Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Potassium isooctadecanoate
EC Number:
266-369-7
EC Name:
Potassium isooctadecanoate
Cas Number:
66469-15-6
Molecular formula:
C18H36O2.K
IUPAC Name:
potassium 16-methylheptadecanoate
Test material form:
solid: crystalline
Details on test material:
Product description: Potassium isooctadecanoate/potassium isostearate/potassium 16-methylheptadecanoate
Name: Isooctadecanoic acid, potassium salt (1:1)
CAS No.: 66469-15-6
Physical state: off white to yellowish solid at 20 °C
Batch No.: PFS-755-169
Re-certification date of batch: 12 December 2017
Purity: 100 % (mono constituent substance)
pH, 10% in DI water 8.0 - 9.0
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light


Specific details on test material used for the study:
Product description: Potassium isooctadecanoate/potassium isostearate
Name: Isooctadecanoic acid, potassium salt
CAS No.: 66469-15-6
Physical state: off white to yellowish solid at 20 °C
Batch No.: PFS-755-169
Re-certification date of batch: 12 December 2017
Purity: 100 % (UVCB)
pH, 10% in DI water 8.0 - 9.0
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Rat / CD / Crl:CD(SD)

Charles River Laboratories, Research Models and Services, Germany GmbH
Sandhofer Weg 7
97633 Sulzfeld
Germany
Sex:
male/female
Details on test animals or test system and environmental conditions:
For this study CD rats bred by Charles River Laboratories Germany GmbH were used. The healthy nulliparous adult animals were randomised and assigned to the treatment groups and cages. The body weight range did not exceed 20% of the mean weight for each sex at the time of selection. The animals were held for 5 days for adaptation. Health checks were performed on the day of delivery and at first administration.

Body weight (at 1st administration, TD15)
Males: 383.7 g - 468.9 g
Females: 257.5 g - 302.0 g

Age (at 1st administration)
Males and Females: 80 days

Selection of species: The rat is a commonly used rodent species for such studies.

Housing and feeding

Diet: A certified commercial diet (ssniff® R/Z V1324) served as food. This food was offered ad libitum. Food residue was removed and weighed.
Samples of the food are analysed for contaminants based on EPA/USA by LUFA-ITL at least twice a year. Certificates of analysis of the composition and for contaminants are provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.

Drinking water: Tap water was offered ad libitum. Samples of the drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung, Bundesgesetzblatt 2001' [German Regulations on drinking water, public notice of the law, 2001]. In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the "Deutsche Trinkwasserverordnung 2001, Anlage 1".

Housing: With the exception of the mating period, the males and females (F0-Generation) were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. The room temperature was 22°C ± 3°C (maximum range) and the relative humidity was 55% ± 15% (maximum range).

Granulated textured wood released for animal bedding (Granulat A2) was used as bedding material in the cages. The cages were changed and cleaned once a week. The rooms were alternately lit (about 150 lux at approximately 1.5 m room height) and darkened in a 12 hours dark/12 hours light cycle. The ventilation rate of the animal room was between fifteen to twenty air changes per hour.



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, samples of approximately 5 mL were taken at the following times and stored at ≤ -20°C until analysis.

At start of the treatment period (first dosing day):
Analysis of stability and concentration

Immediately after preparation of the test item-vehicle formulation as well as after 8 and 24 hours storage at room temperature
(3 samples / dose level group; groups 2 - 4).
Number of samples: 3 x 3 = 9

Towards the end of the treatment period (when the majority of animals was dosed):
Analysis of concentration

During treatment always before administration to the last animal/dose level group
(1 sample / dose level group; groups 2 - 4).
Number of samples: 3 x 1 = 3

Sum of all samples: 12

The samples were labelled with the study number, species, type of sample, concentration, test day, sampling time and date.

