Reaction mass of sodium hydrogen N,N-dibutyl-10-(sulphonatooxy)octadecanamidate and sodium sulphooctadecanoate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a reproduction toxicity screening study according to OECD 421 in the rat, following NOAELs were determined:

NOAEL for reproductive performance of male/female rats: 700 mg/kg bw/day

NOAEL for F1 offspring development: 700 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-05-25 to 2021-25-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

dose range finding study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The dose levels were chosen on the basis of the results of a preliminary repeated dose toxicity study with the test item in rats. In this dose range finding study with a 14-day treatment period (study no. 805-400-5441), erosion and inflammatory cell infiltrates occurred in some animals at 800 mg/kg bw/day, indicating potential inflammation or ulceration for longer exposure times.
The high dose was therefore chosen with the aim of inducing toxic effects but no mortality or severe suffering of the animals.
The low dose was chosen to induce no toxic effect.
The mid dose was interpolated geometrically.
The dose spacing was chosen according to specifications in OECD 421.

Species:

rat

Strain:

Wistar

Remarks:

Han:WIST SPF of Wistar origin

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: male animals: 85 – 89 days; female animals:85 – 89 days
- Weight at study initiation: 340 – 404 g for male animals, 203 – 243 g for female animals
(The weight variation in animals involved at the starting point of the study did not exceed ± 20 % of the mean group weight of each sex).
- Fasting period before study: no
- Housing:
Before mating: 2 animals of the same sex/cage, Mating: 1 male and 1 female/cage, Pregnant females were housed individually, Males after mating: 2 animals/cage; in Type III polypropylene/polycarbonate cages with certified laboratory wood bedding.
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water from municipal supply
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): Above 10 air-exchanges / hour by central air-condition system
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 200, 60 and 20 mg/mL. Formulations were prepared in the formulation laboratory of Test Facility beforehand not longer than for three days and stored at room temperature until use.

VEHICLE
- Concentration in vehicle: 200, 60 and 20 mg/mL
- Amount of vehicle: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Females will remain with the same male until copulation occurs or 14 days have elapsed.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male not mated male from same group (to obtain 8 pregnant females)
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (verification of concentrations and homogeneity) was performed in the Analytical Laboratory of Test Facility twice during the study. Five aliquots of 5 mL (first occasion) of each formulation and five aliquots of 5 mL control substance (vehicle) were taken and analyzed.
Concentration of the test item in the dosing formulations varied in the range of 90.6 and 100 % of the nominal values at both analytical occasions.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front.
The recovery of the test item from the vehicle was within the acceptance criteria (92 and 96 % relative to nominal concentrations) at 20 mg/mL and at 500 mg/mL, respectively.
Fatty acids, C18 (linear and branched, unsaturated) and fatty acids C18 (linear and branched, unsaturated) -N,N-dibutylamide, monosulphated, sodium salts proved to be stable in distilled water at the intended concentrations at room temperature for three days.

Duration of treatment / exposure:

Males were dosed for at least 28 days (14 days pre-mating and 14 days mating plus approx. 14 days post mating period).
Females were dosed for 14 days pre-mating, through 14 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice.

Frequency of treatment:

once per day, 7 days per week

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

700 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 animals/sex in the control and dose groups (at least 8 pregnant female animals per group were expected)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. In case of severe signs of toxicity high dose will be reduced.
- Rationale for animal assignment: was done randomly
- Fasting period before blood sampling for clinical biochemistry: no

Positive control:

not performed

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: behavioral changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. When signs of toxicity were observed, animals were observed more frequently.

BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (day 0), at least weekly thereafter and at termination. Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4, 7 days post-partum and at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The food consumption was determined weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment period except mating phase as follows: premating Days 0, 7 and 13 and by weekly interval during post-mating period for male animals; premating Days 0, 7 and 13, gestation days 0, 7, 14 and 21, lactation days 0, 7, 13 for female animals

Other:
- serum levels of thyroid hormones (T4, TSH) were measured from all dams on lactation day 13 and from all parent male animals at termination and non-pregnant, not mated, not delivered female animals at termination

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears before the treatment started from each animal being considered for study for two weeks. Animals exhibiting typical 4-5 days cycles were included in the study preferably. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation. Vaginal smear will also be prepared on the day of the necropsy.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
The testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole, of all male adult animals were trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying.

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 discarded.

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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was determined for pups born or found dead, if possible to identify, particular attention was paid to the external reproductive genitals

Other:
- serum levels of thyroid hormones (T4, TSH) were measured from at least two pups per litter on post-natal day 4 and 13

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after mating period (not earlier than Day 28) or after the optionally extended post-mating period
- Maternal animals: All surviving animals: Dams: on post-partum day 13 or shortly thereafter; Not mated female animals: shortly after the mating period; Non-pregnant (but mated) female animals: 24-26 days after positive vaginal smears were observed (or shortly thereafter)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examination were performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure; on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary as well as the epithelial capsule and ovarian stroma. In addition, these organs were processed and examined histologically in not mated, non-pregnant or not delivered females and males these females cohabited with in the low and middle dose groups. All gross lesions were also be examined histologically. Organ weights were determined for brain, testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole.

Postmortem examinations (offspring):

SACRIFICE
- Pups were sacrificed on post-natal day. Excess pups were sacrificed as specified above.
- Pups were subjected to postmortem examinations. Excess pups were not examined.

GROSS NECROPSY
- Gross necropsy consisted of external examinations. Particular attention was paid to the external reproductive genitals. Any pups showing abnormalities in structure or behavior were subjected to necropsy by macroscopic examination. The probable cause of death of dead pups were recorded, if it was identified.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant results at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons was performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Reproductive indices:

Following indices were calculated:
- Copulatory Index (Measure of animals’ ability to mate) for male and female animals
- Fertility Index (Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant) for male and female animals
- Gestation Index (Measure of pregnancy that provides at least one live pup) for female animals

Offspring viability indices:

Following indices were calculated:
- Post-implantation mortality
- Post-natal mortality
- Survival Index
- Sex ratio

