show relevant gross lesions. Female No 118, which showed implantations on GD 0 did not show any gross internal lesions.

Neuropathological findings:

not examined

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. In high-dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

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.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

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.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

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.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The mean duration of gestation was 22.0 days in test groups 0 and 2, 22.1 days in test group 1 and 22.2 days in test group 3.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The mean duration of gestation was 22.0 days in test groups 0 and 2, 22.1 days in test group 1 and 22.2 days in test group 3.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

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.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

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.

Basis for effect level:

other: No adverse effects observed.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body 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.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): 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.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

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

Changes in sex ratio:

no effects observed

Description (incidence and severity):

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.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

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

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

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.

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Gross pathology
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.

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.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental 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 Sex ratio of live F1 pups

PND 0	Test group 0 (0 mg/kg bw/d)	Test group 1 (100 mg/kg bw/d)	Test group 2 (300 mg/kg bw/d)	Test group 3 (1000 mg/kg bw/d)
Live males [%]	52.8	50.7	49.1	47.1
Live females [%]	47.2	49.3	50.9	52.9
PND 13
Live males [%]	51.2	48.6	48.8	50.0
Live females [%]	48.8	51.4	51.2	50.0

 

Table 3 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

 

Table 4 Summary of pup body weights and body weight changes

		Test Group 0 (F) 0 mg/kg bw/d	Test Group 1 (F) 100 mg/kg bw/d	Test Group 2 (F) 300 mg/kg bw/d	Test Group 3 (F) 1000 mg/kg bw/d
Day 1 Males + Females	Mean	6.6 n	6.7	6.1	6.4
	S.D.	0.8	0.6	0.5	1.0
	N	10	9	10	8
	Deviation Vs Control [%]		1.3	-5.5	4.2
Day 4 Males + Females	Mean	10.1 n	10.5	9.7	10.5
	S.D.	1.5	1.1	0.9	1.6
	N	10	9	10	8
	Deviation Vs Control [%]		3.6	-4.6	4.0
Day 7 Males + Females	Mean	16.3 n	17.1	16.2	17.2
	S.D.	1.8	1.0	1.2	1.9
	N	10	9	10	8
	Deviation Vs Control [%]		4.9	-0.4	5.6
Day 13 Males + Females	Mean	30.1 n	31.8	31.1	31.4
	S.D.	2.4	1.4	1.5	2.8
	N	10	9	10	8
	Deviation Vs Control [%]		5.5	3.2	4.1
D 1 -> 4 Males + Females	Mean	3.5 n	3.8	3.4	3.8
	S.D.	0.8	0.5	0.4	0.7
	N	10	9	10	8
D 4 -> 7 Males + Females	Mean	6.2 n	6.6	6.5	6.6
	S.D.	0.5	0.5	0.5	0.6
	N	10	9	10	8
D 7 -> 13 Males + Females	Mean	13.8 n	14.7	14.9	14.2
	S.D.	1.2	1.0	1.2	1.1
	N	10	9	10	8
D 1 -> 13 Males + Females	Mean	23.5 n	25.0	24.8	24.7
	S.D.	1.8	1.3	1.5	2.1
	N	10	9	10	8

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