Analysis of the test item in the test item formulations

The analytical method was validated by LPT, whereby the following parameters were determined:
- Linearity
- Accuracy
- Precision
- Sensitivity
- Specificity
- Stability

The investigation of the above mentioned validation parameters confirmed that the method employed was suitable for the determination and quantification of the test item Potassium isostearate.

Validation of the method
The analytical method was successfully validated by with HPLC-UV detection.
Analysis of the test item-formulation

The results of the test item-formulation analyses for the investigated parameters are listed in the table below:

Parameter Sampling / Dealing Percent of nominal concentration [%]

Concentration immediately after preparation on test day 15 101.5 % - 103.3 %
Stability left at room temperature after preparation for 8h or 24 h on test day 15 102.7 % - 105.2 %
Concentration before administration to the last animal per group on test day 58 99.7 % - 104.5%

These results indicated correctly prepared test item vehicle mixtures, which were stable for at least 24 hours.
Duration of treatment / exposure:
The study animals were treated during the following periods:

Males: 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30# until test day 33# at maximum) and during the post-mating period until test day 47 (one day before sacrifice).

Females: 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30# until test day 33# at maximum) and during the lactation period until test day 64 to 68 (corresponding to lactation days 13 to 15). The last dosing was always one day before sacrifice.

Frequency of treatment:
once daily
Details on study schedule:
The study animals were treated during the following periods:
Males: 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30# until test day 33# at maximum) and during the post-mating period until test day 47 (one day before sacrifice).

Females: 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30# until test day 33# at maximum) and during the lactation period until test day 64 to 68 (corresponding to lactation days 13 to 15). The last dosing was always one day before sacrifice.

Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
negative control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
(low dose)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
(intermediate dose)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
(high dose)
No. of animals per sex per dose:
10 male and 10 female rats
Control animals:
yes
Details on study design:
The dose levels were selected by the sponsor based on available toxicological data and a 14-day dose range finding study. In the 14-day dose range finding study, Potassium isostearate was administered orally to male and female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day for 2 weeks.
Changes in behaviour or the external appearance were noted in all test item treated groups (100, 300 or 1000 mg/kg b.w./day) in the form of salivation and nose discharge. Furthermore, a reduced motility and gasping were noted for 2 or 1 animal at the high dose level (1000 mg/kg b.w./day). No test item-related changes were noted on body weight and body weight gain, whereas slight reductions in food consumption where noted during the first and the second test week (at maximaum 20 % below the value of the control group; p ≤ 0.01). Based on the data obtained in this dose range finding study, dose levels of 100, 300 and 1000 mg Isooctadecanoic acid, potassium salt /kg b.w./day were selected for the main study in agreement with the Sponsor.
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
Reproductive performance

The following parameters and indices were evaluated:
Reproductive parameters

Pre-coital time and gestation length
- number of pregnant females
- duration of pre-coital time
- gestation length

(The duration of gestation was calculated from gestation day 0 (day of positive sperm detection) until (but not including) lactation day 1 (lactation day 1: morning after littering when no signs of littering were noted anymore)).

Implantation sites

- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group

Oestrous cyclicity (parental animals):
The oestrus cycle was monitored during the pre-mating and mating period from test day 15 (start of treatment) until one day before the first sign of mating was noted. No differences were noted in the mean number of oestrus cycles per dam during the pre-mating and mating period between the female animals of the control group and the female animals of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Sperm parameters (parental animals):
Sperm viability and morphology (spermiogram)

The right epididymis and the right testis of the 5 selected male animals from each group were used for sperm count and the examination of sperm viability and sperm morphology. The examinations were performed according to the methods described by L. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).
Litter observations:
Number of pups absolute:
- at birth (alive and dead)
- after 4 and 13 days of life

Number of pups per dam:
- at birth (alive and dead)
- after 4 and 13 days of life

Number of male and female pups:
- at birth
- after 4 and 13 days of life

Number of stillbirths:
- absolute
- per dam

Number of pups with malformations:
- absolute
- per dam

Examination of the pups

As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities. Any abnormal behaviour of the offspring would have been recorded. However, no abnormal behaviour was noted for the pups.
The following examinations/observations were done for the offspring.