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 100, 300 or 700 mg/kg bw/day at the daily clinical observations. However, observations on the bedding material (wet and slightly yellow) referred to enhanced urination at 300 and 700 mg/kg bw/day (male and female).
Dead animals
The behavior and physical condition of dead animals were normal during the days prior to their death. Death of animals occurred after the working hours (late afternoon or at night). Therefore, recording of agonal signs was not feasible.
Surviving animals
There were no clinical signs in male animals in the control, 100 or 300 mg/kg bw/day groups during the entire observation period (pre-mating, mating and post-mating periods).
At 700 mg/kg bw/day, sanguineous hairs and around the nose and noisy breathing were noted for one male animal (1/12) on Day 0 and alopecia was observed on the skin of the left side shoulder in one other (1/12) between Days 6 and 36.
Slightly wet, yellowish bedding material was detected in cages of male animals at 300 mg/kg bw/day (6/6) and at 700 mg/kg bw/day (6/6) from Day 6 up to the termination of the study.
In surviving female animals, dermal changes (scars and alopecia) were seen as follows:
100 mg/kg bw/day:
scar on the skin of the back from gestation day 12 (1/12) up to termination on lactation day 13 (1/11);
alopecia on the neck and abdomen (1/11) between lactation day 12-14;
300 mg/kg bw/day:
scar on the entire body, shoulder or neck during premating (2/12), gestation (2/11) and lactation (2/11) periods;
Slightly wet, yellowish bedding material was detected in cages of animals at 300 mg/kg bw/day (6/6 during pre-mating period, 11/11 during gestation and lactation period) and at 700 mg/kg bw/day (6/6 during pre-mating period, 11/11 during gestation and lactation period) from Day 6 up to the termination of the study.
Clinical signs of single male animal at 700 mg/kg bw/day (sanguineous hairs around the nose and noisy breathing) were judged to be related to the administration procedure regarding the transient occurrence (Day 0).
Dermal changes (alopecia, scar) are common in experimental rats of this strain occurring also in non-treated animals. The mentioned signs were detected with low incidence, independently from the doses. Therefore, these clinical signs were judged to be toxicologically not relevant in the present study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three female animals died during the course of the study: 1/12 at 300 mg/kg bw/day on Day 9, 1/12 at 700 mg/kg bw/day on Day 13 and 1/11 at 700 mg/kg bw/day on lactation day 6. There were no preceding clinical signs or changes in the body weight development in any of these animals. All these three animals were found dead at the morning inspection. Based on macroscopic and microscopic findings in the lungs, non-intended and incidentally secondary exposure by aspiration may have led to suffocation as cause of death of these animals. In addition, due to findings in the thoracic cavity in one high dose female found dead on Day 13, a mis gavage cannot be excluded completely.
There was no mortality in the control, 100, 300 and 700 mg/kg bw/day groups during the course of study (pre-mating, mating and post-mating period) in male animals.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight development was not adversely influenced in male or female animals at 100, 300 or 700 mg/kg bw/day during the entire treatment period. The mean body weight gain was dose-dependently reduced in male animals reaching statistical significance at 700 mg/kg bw/day. The reduction resulted only in slight changes in the mean body weight (≤5 % reduction relative to control) and therefore this change was considered to be not an adverse effect of toxicological relevance.
There were no statistically significant differences between the control and test item administered male animals (100, 300 and 700 mg/kg bw/day) in the mean body weight from Day 0 up to and including Day 34.
Statistical significance with respect to the control was observed at the slightly lower mean body weight gain of male animals at 300 mg/kg bw/day between Days 7-13, at 700 mg/kg bw/day between Days 0 and 7 and if summarized (between Days 0 and 34). These minor changes however had no significant influence on the mean body weight. Therefore, these findings in male animals were considered to have no toxicological relevance.
In the female animals, the mean body weight and body weight gain were comparable to their control at 100 mg/kg bw/day during the pre-mating, gestation and lactation periods.
In female animals at 300 mg/kg bw/day, the mean body weight exceeded the control on gestation days 0 and 7 and on lactation day 0.
At 700 mg/kg bw/day, statistical significance was detected at the higher mean body weight on pre-mating day 13, on gestation days 0 and 7, as well as at the higher mean body weight gain between pre-mating days 7-13 and 0-13 and between lactation days 4 and 13 in female animals.
These statistically significant differences with respect to the control were considered to be toxicologically not relevant but indicative of biological variation in the female animals because of the low degree, sporadic occurrence or in the lack of dose dependency.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

Description (incidence and severity):

There were no test item related adverse changes in the food consumption of male or female animals at any dose level (100, 300 or 700 mg/kg bw/day).

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Necropsy did not reveal any adverse effects.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

The thyroid hormone (FT3, FT4 and TSH) levels were not adversely affected in parental male animals or in PND13 offspring at any dose levels.
No statistically significant differences were observed between the control and test item administered parental male animals in the concentrations of FT3, FT4 and TSH.
In the PND13 pups, the mean FT4 concentrations were slightly above the mean control value at 100 and 700 mg/kg bw/day independently from doses. Although, the individual values at 100, 300 and 700 mg/kg bw/day were within the historical control ranges.
Therefore, these minor changes in the FT4 level were judged to be of no toxicological significance in the lack of related changes in the FT3 and TSH levels. This assessment is further justified as the values in the control were slightly below the mean values of the historical control data. Thus, the observed statistically significance difference is considered to be just a calculation phenomenon rather than a biological or toxicologically relevant effect.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

see 'clinical signs'

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

see 'clinical signs'

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Squamous cell hyperplasia and erosion in the mucous membrane of forestomach was detected in male and female animals at 300 and 700 mg/kg bw/day.

Dead animal
Histological examination revealed diffuse alveolar emphysema, alveolar edema (1/1 at 300 mg/kg bw/day, 2/2 at 700 mg/kg bw/day, both lesions) and catarrhal infiltration in the lungs (1/1 at 300 mg/kg bw/day) in dead animals.
These lesions refer to a possible treatment related suffocation (due to non-intended and incidentally secondary exposure or mis-gavage) as the cause of the death of these animals. No toxic lesions (degeneration, necrosis etc.) were observed in the investigated organs. There were no histological findings in accordance with macroscopic observation in the stomach (thin mucous membrane). The stomach showed normal morphology histologically in all dead animals.
Surviving animals
The investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histological normal and characteristic on the sexually mature organism in the male animals in control (12/12) and in 700 mg/kg bw/day (12/12) groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals.
The histological picture of epididymides, prostate, seminal vesicles, and coagulating glands was normal in all cases as well.
In the female animals in the control (12/12), 300 mg/kg bw/day (1/1 dead) and 700 mg/kg bw/day groups (2/2 dead and 10/10 survivors), the ovaries, uterus, cervix, vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle in the most cases. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
Dilatation of the uterine horn was detected in connection with the normal sexual cycle (pro-estrus phase) of uterus in two female animals (2/2) at 300 mg/kg bw/day and in one of the dead animals (1/2) at 700 mg/kg bw/day. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and is considered to be without pathological significance. Fibroma in the uterine horn (1/1 not delivered pregnant female animal) was an individual disorder occurring also in not treated animals of this strain.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male and female animals.
Focal squamous cell hyperplasia was seen in the mucous membrane of non-glandular stomach (forestomach) in several animals: - 2/2 male and 2/2 female at 300 mg/kg bw/day and
- 12/12 male and 9/10 female at 700 mg/kg bw/day.
Additionally, hemorrhages were detected at the cardia in female animals: 1/2 at 300 mg/kg bw/day and 1/10 at 700 mg/kg bw/day.
These gastric lesions were not accompanied with marked infiltration of inflammatory cells in the lamina propria or ulceration of mucous membrane.
The squamous cell hyperplasia in mucous membrane of non-glandular stomach is most likely related to the irritative/corrosive properties of the 300 and 700 mg/kg bw/day doses of the test item and thus considered to be a local effect.
Subacute focal hemorrhage in the mucous membrane of stomach occurred sporadically. The findings may be related to the irritative/corrosive properties t of the 300 and 700 mg/kg bw/day dose of the test item, however, a mechanical origin cannot be excluded entirely.
One or both sided pyelectasia (4/4, 3/3, 2/2 and 4/4 male in the control, 100, 300 and 700 mg/kg bw/day, respectively, and 1/1 female at 700 mg/kg bw/day. Pyelectasia without degenerative, inflammatory or other histological (fibrotic etc.) lesions is considered as a common finding in laboratory rats and is judged to be of no toxicological significance.
Atrophy of hair follicles (1/1 male at 700 mg/kg bw/day, 1/2 female at 100 mg/kg bw/day and 1/2 female at 300 mg/kg bw/day) and subacute dermatitis (1/2 female at 100 mg/kg bw/day and 1/2 female at 300 mg/kg bw/day) accompanied with focal or multifocal alopecia or scars occurred in some animals and these are common findings in laboratory rats and are considered as individual disease.
No morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, the pancreas, the cardiovascular system, the respiratory system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the central, or peripheral nervous system, the eyes, the integumentary system, the reproductive system was observed.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Slight variations were detected at the examined parameters of estrous cycle (percentage of animals with regular/ irregular cycle, number of cycles, length of cycles, number of days in pro-estrous, estrous or diestrous) at 700 mg/kg bw/day during the pre-mating period. However, a test item influence on the estrous cycle can be excluded as the delivery and pregnancy data were unaffected and values mostly met well (i.e., were within or were near to) the historical control data.
All examined parameters of estrous cycle – percentage of animals with regular/ irregular cycle, mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrous – were comparable in animals appointed for the control and test item dosed groups during the pre-treatment period.
There were no significant differences between the control and test item treated animals in the investigated parameters of estrous cycle at 100 and 300 mg/kg bw/day during the two weeks pre-mating period.
Statistical significance with respect to the control was detected at the lower percentage of animals with regular cycle, less mean number of cycle, longer mean length of cycle, lower mean number of days in pro-estrous and estrous and higher mean number of days in diestrous at 700 mg/kg bw/day.
Overall, there was no test item related adverse or biologically relevant impairment of estrous cycle.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The weight of examined organs – testes, epididymides, seminal vesicle with coagulating gland or prostate as a whole (absolute and relative to body and brain weights – were comparable in the control, 100, 300 and 700 mg/kg bw/day groups.
Statistical significance with respect to the control was detected at the slightly lower mean fasted body weight of male animals at 700 mg/kg bw/day.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences in the evaluated parameters of pregnancy and delivery of female animals between the control and test item treated groups (100, 300 or 700 mg/kg bw/day).
There were no significant differences between the control and test item treated groups in the mean number if implantation, pre- and post-natal loss or in total loss, number of pregnant animals, dams delivered, mean duration of pregnancy, mean number of births (total, live and stillborn), in the live birth indices, in the mean of.
The reproductive performance was not affected by the test item at 100, 300 or 700 mg/kg bw/day in male or female animals based on the examined parameters.
Statistical significance with respect to the control was detected at the slightly lower copulatory index (lower percentage of mated male animals) at 100 and 700 mg/kg bw/day as one pair of both groups failed to mate. Data met well historical control value and in absence of related findings in the reproductive parameters, this minor difference was considered to be toxicologically not relevant.
Statistically significant difference with respect to the control was detected at the lower percentage of delivered pregnant female animals – i.e., gestation index – at 100 mg/kg bw/day. The percentage of pregnant females and of females with live offspring, the pre-coital interval, number of conceiving days, copulatory and fertility indices were comparable in the control and 100, 300 and 700 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