Counting
Live pups were counted, sexed and weighed on post-natal days 1, 4 and 13.

Ano-genital distance
On post-natal day 4 before litter adjustment the ano-genital distance (AGD) of all pups was determined using a scale.

Litter adjustment on PND 4
After counting on PND 4, the litters were adjusted to 10 pups per litter by eliminating (culling) surplus pups following a randomization scheme.

Blood sampling for thyroid hormone (T4) determination
On PND4, blood samples for T4 hormone level determination were taken from 2 of the culled surplus pups. If possible, sampling was performed on one male and one female pup.

Male nipples counting
Nipples were counted in all male pups on PND 13 (shortly before scheduled sacrifice).
Postmortem examinations (parental animals):
Gross necropsy

Vaginal smears were examined on the day of necropsy to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs. The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
males - on test day 49 (after a dosing period of at least 4 weeks)
pups - on PND 13
dams - on lactation day 14, 15 or 16.

Dissection of adult animals

At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
During necropsy the number of implantation sites was recorded in the female animals.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to Salewski.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

Organs to be weighed

The weight of the following organs of all adult male and female animals was determined before fixation (where applicable):
Adrenal gland (2), Brain, Epididymis (2), heart, kidney (2), liver, ovary (2), prostate, seminal vesicles with coagulating glands, thyroid, spleen testicle (2), thymus, uterus including cervix.

Thyroid weight was determined after fixation; paired organs were weighed individually and identified as left or right.

Organs for histopathology
Fixed organs from the 5 randomly selected male and female animals per group.
Fixative: Davidson'solution
Eye with optic nerve (2) - Fixative: modified Davidson'solution
Epididymis (left) - Fixative: 7 % buffered formalin
Adrenal gland (2)
Bone
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem (pons))
Gross lesions observed
Heart (3 levels: right and left ventricle, septum)
Intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method)
Intestine, large (colon, rectum)
Kidney and ureter (2)
Liver
Lungs (with mainstem bronchi and bronchioles), preserved by inflation with fixative and then immersion
Lymph node (1, cervical)
Lymph node (1, mesenteric)
Mammary gland
Muscle (skeletal)
Nerve (sciatic)
Oesophagus
Pituitary
Ovary and oviduct
Prostate and seminal vesicles with coagulating glands
Spinal cord (3 sections)
Spleen
Stomach
Testicle (left) - Fixative: 7 % buffered formalin
Thyroid (including parathyroids)
Tissue masses or tumors including regional lymph nodes)
Thymus
Tongue (including base)
Trachea (including larynx)
Urinary bladder
Uterus (including cervix)
Vagina

Any other organs displaying macroscopic changes were also preserved.

Full histopathology was performed on the preserved organs of the selected parental animals of groups 1 and 4, and the thyroid of the selected pups. The organs listed above were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they are noted were grossly enlarged. In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O and examined histologically. Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected males of groups 1 and 4 following H-E and PAS staining.

Sperm viability and morphology (spermiogram)

The right epididymis and the right testis of the 5 selected male animals from each group were used for sperm count and the examination of sperm viability and sperm morphology. The examinations were performed according to the methods described by L. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).


Postmortem examinations (offspring):
Gross necropsy

The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
Pups on PND 13

Examination of the pups

Dead pups and pups sacrificed at day 13 post-partum were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
The thyroid of 1 male and 1 female pup from each litter was fixed in 7% formalin. Thyroid weight was determined after fixation.
Statistics:
Parametrical data

The statistical evaluation of the parametrical values was done by Provantis (Provantis® integrated preclinical software, version 9.4.0.1, Instem LSS Ltd) using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data

The statistical evaluation of non-parametrical values was done by comparison of the group values using the FISHER or the Chi2 test with the following software:
- Using Provantis: Statistical evaluation of histopathology findings
- Using StatXact 4.0.1: Statistical evaluation of the fertility index, the gestation index, the birth and live birth index, the viability indices and the post-implantation loss
Reproductive indices:
Reproductive indices

The following indices were calculated for each group:

Female Fertility Index [%] = Number of pregnant rats / Number of rats used x 100

The female fertility index reflects the total number of dams that had achieved pregnancy, including dams which delivered at term, aborted or had fully resorbed litters.

Gestation Index [%] = Number of dams with live pups / Number of pregnant rats x 100
Offspring viability indices:
For each litter and group the following indices were determined:

Birth Index [%] = Total number of pups born (alive + dead) / Number of implantation scars x 100

Live Birth Index [%] = Number of pups alive on day 0/1 of lactation / Total number of pups (alive + dead) x 100

Viability Index [%] pre-cull = Number of pups alive on day 4 (pre cull) / Number of pups alive on day 0/1 x 100

Viability Index [%] post cull = Number of pups alive on day 13 / Number of pups alive on day 4 (post cull) x 100

Post-implantation loss [%] = Implantations - living fetuses / Implantations x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males and females: At 300 or 1000 mg test item/kg b.w./day, a clear post-dosing salivation was noted in a dose related manner for the male and female animals. The saliva was reddish discoloured for one observation at the intermediate dose level and a few observations at the high dose level. The observation of salivatio disappeared again after several minutes. This transient form of salivation maybe related to the taste or irritant properties of the test item rather than an indication of adverse toxicity.
Males: At 1000 mg test item/kg b.w./day nose discharge was noted for several male animals (reddish discoloured for a few occasions).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Males: No test item-related premature death was noted for the animals treated with 100, 300 or 1000 mg test item/kg b.w./day.
Females: No test item-related premature death was noted for at the low and the intermediate dose level (100 or 300 mg test item/kg b.w./day).
At 1000 mg test item/kg b.w./day 2 of 10 pregnant females died on their gestation days 17 or 18. The deaths were considered as test item-related. Test item-related changes for both prematurely deceased animals were noted during the macroscopic examination at necropsy (dark-red discoloured lungs, reddened thymus, haemorrhagic nose/snout, enlarged adrenal glands) and during the histopathological examination (marked congestion in the lungs, adrenal cortical hypertrophy, thymus atrophy).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: No test item-related influence on body weight and body weight gain was noted for the male animals.
Females: At 1000 mg test item/kg b.w./day slight reductions in body weight were noted at the end of the gestation period and during the lactation period (5.4 % below the value of the control group on gestation day 20 and 6.7 % or 4.9 % below the values of the control group on lactation days 4 and 13). None of these changes were statistically significant. Body weight gain of the female animals of the high dose group was reduced during the gestation period (39.8% in comparison to 52.1% in the control group).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males and females: No test-item related changes were noted.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was noted on the haematological parameters of the male and female animals in any of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) for the haemoglobin content, the number of erythrocytes, the number of leucocytes, the number of reticulocytes and platelets, the haematocrit value, the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH), the mean corpuscular haemoglobin concentration (MCHC) and the parameters of coagulation (TPT, aPTT). No test item-related changes were noted in the relative and absolute differential blood counts. However, statistically significantly decreased values in comparison to the control group were noted for the haemoglobin concentration, the number of reticulocytes and the haematocrit value at the intermediate and or the high dose level (see table below). As no dose response relationship was noted and no statistically significant changes were noted for the female animals, the statistically significant changes that were noted for the male animals were considered as spontaneous and not as test item related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males and females: No test item related influence was noted for the examined plasma levels of the biochemical parameters in any of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day), i.e. levels of albumin, globulin, the albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea in blood, calcium, chloride, potassium, sodium, sodium/potassium ratio, urea/creatinine, the activity of the alanine aminotransferase (ALAT), of the alkaline phosphatase (aP), of the aspartate aminotransferase (ASAT) and the lactate dehydrogenase (LDH). However, slightly decreased protein concentrations were noted for the male animals of the intermediate and the high dose group (4.6 % or 6.3 % below the value of the control group, statistically significant at p ≤ 0.05 at the high dose group). For the female animals slightly decreased protein levels were noted for all treatment groups (between 5.5 % and 6.4 % below the value of the control group, statistically not significant. This led to decreased globulin concentrations and increased albumin/globulin ratios for the male and the female animals (statistically significant or not, see table below).
The decreased protein concentration was due to slightly increased mean values of the protein concentrations of the male and the female animals of the control group (60.8 g/L for the males and 65.2 g/L for the females). For both, the male and the female animals the mean protein concentrations of the high dose group (57.0 g/L for the males and 61.6 g/L for the females) were at the level of the mean protein concentration of LPT background data (57.8 g/L for the males and 61.2 g/L for the females). Therefore, the observations were considered as spontaneous and not as test item-related.