Test item related clinical signs were not detected in the offspring between post-natal days 0 and 13.
The percentage of offspring with signs was not related to doses in the control, 100, 300 and 700 mg/kg bw/day groups.
The percentage of cold, dark colored visceral organs with white pattern, hemorrhage, smaller than others or missing pups was low (1 or 2 %) and independent from doses.
The percentage of not suckled pups was the highest in offspring at 100 mg/kg bw/day.
These signs and random differences with respect to the control were considered to be toxicologically not relevant as the signs were transient and were not associated with depression of the development of the offspring.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no test item related effect on offspring’s extra uterine mortality at 100, 300 or 700 mg/kg bw/day.
The mean number of live pups per litter and the mean number of viable pups per litter were similar in all groups on post-natal days 0 and 4.
Statistical significance with respect to the control was observed at the slightly higher mean number of viable pups on post-natal day 13. This minor difference was presumably due to the slightly lower mean number of euthanized pups at 700 mg/kg bw/day on postnatal day 4 as the mortality between post-natal days 4 and 13 were similar in the control and high dose groups. Moreover, the survival indices were comparable in the control and test item treated (100, 300 or 700 mg/kg bw/day) groups on post-natal day 13.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development of offspring was slightly depressed in offspring at 700 mg/kg bw/day between post-natal days 0 and 4. During the further course, the body weight development improved and the initially transient reduction in body weight was partially normalized by the end of the observation period. Therefore, this observation was considered to be toxicologically not relevant. Moreover, the values were within the individual ranges of the historical control data.
The mean litter weights and litter weight gain were comparable in the control and at 100, 300 and 700 mg/kg bw/day on post-natal days 0, 4 and 13.
Statistical significance at 700 mg/kg bw/day was detected at the lower mean body weight of offspring on post-natal days 0 and 4 and at the lower mean body weight gain compared to the control between post-natal days 0-4. If evaluated male and female pups separately, statistical significance with respect to the control was detected at the lower mean body weight of male and female pups at 700 mg/kg bw/day on post-natal day 4. However, as this statistical difference occurred only initially, improved over the course of the study and the deviations values were well within the individual ranges of the historical control data, these findings were considered to be toxically not relevant.
Overall, the body weight data were considered to be not adversely impaired.

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

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Statistical significance was observed at the longer mean normalized anogenital distance in male offspring at 700 mg/kg bw/day when compared to the control.
In the female offspring, the absolute anogenital distance was slightly shorter at 700 mg/kg bw/day with respect to the control group.
However, the changes in anogenital distances were of minor degree – i.e., were within or close to the historical control – therefore these were considered to neither biologically relevant nor toxicologically adverse impaired.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Nipples/areoles were not visible in any of the examined male offspring in the control or 100, 300 or 700 mg/kg bw/day groups on post-natal day 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Sex distribution:

There were no test item related differences between the control and test item treated groups in the ration or in the litter means of genders on post-natal days 0, 4 or 13.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Treatment related:

no

Conclusions:

Under the conditions of the present oral exposure study, Fatty acids, C18 (linear and branched, unsaturated) and fatty acids C18 (linear and branched, unsaturated)-N,N-dibutylamide, monosulphated, sodium salts did not adversely influence reproductive performance (gonad function, mating behavior, conception, parturition) and did not cause primary systemic toxicity nor intended exposure related deaths in parental male and female Han:WIST rats at 100, 300 and 700 mg/kg bw/day as far as investigated in this study.

Local effects in the form of gastric irritation were detected macroscopically and microscopically in the forestomach as port of entry in male and female animals at 300 and 700 mg/kg bw/day. This local gastric effect did not induce adverse systemic changes.

The development of the F1 offspring was not impaired from birth up to post-natal day 13 although, the offspring’s body weight development was slightly but not adversely reduced during the first five days of observation period after repeated oral administration of dams at 100, 300 and 700 mg/kg bw/day

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:


NOAEL for systemic toxicity of male/female rats: 700 mg/kg bw/day

NOAEL for local gastric irritation of male/female rats: 100 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 700 mg/kg bw/day

NOAEL for F1 offspring development: 700 mg/kg bw/day

Executive summary:

The objective of this study was to obtain initial information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 100, 300 and 700 mg/kg bw/day (calculated by active ingredient) compared to control animals according to OECD 421. The guideline is designed for use with the rat, which is the preferred rodent species for toxicity and reproduction toxicity testing.

As a screening test, it intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to post-natal day 13 associated with administration of repeated maternal doses. The dose setting was agreed with the Sponsor. Doses are selected with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.

Four groups of Han:WIST rats consisting of 12 animals per group and sex were administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 700 mg/kg bw/day doses in concentrations of 20, 60 and 140 mg/mL corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (distilled water) treated animals (n= 12/sex) served as a control. The suitability of the vehicle for the test item was analytically verified up front.

The concentration of the test item in the dosing formulations administered to animals was checked two times during the study. Colemanite concentrations in the dosing formulations varied in the acceptable range between 90.6 % and 100 % of the nominal values and confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 37 days). Dams were additionally exposed through the gestation period and up to lactation days 13-15, i.e., up to the day before necropsy (altogether for 51, 54, 63 or 65 days). Not delivered pregnant female animal was administered for 40 days.

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating as well as on the day of the necropsy. Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 2-6 pups per litter (where it was feasible) on post-natal day 4, from all dams and 2-7 pups per litter on post-partum/ post-natal day 13, from not delivered pregnant female on Day 40 and from all parental male animals on Day 37.

The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.

All parental animals were subjected to gross pathology one day after the last treatment. The sexual organs (ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands) and all organs showing macroscopic lesions of all adult animals were preserved. Based on the necropsy observations, the stomach of all male and female animals in each group were also fixed in 4 % buffered formaldehyde solution for further possible histological examinations.

Thyroid gland was preserved from all adult males and females and one male and one female pup per litter for the possible subsequent histopathological examination.

Gross necropsy was performed for offspring selected for thyroid gland preservation.

The body weight, brain weight and weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined.

Histopathology examination was performed on ovaries, uterus with cervix and fallopian tube, testes, epididymides, prostate, seminal vesicles with coagulating gland in the control and high dose groups. The stomach was evaluated in all male and female animals at 700 mg/kg bw/day based on necropsy observations. Additionally, organs with macroscopic findings were processed and evaluated histologically (stomach, kidneys, skin).