Urinalysis findings:
no effects observed
Description (incidence and severity):
Males and females: No test-item related changes were noted.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Males and females: No test-item related changes were noted.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males and females: No changes were noted during the neurological screening.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Males and females: No test-item related changes were noted for the 5 selected males and the 5 selected female animals from the surviving 8 animals of the high dose group.

Microscopic examination of the reproductive organs:
No test item-related microscopic changes were observed in the reproductive organs for group 4 males and females that were examined microscopically.
The histopathological examination performed on one testicle and one epididymis of the selected males of groups 1 and 4 (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure), did not reveal any test izem-related effects. The mammary glands of the observed female animals showed prominent mammary development. No test item-related microscopic changes could be seen in the reproductive organs of the female animals of group 4.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Males and females: No test-item related changes were noted.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
T4 Hormone Determination

Male and female animals: No toxicologically relevant changes were noted for the T4 levels of all treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
The statistically significantly decreased T4 levels that were noted at the intermediate and the high dose level were not considered to be of toxicological relevance (see assessment below).

TSH Hormone Determination

Male animals: No test item-related changes were noted between the TSH levels of the male animals of the control group and the male animals of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Asssessment of the maternal T4 / TSH levels

As in the present study no influence of the decreased T4 level was noted on the TSH level and no changes were noted in the thyroid weight and the histopathology of the thyroids, the decreased T4 levels that were noted for the maternal animals were not considered as of toxicological relevance for the maternal animals and were not considered for the determination of the NOAEL. This is in accordance with a published 90 day rat toxicity study.[1] In this study dose levels with significant changes for the T4 and the TSH level, but without changes of the thyroid (weight and histopathology) or other indicators of toxicity (body weight, adverse clinical signs) were not considered for the NOAEL.
The mode of toxicological action on the thyroid gland by several substances is quite similar, independently of the mechanism that led to the reduction of the T4 level. As a response to a decreased circulating T4 level an increased release of TSH from the pituitary gland is observed. The increased TSH level results in thyroid follicular cell hyperplasia and hypertrophy.[2] It is generally known that histopathological examination of the thyroid is usually more sensitive than thyroid weight and hormone levels.[3] The validation report of OECD 407 states that “thyroid histopathology was consistently the most reliable and most sensitive endpoint for the detection of thyroid modulation. Thyroid weigth was reliable, but was somewhat less sensitive when compared to thyroid histopathology. Circulating thyroid hormone levels (T3, T4, and TSH) were not always reliable and sensitive, but the standard operating procedures for blood sampling and for thyroid hormone analyses were not standardized to reduce stress induced variability and to reduce analytical variability, respectively. Circulating T4 levels were the most promising of the three thyroid hormonal values”.[3]