 

Three female animals were found dead at the morning inspection during the course of the study: 1/12 at 300 mg/kg bw/day, 2/12 at 700 mg/kg bw/day on Day 9, Day 13 and on lactation day 6, respectively. There were no preceding clinical signs or changes in the body weight development in any of these animals. Based on macroscopic and microscopic findings in the lungs, non-intended and incidentally secondary exposure by aspiration in most of the cases that may have led to suffocation as cause of death of these animals. In addition, due to findings in the thoracic cavity in one high dose female found dead on day 13, a mis-gavage cannot be excluded completely.

Parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 100, 300 or 700 mg/kg bw/day at the daily clinical observations. However, observations on the bedding material (wet and slightly yellow) referred to slightly enhanced urination at 300 and 700 mg/kg bw/day (male and female).

The body weight development was not adversely influenced in male or female animals at 100, 300 or 700 mg/kg bw/day during the entire treatment period.

There was a dose-dependent reduction of the mean body weight gain in male animals reaching statistical significance at 700 mg/kg bw/day. However, the reduction resulted only in slight changes in the mean body weight (5 % reduction relative to control) and therefore this change was considered to be not an adverse effect of toxicological relevance.

The mean daily food consumption was not adversely affected by the test item in male or female animals at 100, 300 or 700 mg/kg bw/day during the entire study (pre-mating, post-mating, gestation and lactation periods).

There was no test item related adverse or biologically relevant impairment of estrous cycle.

Slight variations were detected at the examined parameters of estrous cycle (percentage of animals with regular/ irregular cycle, number of cycles, length of cycles, number of days in pro-estrous, estrous or diestrous) at 700 mg/kg bw/day during the pre-mating period. However, a test item influence on the estrous cycle can be excluded as the delivery and pregnancy data were unaffected and values mostly met well (i.e., were within or were near to) the historical control data.

There were no differences in the evaluated parameters of pregnancy and delivery of female animals between the control and test item treated groups (100, 300 or 700 mg/kg bw/day).

The reproductive performance was not affected by the test item at 100, 300 or 700 mg/kg bw/day in male or female animals based on the examined parameters.

The thyroid hormone (FT3, FT4 and TSH) levels were not adversely affected in parental male animals or in PND13 offspring at any dose levels.

Gross necropsy revealed test item and oral exposure related local macroscopic findings in the stomach of male and female animals at 300 and 700 mg/kg bw/day (thickened mucous membrane and hemorrhage at cardiac part of the stomach) in animals survived until termination. In females found dead at 300 and 700 mg/kg bw/day additional findings predominantly in the lungs, thoracic cavity (single case at 700 mg/kg bw/day) and stomach were noted. Overall, these findings were predominantly considered as related to non-intended and incidentally secondary exposure to the lungs leading to the premature death. Aspiration may be a likely cause of death. Except for one high dose female found dead on day 13, where findings in the thoracic cavity may indicate mis-gavage that cannot be excluded completely.

The weight of examined organs – testes, epididymides, seminal vesicle with coagulating gland or prostate as a whole (absolute and relative to body and brain weights – were comparable in the control, 100, 300 and 700 mg/kg bw/day groups.

Histopathological investigation did not reveal toxic or other test item related lesions detectable by microscopic examination of the investigated reproductive organs of experimental male and female animals at 700 mg/kg bw/day.

Gastric irritation in the form of squamous cell hyperplasia and erosion in the mucous membrane of forestomach were detected in male and female animals at 300 and 700 mg/kg bw/day as indication for local toxicity at the port of entry.

In dead animals, pulmonary lesions refer to a possible treatment related suffocation (due to non-intended and incidentally secondary exposure or mis-gavage) as the cause of the death of these animals.

Gastric irritation in the form of squamous cell hyperplasia and erosion in the mucous membrane of forestomach were detected in male and female animals at 300 and 700 mg/kg bw/day as indication for local toxicity at the port of entry.

In dead animals, pulmonary lesions refer to a possible treatment related suffocation (due to non-intended and incidentally secondary exposure or mis-gavage) as the cause of the death of these animals.

The offspring’s development was not impaired at 100, 300 or 700 mg/kg bw/day from birth up to post-natal day 13. The offspring’s body weight was initially slightly but not adversely reduced on postnatal days 0 and 4 as well as the corresponding body weight gain. As both parameters improved over time, were partially normalized by the end of the observation period and were within the individual ranges of the historical control data, no toxicological relevance was given. No adverse effect on mortality, clinical signs or necropsy were detected in the offspring terminated as scheduled. Anogenital distance was not adversely impaired and there was no nipple retention (male offspring).

Under the conditions of the present oral exposure study, the test item did not adversely influence reproductive performance (gonad function, mating behavior, conception, parturition) and did not cause primary systemic toxicity nor intended exposure related deaths in parental male and female Han:WIST rats at 100, 300 and 700 mg/kg bw/day as far as investigated in this study.

Local effects in the form of gastric irritation were detected macroscopically and microscopically in the forestomach as port of entry in male and female animals at 300 and 700 mg/kg bw/day. This local gastric effect did not induce adverse systemic changes.

The development of the F1 offspring was not impaired from birth up to post-natal day 13 although, the offspring’s body weight development was slightly but not adversely reduced during the first five days of observation period after repeated oral administration of dams at 100, 300 and 700 mg/kg bw/day

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

 

NOAEL for systemic toxicity of male/female rats: 700 mg/kg bw/day

NOAEL for local gastric irritation of male/female rats: 100 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 700 mg/kg bw/day

NOAEL for F1 offspring development: 700 mg/kg bw/day

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Dose descriptor:

NOAEL

700 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive toxicity screening study

The objective of this study was to obtain initial information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 100, 300 and 700 mg/kg bw/day (calculated by active ingredient) compared to control animals according to OECD 421. The guideline is designed for use with the rat, which is the preferred rodent species for toxicity and reproduction toxicity testing.

As a screening test, it intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to post-natal day 13 associated with administration of repeated maternal doses. The dose setting was agreed with the Sponsor. Doses are selected with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.

Four groups of Han:WIST rats consisting of 12 animals per group and sex were administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 700 mg/kg bw/day doses in concentrations of 20, 60 and 140 mg/mL corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (distilled water) treated animals (n= 12/sex) served as a control.

The suitability of the vehicle for the test item was analytically verified up front.

The concentration of the test item in the dosing formulations administered to animals was checked two times during the study. Colemanite concentrations in the dosing formulations varied in the acceptable range between 90.6 % and 100 % of the nominal values and confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 37 days). Dams were additionally exposed through the gestation period and up to lactation days 13-15, i.e., up to the day before necropsy (altogether for 51, 54, 63 or 65 days). Not delivered pregnant female animal was administered for 40 days.

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating as well as on the day of the necropsy. Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from 2-6 pups per litter (where it was feasible) on post-natal day 4, from all dams and 2-7 pups per litter on post-partum/ post-natal day 13, from not delivered pregnant female on Day 40 and from all parental male animals on Day 37.

The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.

All parental animals were subjected to gross pathology one day after the last treatment. The sexual organs (ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands) and all organs showing macroscopic lesions of all adult animals were preserved. Based on the necropsy observations, the stomach of all male and female animals in each group were also fixed in 4 % buffered formaldehyde solution for further possible histological examinations.

Thyroid gland was preserved from all adult males and females and one male and one female pup per litter for the possible subsequent histopathological examination.

Gross necropsy was performed for offspring selected for thyroid gland preservation.

The body weight, brain weight and weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined.

Histopathology examination was performed on ovaries, uterus with cervix and fallopian tube, testes, epididymides, prostate, seminal vesicles with coagulating gland in the control and high dose groups. The stomach was evaluated in all male and female animals at 700 mg/kg bw/day based on necropsy observations. Additionally, organs with macroscopic findings were processed and evaluated histologically (stomach, kidneys, skin).

 

The results of this study were summarized as follows:

 

Mortality

Three female animals were found dead at the morning inspection during the course of the study: 1/12 at 300 mg/kg bw/day, 2/12 at 700 mg/kg bw/day on Day 9, Day 13 and on lactation day 6, respectively. There were no preceding clinical signs or changes in the body weight development in any of these animals. Based on macroscopic and microscopic findings in the lungs, non-intended and incidentally secondary exposure by aspiration in most of the cases that may have led to suffocation as cause of death of these animals. In addition, due to findings in the thoracic cavity in one high dose female found dead on day 13, a mis-gavage cannot be excluded completely.

 

Clinical observation

Parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 100, 300 or 700 mg/kg bw/day at the daily clinical observations. However, observations on the bedding material (wet and slightly yellow) referred to slightly enhanced urination at 300 and 700 mg/kg bw/day (male and female).

 

Body weight and body weight gain

The body weight development was not adversely influenced in male or female animals at 100, 300 or 700 mg/kg bw/day during the entire treatment period.

There was a dose-dependent reduction of the mean body weight gain in male animals reaching statistical significance at 700 mg/kg bw/day. However, the reduction resulted only in slight changes in the mean body weight (5 % reduction relative to control) and therefore this change was considered to be not an adverse effect of toxicological relevance.

Food consumption

The mean daily food consumption was not adversely affected by the test item in male or female animals at 100, 300 or 700 mg/kg bw/day during the entire study (pre-mating, post-mating, gestation and lactation periods).

 

Estrous cycle

There was no test item related adverse or biologically relevant impairment of estrous cycle.

Slight variations were detected at the examined parameters of estrous cycle (percentage of animals with regular/ irregular cycle, number of cycles, length of cycles, number of days in pro-estrous, estrous or diestrous) at 700 mg/kg bw/day during the pre-mating period. However, a test item influence on the estrous cycle can be excluded as the delivery and pregnancy data were unaffected and values mostly met well (i.e., were within or were near to) the historical control data.

 

Pregnancy and delivery data of dams

There were no differences in the evaluated parameters of pregnancy and delivery of female animals between the control and test item treated groups (100, 300 or 700 mg/kg bw/day).

 

Reproductive performance

The reproductive performance was not affected by the test item at 100, 300 or 700 mg/kg bw/day in male or female animals based on the examined parameters.

 

Serum thyroid hormones

The thyroid hormone (FT3, FT4 and TSH) levels were not adversely affected in parental male animals or in PND13 offspring at any dose levels.

 

Necropsy

Gross necropsy revealed test item and oral exposure related local macroscopic findings in the stomach of male and female animals at 300 and 700 mg/kg bw/day (thickened mucous membrane and hemorrhage at cardiac part of the stomach) in animals survived until termination. In females found dead at 300 and 700 mg/kg bw/day additional findings predominantly in the lungs, thoracic cavity (single case at 700 mg/kg bw/day) and stomach were noted. Overall, these findings were predominantly considered as related to non-intended and incidentally secondary exposure to the lungs leading to the premature death. Aspiration may be a likely cause of death. Except for one high dose female found dead on day 13, where findings in the thoracic cavity may indicate mis-gavage that cannot be excluded completely.

 

Organ weight

The weight of examined organs – testes, epididymides, seminal vesicle with coagulating gland or prostate as a whole (absolute and relative to body and brain weights – were comparable in the control, 100, 300 and 700 mg/kg bw/day groups.

 

Histopathology

Histopathological investigation did not reveal toxic or other test item related lesions detectable by microscopic examination of the investigated reproductive organs of experimental male and female animals at 700 mg/kg bw/day.

Gastric irritation in the form of squamous cell hyperplasia and erosion in the mucous membrane of forestomach were detected in male and female animals at 300 and 700 mg/kg bw/day as indication for local toxicity at the port of entry.

In dead animals, pulmonary lesions refer to a possible treatment related suffocation (due to non-intended and incidentally secondary exposure or mis-gavage) as the cause of the death of these animals.

Gastric irritation in the form of squamous cell hyperplasia and erosion in the mucous membrane of forestomach were detected in male and female animals at 300 and 700 mg/kg bw/day as indication for local toxicity at the port of entry.

In dead animals, pulmonary lesions refer to a possible treatment related suffocation (due to non-intended and incidentally secondary exposure or mis-gavage) as the cause of the death of these animals.

 

Offspring

The offspring’s development was not impaired at 100, 300 or 700 mg/kg bw/day from birth up to post-natal day 13. The offspring’s body weight was initially slightly but not adversely reduced on postnatal days 0 and 4 as well as the corresponding body weight gain. As both parameters improved over time, were partially normalized by the end of the observation period and were within the individual ranges of the historical control data, no toxicological relevance was given. No adverse effect on mortality, clinical signs or necropsy were detected in the offspring terminated as scheduled. Anogenital distance was not adversely impaired and there was no nipple retention (male offspring).

 

Under the conditions of the present oral exposure study, the test item did not adversely influence reproductive performance (gonad function, mating behavior, conception, parturition) and did not cause primary systemic toxicity nor intended exposure related deaths in parental male and female Han:WIST rats at 100, 300 and 700 mg/kg bw/day as far as investigated in this study.

Local effects in the form of gastric irritation were detected macroscopically and microscopically in the forestomach as port of entry in male and female animals at 300 and 700 mg/kg bw/day. This local gastric effect did not induce adverse systemic changes.

The development of the F1 offspring was not impaired from birth up to post-natal day 13 although, the offspring’s body weight development was slightly but not adversely reduced during the first five days of observation period after repeated oral administration of dams at 100, 300 and 700 mg/kg bw/day

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

 

NOAEL for systemic toxicity of male/female rats: 700 mg/kg bw/day

NOAEL for local gastric irritation of male/female rats: 100 mg/kg bw/day

NOAEL for reproductive performance of male/female rats: 700 mg/kg bw/day

NOAEL for F1 offspring development: 700 mg/kg bw/day

Effects on developmental toxicity

Description of key information

In a developmental toxicity study according to OECD 414 in the rat, follwing NOAELs were determined:

NOAEL for maternal toxicity: 300 mg/kg bw/day

NOAEL for developmental toxicity: 1000 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-09-07 to 2022-10-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han: WIST

Details on test animals or test system and environmental conditions:

EST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: females: ca. 10 weeks of age at start of the mating period; males: ca. 31 – 32 weeks of age at start of the mating period
- Weight at study initiation: females: The group averages of the body weight of the females were as similar as possible on the first day of gestation
- Fasting period before study: no
- Housing:
before mating: 2 females/cage, 2 maless/ cage; during mating: 1 male and 1 – 2 females/cage; during gestation: 2 sperm positive females/ cage, if not possible 1 sperm positive female/ cage;
cages: Type II polypropylene/polycarbonate;
bedding: Certified laboratory wood bedding (Safe 3/ 4-S-FASERN produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany)
- Diet: ad libitum, ssniff SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 24.3
- Humidity (%): 53 – 69
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2022-09-07 To: 2022-10-05

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (distilled water). Formulations were prepared beforehand not longer than for three days and stored at room temperature until use.

VEHICLE
- Justification for use and choice of vehicle: Water (Aqua purificata) has been shown to be a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 20, 60 and 200 mg/mL (calculated by the concentration of active ingredient)
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 2206-8278

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (verification of concentrations and homogeneity) was performed twice during the study. Five aliquots of 5 mL (first occasion) of each formulation and five aliquots of 5 mL of the control substance (vehicle) were taken and analyzed. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria (92 and 96 % relative to nominal concentrations) at 20 mg/mL and at 500 mg/mL, respectively.
Fatty acids, C18 (linear and branched, unsaturated) and fatty acids C18 (linear and branched, unsaturated) -N,N-dibutylamide, monosulphated, sodium salts proved to be stable in distilled water at the intended concentrations at room temperature for three days.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: one male/ one to three females
- Length of cohabitation: 2 - 4 h
- After unsuccessful pairing replacement of first male by another male with proven fertility: not performed
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

14 days, from gestational day (G.D.) 5 to 19

Frequency of treatment:

daily

Duration of test:

The animals were sacrificed by anaesthesia on gestation day 20.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

A total of 104 females were used for mating to achieve the sufficient number of females per group of at least 26 spermium positive females per group.