[1] Siglin JC et al.: A 90-day drinking water toxicity study in rats of the environmental contaminant ammonium perchlorate, Toxicol Sci (2000) 57(1): 61-74.
[2] G. Coelho-Palermo Cunha and B. van Ravenzwaay: Evaluation of mechanisms inducing thyroid toxicity and the ability of the enhanced OECD Test Guideline 407 to detect this changes, Arch Toxicol (2005) 79: 390 - 405.
[3] Manon Beekhuijzen et al.: Update of OECD DART guidelines with endocrine disruptor relevant endpoints: Practical considerations, Reproductive Toxicology (2016) 64: 64 - 71.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The oestrus cycle was monitored during the pre-mating and mating period from test day 15 (start of treatment) until one day before the first sign of mating was noted. No differences were noted in the mean number of oestrus cycles per dam during the pre-mating and mating period between the female animals of the control group and the female animals of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm count
No test item-related differences were noted for the number of spermatids in the right testis between the rats of the control group and the rats of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Sperm motility
The examination of the spermatids from the cauda of the right epidimydes revealed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Sperm morphology
The morphological analysis of the spermatids from the cauda of the right epidimydes revealed no test item-related differences in the number of malformed spermatids between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Reproductive performance:
no effects observed
Description (incidence and severity):
No test item-related differences were noted for the fertility index of the female rats treated with 100, 300 or 1000 mg test item/kg b.w./day.
All female rats which were used for mating were successfully mated by their male partners (confirmed by sperm detection).

Details on results (P0)

Monitoring of oestrus cycle

The oestrus cycle was monitored during the pre-mating and mating period from test day 15 (start of treatment) until one day before the first sign of mating was noted. No differences were noted in the mean number of oestrus cycles per dam during the pre-mating and mating period between the female animals of the control group and the female animals of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Female fertility

No test item-related differences were noted for the fertility index of the female rats treated with 100, 300 or 1000 mg test item/kg b.w./day.
All female rats which were used for mating were successfully mated by their male partners (confirmed by sperm detection).

Gestation index

No test item-related influence on the gestation index was noted for the female rats treated with 100, 300 or 1000 mg test item/kg b.w./day.

Pre-coital time

No test item-related differences were noted in the length of the pre-coital time between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Gestation length

No test item-related differences were noted for the length of the gestation period between the rats of the control group and the rats of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Birth indices, pre- and post-implantation loss

No test item-related differences were noted for the mean number of implantation sites, the mean number of pups born (alive and dead) and the mean number of live born pups between the control group and the low and the intermediate dose group (100 or 300 mg test item/kg b.w./day). Correlating the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the low and the intermediate dose group (100 or 300 mg test item/kg b.w./day). A statistically significantly decreased birth index (group level) and thus a statistically significantly increased post-implantation loss (group level) (both at p ≤ 0.05) were noted at the high dose level (1000 mg test item/kg b.w./day).


Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
other: adverse effects on prenatal development (conceptus to birth)

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Mortality:
mortality observed, treatment-related

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Viability index

No test item-related differences were noted between the viability index (pre- and post cull) of the pups from the dams of the control group and the pups from the dams of the low and the intermediate dose group (100 or 300 mg test item/kg b.w./day).
A statistically significantly decreased viability index for the period between lactation day 0/1 and lactation day 4 (pre-cull) was noted for the pups of the high dose group (1000 mg test item/kg b.w./day)
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight of pups
No test item-related difference was noted between the mean body weight of the pups from the dams of the control group and the mean body weight of the pups from the dams of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) on lactation days 1, 4 and 13.
However, a slightly reduced pup body weight was noted at the high dose level on lactation day 13 (8.9 % below the value of the control group for the male and female pups combined, statistically not significant).