The number of mated and of pregnant females was as follows:
Control: mated: 26, pregnant: 22
300 mg/kg bw/d: mated: 26, pregnant: 22
700 mg/kg bw/d: mated: 26, pregnant: 22
1000 mg/kg bw/d: mated: 26, pregnant: 20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen on the basis of the results of “Dose Range Finding Prenatal Developmental Toxicity Study with Fatty acids, C18 (linear and branched, unsaturated) and fatty acids C18 (linear and branched, unsaturated)-N,N-dibutylamide, monosulphated, sodium salts in the Rat by Oral (Gavage) Administration (please refer to 'Cross-reference'). In this dose range finding study, death of two animals in the 300 and one in the 700 mg/kg bw/day group, as well as moribund state of one animal in the 700 mg/kg bw/day group was attributed to a technical reason (secondary exposure by aspiration). In case of one of the 300 mg/kg bw/day dam, mis-gavage was assumed. None of the dams died at 1000 mg/kg bw/day.

- Rationale for animal assignment:
The sperm positive females were allocated to each experimental group on each mating day in such a way that the group averages of the body weight were as similar as possible on the first day of gestation. Females paired with the same male were allocated to different groups on the same mating day.

- Fasting period before blood sampling for dam thyroid hormones: No fasting period described.
- Time of day for dam blood sampling: in the morning on the day of necropsy

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day from G.D. 0 to 20, on treatment days after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. When signs of toxicity were observed, animals were observed more frequently.
Individual observation included the check of behavior and general condition.
Duration and severity of the clinical signs were recorded.
Observations for signs of morbidity and mortality were made twice daily from G.D. 1 to 19, at the beginning and end of the working period, and once on G.D. 0 and 20.

DETAILED CLINICAL OBSERVATIONS: Yes, see above.

BODY WEIGHT: Yes for females; No for males
- Time schedule for examinations: Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g).

FOOD CONSUMPTION: Yes
- Food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: 20
- Organs examined: The uterus with cervix was removed and weighed. The ovaries were placed into a Petri dish after removal. After removing the uterus, gross pathology of dams’ viscera was performed. Thyroid glands of all sperm positive females were weighed and histological examination was performed on the thyroid glands of all females.
Organs and tissues with undiagnosed macroscopic findings were fixed in 4 % (v/v) buffered formalin solution at necropsy for possible histological examination. Control organs were fixed in 4 % (v/v) buffered formalin solution and preserved for comparison.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Live fetuses and fetal death were counted.

Blood sampling:

Blood samples collected for TSH and Thyroid Hormones (FT3 and FT4) measurements were drawn from all sperm positive females from the sublingual vein in the morning on the day of necropsy.
- Plasma: No
- Serum: Yes
- Volume collected: at least 2.5 mL blood

Fetal examinations:

- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: Yes, half per litter
- Anogenital distance of each fetus was determined.

Statistics:

Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually.
Current historical control data were used to enhance interpretation of study results (please refer to 'Overall remarks, attachments'). Means and standard deviations and/or percentages were calculated.
Statistical analysis were performed with IBM SPSS 25 software.
The heterogeneity of variance between groups was checked by Levene homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test. Chi2 test was performed if feasible.

Indices:

- Pre-implantation loss: ((Number of corpora lutea - Number of implantations)/Number of corpora lutea)*100
- Post-implantation loss: ((Number of implantations - Number of live fetuses)/Number of implantations)*100
- Sex distribution: (Number of Male (Female) fetuses/Number of fetuses)*100
- External- (including placentas), visceral and skeletal abnormalities abnormalities/litter: (Number of fetuses with abnormality/Number of fetuses)*100

Historical control data:

Historical control data on prenatal developmental toxicity from studies in the rat strain conducted in the same laboratory from 2017-2021 were included in the study report and used as reference (please refer to 'Overall remarks, attachments').

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The most common clinical sign on different days of gestation for the survived females was reduced activity of 2, 4 and 7 animals in the 100, 300 and 1000 mg/kg bw/day dose groups (together with the excluded animals). In most cases this clinical sign was accompanied with piloerection (1, 3 and 7 animals in the 100, 300 and 1000 mg/kg bw/day dose groups respectively). In addition, noisy respiration was recorded for one animal in the 300 and one in the 1000 mg/kg bw/day group, as well as hunched back for one non-pregnant in the 1000 mg/kg bw/day dose group. The clinical signs lasted one to six days long.
Reflux of the test item directly after treatment was observed in three females from four with clinical signs in the 300 and for five from seven with clinical signs in the 1000 mg/kg bw/day group.
In the cases where no reflux of the test item was seen directly after treatment, but clinical signs occurred (2 from 2, 2 from 4 and 1 from 7 in the 100, 300 and 1000 mg/kg bw/day dose groups respectively), also aspiration was assumed based on the similar clinical signs.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1000 mg/kg bw/day: 1/20 (G.D. 20);
No clinical sings were recorded priorly and blood around the snout was observed. It was assumed to be due to a technical reason and test item relation could not be proven.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slightly lower body weight was observed in the 1000 mg/kg bw/day dose group from the first treatment which was statistically significant (p<0.05) on G.D. 8 and 11.
Weight loss was indicated in the 1000 mg/kg bw/day dose group between G.D. 5 and 8 (p<0.01) and statistically significantly (p<0.05) lower body weight gain was seen if calculated from the first treatment to up to the end of the in-life phase (G.D. 5 to 20). Also, the for gravid uterine weight corrected body weight gain was statistically significantly lower (p<0.01) in the 1000 mg/kg bw/day group and lower corrected body weight was observed without a statistical significance.
Statistically significant increases were observed in the body weight gain (p<0.05 and p<0.01) in the 100 and 300 mg/kg bw/day groups respectively, as well as if calculated for between G.D. 5 and 20 (p<0.05) in the 300 mg/kg bw/day group. They were presumed to be of no toxicological relevance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced food consumption (-13 to -38% versus control, p<0.01) was observed in the 1000 mg/kg bw/day group between G.D. 5 and 14 (for all three days periods (5 to 8, 8 to 11 and 11 to 14). Also, if the mean values were evaluated, the reduced food consumption in the 1000 mg/kg bw/day dose group (-16%) was statistically significant (p<0.05) between G.D. 5 and 20.
Statistical significances were revealed for the slightly lower food consumption in the 300 and 100 mg/kg bw/day group (p<0.01, -10 and -6 % respectively).
Based on the slight manner this was not considered as adverse, which was supported by the body weight and corrected body weight/gain parameters which were unaffected.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

please refer to 'Endocrine findings'

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

The mean FT4 level was statistically significantly lower (p<0.05) in the 300 mg/kg bw/day group without a dose response, hence was not considered as adverse. There were no significant differences in the FT3 and TSH levels.

There were no significant differences in the mean values of thyroid weight in the experimental groups.

No lesion of the thyroid tissue in any dam was detected at histological examination.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The gravid uterine and thyroid weights (please refer to 'Endocrine findings') were comparable in all groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Thicker cardia was observed in 10 of 19 evaluated as well as in 4 of 7 excluded females in the 1000 mg/kg bw/day dose group. Dark discoloration in the stomach mucosa was seen in one, and thicker cardia mucosa in another female in the 1000 mg/kg bw/day group.
These finding were absent in the lower dose groups and were attributed to the irritative effect of the test item.

Dilated renal pelvis was observed in one pregnant female in the 100 and in one non-pregnant in the 1000 mg/kg bw/day dose group. Hydrometra was recorded for one female in the control group. These findings were not attributed to the treatment based on the low incidence and lack of dose response or occurrence in the control group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

For thyroid results, please refer to 'Endocrine findings'.

Histopathological examination of deceased animal wil be included in an update.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

There was no statistically significant or dose related increase indicated.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no statistically significant increase indicated in the pre- implantation loss.
There were two females with total post-implantation loss, one in the 100 (No.: 188) and one in the 1000 (No.: 227) mg/kg bw/day dose group. Considering the lack of dose response, this was not proved to be test item related.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There was no statistically significant increase indicated.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was no statistically significant increase indicated in the early- and late embryonic death.