Litter weight
The litter weights from the dams of the control group and those from the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) revealed no test item-related differences on lactation days 1, 4 and 13.
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
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Pups - Weight of the thyroid gland

The weight of the thyroid glands of the pups revealed no test item-related differences between the control group and the test item-treated groups (100, 300 or 1000 mg test item/kg b.w./day). However, a statistically not significantly decreased weight of the left thyroid was noted for the pups of all treatment groups in comparison to the control group. As no decrease was noted for the organ weight of the right thyroid, the decreased weight that was noted for the left thyroid was considered as spontaneous and not test item-related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
External examination of the pups

No gross abnormalities (e.g. malformations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 100, 300 or 1000 mg test item/kg b.w./day after terminal sacrifice on lactation day 13 or for the pups that died during the lactation period.
Histopathological findings:
no effects observed
Description (incidence and severity):
Pups - Microscopic examination of the thyroid glands

No test item-related changes were noted for the thyroid glands from the pups of the control group and the pups from the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio

No test item-related differences were noted for the male to female ratio on lactation days 1 and 4.

Number of live pups

No test item-related differences in the number of live pups on lactation days 1, 4 and 13 were noted between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Ano-genital distance

No test item-related difference was noted for the ano-genital distance of the male and the female pups between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) for the relative and the absolute ano-genital distance.

Examination of the male pups for nipples

No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

External examination of the pups

No gross abnormalities (e.g. malformations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 100, 300 or 1000 mg test item/kg b.w./day after terminal sacrifice on lactation day 13 or for the pups that died during the lactation period.

Pups - T4 hormone determination
No toxicologically relevant changes were noted for the T4 levels of the male and female pups of all treatment groups (100, 300 or 1000 mg test item/kg b.w./day) on lactation days 4 and 13.

Pups - TSH hormone determination
No test item-related changes were noted between the TSH levels of the male animals of the control group and the male animals of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

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:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
other: adverse effects on postnatal development (pup)

Overall reproductive toxicity

Key result
Reproductive effects observed:
not specified
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Birth Indices and Post Implantation loss Rat - Values per Dam - Summary

















































































 Sex: FemaleReproductive indices                         
 

 



Implant.


left [a]



Implant.


right [a1]



Implant.


left+right


[a2]



Born


alive+dead


(Male+Fem


ale)



Born


alive


(Male+Fem


ale)



Birth


Index


[a2]



Live Birth


Index


[a2]



Post-impl.


loss


[a2]


   NecropsyNecropsy Necropsy LD 0/1LD 0/1 LD 0/1 LD 0/1Necropsy
Gr. 1: control

Mean


SD


N



7.5


2.1


10



7.4


2.5


10



14.9


2.5


10



14.0


2.9


10



14.0


2.9


10



93.60


8.03


10



100.00


0.00


10



6.40


8.03


10


Gr. 2: 100 mg/kg

Mean


SD


N


%Diff



6.2


1.5


10


-17.3



8.3


1.2


10


12.2



14.5


1.6


10


-2.7



13.6


2.0


10


-2.9



13.1


1.9


10


-6.4



93.57


3.93


10


.



96.49


5.20


10


.



9.65


7.00


10


.



Gr. 3: 300 mg/kg 



Mean


SD


N


%Diff



7.3


2.4


10


-2.7



6.9


2.3


10


-6.8



14.2


1.3


10


-4.7



13.1


1.2


10


-6.4



13.0


1.2


10


-7.1



92.77


9.57


10


.



99.29


2.26


10


.



7.90


9.76


10


.



Gr. 4: 1000 mg/kg 



Mean


SD


N


%Diff



6.4


1.8


8


-15.0



8.1


1.7


8


9.8



14.5


1.2


8


-2.7



12.5


3.3


8


-10.7



12.4


3.3


8


-11.6



85.88


19.86


8


.



98.96


2.95


8


.



15.09


19.69


8


.