Dead fetuses:

no effects observed

Description (incidence and severity):

There was no statistically significant increase indicated in the number of dead fetuses.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number and percentage of pregnant females compared to mated females showed similar values in all groups.
Control: mated: 26, pregnant: 22 (84.6%)
100 mg/kg bw/day: mated: 26, pregnant: 22 (84.6%)
300 mg/kg bw/day: mated: 26, pregnant: 22 (84.6%)
1000 mg/kg bw/day: mated: 26, pregnant: 20 (76.9 %)

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the fetal weights.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the number of viable fetuses.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The distribution of the two sexes was similar in the groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in litter size and weights.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in ano-genital distance.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of evaluated fetuses was 213, 214, 247 and 181 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.
At external examinations there were 2/2, 0/0, 1/1 and 1/1 fetuses/litters found in the control, 100, 300 and 1000 mg/kg bw/day group respectively.
One fetus in the 1000 mg/kg bw/day dose group (No.: 0617179/12) had microcephalia, microphthalmia and absence of mandible.
One hypoplastic mandible was found in the 300 mg/kg bw/day dose group (No.: 1250123/8). Also, absence of mandible was recorded for one control fetus (No.: 2073101/2).
Considering the low incidence, these malformations were not judged to be treatment related. Moreover, hypoplastic or absent mandible was present in the control group, and absent or hypoplastic mandible (agnathia or retrognathia) and also microphthalmia may occur in control fetuses according to the Background Pregnancy and Fetal Data (please refer to 'Overall remarks, attachments').
Umbilical hernia was found in one control fetus (No.: 01402127/10) as an incidental finding.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of examined fetuses was 108, 105, 123 and 190 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.
The number of malformed fetuses/litters was 2/2, 0/0, 3/2 and 3/3 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.

In the 1000 mg/kg bw/day dose group fetus No. 0104177/1 and 1046104/12 had hemicentric ossification of the thoracic vertebra IX as a malformation. Fetus No. 0410141 had an interrupted rib and the 2nd rib was not connected to the sternum.

In the 300 mg/kg bw/day dose group fetus No. 1250123/1 had three misshapen thoracic vertebrae and two of these were fused and inclined. Fetus No. 1250123/8 in the same litter, which was found with a markedly hypoplastic mandible, was observed with absence of mandible and in addition cleft palate. Fetus No. 1457170/7 in another litter had a misshapen- and cleft palate.

There were no skeletal malformations found in the 100 mg/kg bw/day group.

In the control group fetus No. 0411154/5 had a split xiphoid of the sternum. Absence mandible of fetus No. 2073101/2 was confirmed by skeletal examination and in addition misshapen, cleft palate and mal-positioned tympani were observed in the same fetus.

The skeletal malformations in the 1000 and 300 mg/kg bw/day dose group were not proved to be test item related, based on the low incidence or presence of the malformation also in the control group (absent or hypoplastic mandible (agnathia, retrognathia) and cleft palate. Moreover, malformation of vertebrae (including hemicentric ossification), as well as if the second rib is not connected to sternum or if a rib is interrupted may occur in control fetuses according to the Background Pregnancy and Fetal Data (please refer to 'Overall remarks, attachments').

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of examined fetuses was 105, 109, 124 and 91 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.
The number of malformed fetuses/litters was 3/3, 0/0, 0/0 and 1/1 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.

Fetus No. 0617179/12 in the 1000 mg/kg bw/day group, which was found as malformed also by external examination, was allocated to visceral examination. Examination of the head and brain revealed dilated lateral brain ventricles, displacement of inner and mid ear, anophthalmia of one eye (eye was not visible, histopathology was not performed to confirm of complete absence), microphthalmia and disposition of the other eye, as well as absence of the nasal region.
In the control group, umbilical hernia (the same as at external examination No.: 01402127/10) and markedly dilated lateral brain ventricles (No.: 1340105/9) as well as malformation of the great arteries (from left carotid artery originated left subclavian artery, and high positioned aortic arch accompanied with the variation short brachiocephalic trunk (No.: 2283112/9) were observed in one fetus each.

There were no visceral malformations found in the 300 and 100 mg/kg bw/day groups.

Considering the single occurrence of the head/brain malformations at soft tissue evaluation of the 1000 mg/kg bw/day dose group fetuses, which were partially present in a control fetus (dilated brain ventricles (No.: 1340105/9) and mal-positioned ear bones (No.: 2073101/2, skeletal examination), as well as that microphthalmia may occur in control fetuses according to the Background Pregnancy and Fetal Data (please refer to 'Overall remarks, attachments'), the malformations in Fetus No. 0617179/12 in the 1000 mg/kg bw/day group were not judged to be test item related.

Other effects:

no effects observed

Description (incidence and severity):

There was no dose related increase in incidence of variations. The incidence of fetuses with overall variations (if external, visceral and skeletal taken into consideration) was statistically significantly higher (p<0.05) in the 300 mg/kg bw/day group which was not attributed to the test item based on lack of dose response.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat. (total fraction)

Sex:

male/female

Basis for effect level:

other: no effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

please refer to 'Overall remarks, attachments'

Conclusions:

Oral treatment of pregnant Han: WIST rats from gestation day 5 up to day 19 (the day before Caesarean section) with Fatty acids, C18 (linear and branched, unsaturated) and fatty acids C18 (linear and branched, unsaturated) -N,N-dibutylamide, monosulphated, sodium salts at the dose level of 1000 mg/kg bw/day has not been proven to cause mortality. Death of one female in the 1000 mg/kg bw/day dose group was attributed to a technical reason (mis-gavage or aspiration of the test item) or to a local effect in the gastrointestinal tract according to the necropsy findings. The test item at the dose level of 1000 mg/kg bw/day caused reduced food consumption and body weight development as well as macroscopic changes in maternal stomachs (thicker cardia) as a local effect. The slight reductions in the food consumption at the dose levels of 300 and 100 mg/kg bw/day were not considered as adverse, based on the unaffected body weight parameters. Clinical signs such as reduced activity, piloerection and or noisy breath or hunched back which occurred in the 1000, 300 and 100 mg/kg bw/day dose groups were in majority with association of reflux of the test item or similar to the observations made in such animals and was assumably attributed to aspiration. The dose levels of 1000, 300 and 100 mg/kg bw/day with the test item had no impact on the maternal FT3, FT4 and TSH hormone levels, the weight of thyroids and it did not cause changes in thyroids which could have been observed at histopathological evaluation. The treatment with the test item had no impact on the intrauterine parameters. There were no test item-related effects proven on external, visceral and skeletal examination of fetuses.

Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity): 300 mg/kg bw/day
NOAEL (developmental toxicity
including teratogenicity): 1000 mg/kg bw/day

Executive summary:

Groups of 26 sperm-positive female Han:WIST rats were treated with the test item in water (Aqua purificata) by oral gavage administration at three dose levels of 100, 300 and 1000 mg/kg bw/day, respectively, from gestation day 5 to 19. A concurrent control group of 26 sperm positive females was treated with the vehicle alone. The dose volume was 5 mL/kg/bw. Dosing formulations were analysed for homogeneity and achieved concentrations of the test item two times during the treatment period using a validated by high performance liquid chromatography coupled with RID detection method. The determined test item concentrations were within 10% of the nominal concentration (90.4 – 102 %) and, therefore, the dosing formulations were considered acceptable. A homogenous distribution of the test item in the vehicle was demonstrated for all analyzed dosing formulations.

For mating, the females were cohoused in the mornings with males of the same strain and source (1 male: 1-2 females) and the day of detection of sperm in the vaginal smear of females was regarded as gestation day 0. During the study, the animals were checked daily twice for mortality and at least once for clinical signs. Body weight and food consumption of the dams were recorded repeatedly. Blood collection for determination of thyroid hormone FT3 (free T3) and FT4 (free T4), as well as TSH level was performed on gestation day 20. A Caesarean section and gross pathology were performed on gestation day 20. Organs of the dams were examined macroscopically. Thyroid glands of all sperm positive females were weighed and histological examination was performed on the thyroid glands of all females.