[a] - Anova & Dunnett


[a1] - Anova & Dunnett(Log)


[a2] - Anova & Dunnett(Rank)


 


Table 2: Dead Pups per Dam and Viability Index - Summary Rat











































































































Sex: FemaleNumber of Pups                               
 

 



 


Alive


on LD4


(Male+Fem


ale)



 


Viability


Index


(%)


[a1]



Post cull


LD4


(Male+Fem


ale)



 


Acci Death


on LD5-13


[a1]


 

 


Found dead


on LD5-13


[a1]


 

 


Cannibal.


on LD5-13


[a1]


 
 

Died


on LD5-13


(summary)


[a1]


  

Died on


on LD


0/1-13


(summary)


 
 

Alive


on LD13


(Male+Fem


ale)



Viability I.


Post cull


[a1]


 
   LD 4  LD 0/1-4  LD4 LD 5-13LD 5-13  LD 5-13 LD 5-13LD 0/1-13  LD 13LD 4-13 

Gr. 1: control



Mean


SD



 13.8


2.8


10



98.70


2.76


10 



9.8


0.6


10



0.0 n


0.0


10



0.0 n


0.0


10


 

0.1


0.3


10 



0.1


0.3


10



0.4


0.7


10



9.6


0.7


10



98.0


4.2


10


 



Gr. 2: 100 mg/kg



Mean


SD


N


%Diff



13.1


1.9


10


-5.1 



96.49


5.20


10


.



10.0


0.0


10


2.0



0.0


0.0


10


.



0.0


0.0


10


.



0.1


0.3


10


0.0



0.1


0.3


10


0.0



0.1


0.3


10


-75.0



9.9


0.3


10


3.1



99.0


3.2


10


.



Gr. 3: 300 mg/kg



Mean


SD


N


%Diff



12.9


1.3


10


-6.5



98.45


3.27


10


.



10.0


0.0


10


2.0



0.0


0.0


10


.



0.0


0.0


10


.



0.0


0.0


10


-100.0



0.0


0.0


10


-100.0



0.1


0.3


10


-75.0



10.0


0.0


10


4.2



100.0


0.0


10


.



Gr. 4: 1000 mg/kg



Mean


SD


N


%Diff



11.5


3.1


8


-16.7



92.32


8.04


8


-



9.3


1.5


8


-5.6



0.0


0.0


8




0.0


0.0


8


.



0.0


0.0


8


-100.0



0.0


0.0


8


-100.0



0.9


1.1


8


118.1



9.3


1.5


8


-3.6



100.0


0.0


8


.



 



 



 



 



 



 



 



 



 



 



 



 


Applicant's summary and conclusion

Conclusions:
The following no-observed adverse-effect levels were established:

Systemic toxicity
NOAEL= 300 mg/kg b.w./day, p.o.

Reproductive toxicity:
reproductive parameters of the parental females: NOAEL > 1000 mg/kg b.w./day, p.o.
adverse effects on pre- and postnatal development NOAEL = 300 mg/kg b.w./day, p.o.
Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item Potassium isostearate was administered orally to rats at dose levels of 100, 300, or 1000 mg test item/kg b.w./day.


 


Reproductive toxicity F0 - Generation


No test item-related influence was noted on the fertility and the gestation indices, the pre-coital time, and the gestation length.


 


F1 - Generation


A test item-related influence on prenatal development was noted at the high dose level in the form of a reduced birth index and an increased post-implantation loss (1000 mg test item/kg b.w./day). During the postnatal development of the pups a reduced viability index for the period after birth until lactation day 4 was noted at the high dose level (1000 mg test item/kg b.w./day).


T4 analyses on lactation days 4 and 13 revealed statistically significantly decreased T4 levels for all dose groups, whereas no changes were noted for the TSH levels, the thyroid weights, and the histopathological examination of the thyroids. Hence, the reduced T4 levels were not considered to be of toxicological relevance. No changes were noted for the pup body weight, the post cull viability index (from lactation day 5 to 13), the anogenital distance, the male nipples counting, and during the external examination for gross abnormalities.


 


Reproductive toxicity:


reproductive parameters of the parental females: NOAEL > 1000 mg/kg b.w./day, p.o.


adverse effects on pre- and postnatal development NOAEL= 300 mg/kg b.w./day, p.o.