The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and external abnormalities. The ano-genital distance was measured. The placentas were weighed and examined externally. The body of about half of each litter was subjected to visceral examination by means of a dissecting microscope after fixation in Sanomiya mixture. The heads were examined by Wilson's free-hand razor blade method. After double staining, the skeletons of the remaining fetuses (not assigned to visceral examination) were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

There were 22 pregnant females in the control, 100 and 300 mg/kg bw/day group as well as 20 in the 1000 mg/kg bw/day group respectively. After deduction of two females with total post-implantation loss, (100 and 1000 mg/kg bw/day) and one female which died assumably due to a technical reason in the 1000 mg/kg bw/day group, the number of evaluated litters was 22 in the control and 300 mg/kg bw/day, 21 in the 100 and 18 in the 1000 mg/kg bw/day respectively.

Death of one female in the 1000 mg/kg bw/day dose group which was found dead on G.D. 20 after lack of any clinical signs priorly, was assumably attributed to a technical reason (administration of the test item) based on the presence of dried blood around the snout. Considering the stomach irritative properties of the test item, a local effect in the stomach and intestines was also not excluded. The recorded clinical signs (one to six days long in different periods of the gestation) such as reduced activity, piloerection, hunched back and noisy breath occurred in most of the cases in animals where reflux of the test item directly after treatment was observed (or in some cases assumed) in the 100, 300 and 1000 mg/kg. Macroscopic observations in the stomach such as thicker cardia in 14 of 26 females (as well as dark discoloration in the stomach mucosa and thicker cardia mucosa in two different females) observed in the 1000 mg/kg bw/day dose group could be in connection with the local effect of the test item based on the similar findings and histopathological results in the DRF study (non GLP). Treatment related slightly lower body weight on G.D. 8 and 11, weight loss between G.D. 5 and 8 as well as lower body weight gain between G.D. 5 and 20, as well as reduced corrected body weight gain in the 1000 mg/kg bw/day dose group was attributed to the treatment. Reduced food consumption (-13 to -38%) in the 1000 mg/kg bw/day group between G.D. 5 and 14 as well as 5 and 20 was judged as treatment related. Statistical significances in the slightly lower food consumption in the 300 and 100 mg/kg bw/day group for between G.D. 5 and 8 (-10 and -6% respectively) were not considered as adverse based on the small difference and the unaffected body weight parameters. There was no test item effect indicated in the above hormone levels. There were no significant differences in the mean values of thyroid weight in the experimental groups. No lesion of the thyroid tissue in any dam was detected at histological examination. There were no statistically significant differences observed in intrauterine mortality, number of viable fetuses and their sex distribution. Based on lack of dose response, occurrence of one female in the 1000 and one in the 100 mg/kg bw/day dose group with total post-implantation loss was not proved to be test item related.

There were no statistically significant differences observed in the body weights of fetuses, placental weights and ano-genital distances. There was no dose related increase in incidence of malformations or variations in the external, visceral and skeletal examination. The malformations and variations were found with low incidences or/and also present in the current control group or and in the historical control data.

 

Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

 

NOAEL (maternal toxicity): 300 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Dose Range Finding Study

 

In this dose range finding study, groups of eleven sperm-positive female Han: WIST rats were treated with Fatty acids, C18 (linear and branched, unsaturated) and fatty acids C18 (linear and branched, unsaturated) -N,N-dibutylamide, monosulphated, sodium salts by oral administration daily at three dose levels of 300, 700 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of eleven sperm positive females was included and the animals were given the vehicle water (Aqua purificata). The treatment volume was 5 mL/kg bw.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day (G.D.) 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were examined externally.

Proceeding from 10, 11, 9 and 8 pregnant females, after death, euthanasia or exclusion due to a technical reason (reflux of the test item formulation), on gestation day 20, a total of 10, 9, 6 and 8 litters in the control, 300, 700 and 1000 mg/kg bw/day groups, respectively, were evaluated.

Two animals died at 300 mg/kg bw/day and one at 700 mg/kg bw/day, as well as one was moribund in latter group. The cause of death was supposed as secondary exposure by aspiration, and in case of one of the 300 mg/kg bw/day dams (No.: 149) mis-gavage was assumed. None of the dams died at 1000 mg/kg bw/day.

The animals which died or were moribund had reflux of the test item formulation related clinical signs such as dyspnoea, reduced activity, hunched back, piloerection and squeling which were observed in most cases after detecting reflux of the test item. Also, three survived females in the 700 mg/kg bw/day group had similar clinical signs.

Reduced food consumption was observed at 700 and 1000 mg/kg bw/day between G.D. 5 11 with a dose response.

At 1000 mg/kg bw/day, the mean body weight of the dams was moderately lower. Slightly lower body weight at 700 mg/kg bw/day at G.D. 8, 17 and 20 was observed.

Weight loss was observed at 1000 mg/kg bw/day from G.D. 5 to 8, as well as the body weight gain was lower which was significant between G.D. 11 and 17, as well as between G.D. 5 to 20 or 0 to 20. The body weight gain was significantly lower at 700 mg/kg bw/day from G.D. 5 to 8.

The corrected body weight/gain was slightly lower than in the control in the test item treated groups at a similar level.

Test item related findings as thicker cardia (2 at 700 and 5 at 1000 mg/kg bw/day) as well as white coated stomach mucosa (1 at 700 mg/kg bw/day) were observed.

Histological examination revealed squamous cell hyperplasia and inflammatory cell infiltrates in the forestomach in the middle and high dose groups.

In this study the development of squamous cell hyperplasia could be in connection with the local effect of 700 mg/kg bw/day and 1000 mg/kg bw/day of the test item.

Other findings like bloody dried fluid at nasal region, compact formation next to salivary gland, point-like bleedings in the thymus, foamy discharge in the esophagus, reddish mottled or dark red lungs, bloody content in the thoracic cavity, yellowish content in the stomach and duodenum and dark red liver with low incidences in the 300 and 700 mg/kg bw/day dose groups were not judged to be a consequence of a direct test item effect.

The mean of preimplantation loss was slightly higher at 1000 mg/kg bw/day, as well as the mean number of implantations was slightly lower in this group. Considering the slight difference, a test item effect was not proved. Post-implantation loss was higher in the test item treated groups without a dose response. None of the dams had total post-implantation loss. The sex ratio of fetuses was similar in all groups. There was no test item effect indicated. There were no fetal variations and malformations observed. There was no dose response in the incidence of body weight retarded (small) fetuses.

In one litter at 300 mg/kg bw/day 6 edemateous placentas were seen (in a litter where the dam had clinical signs and slighter weight loss).

Based on these preliminary observations, the doses for the main Prenatal Developmental Toxicity Study were selected as follows:
Group 1 Vehicle control
Group 2 100 mg/kg bw/day
Group 3 300 mg/kg bw/day
Group 4 1000 mg/kg bw/day

 

 

Prenatal Developmental Toxicity Study (according to OECD 414)

 

Groups of 26 sperm-positive female Han:WIST rats were treated with the test item in water (Aqua purificata) by oral gavage administration at three dose levels of 100, 300 and 1000 mg/kg bw/day, respectively, from gestation day 5 to 19. A concurrent control group of 26 sperm positive females was treated with the vehicle alone. The dose volume was 5 mL/kg/bw. Dosing formulations were analysed for homogeneity and achieved concentrations of the test item two times during the treatment period using a validated by high performance liquid chromatography coupled with RID detection method. The determined test item concentrations were within 10% of the nominal concentration (90.4 – 102 %) and, therefore, the dosing formulations were considered acceptable. A homogenous distribution of the test item in the vehicle was demonstrated for all analyzed dosing formulations.

For mating, the females were cohoused in the mornings with males of the same strain and source (1 male: 1-2 females) and the day of detection of sperm in the vaginal smear of females was regarded as gestation day 0. During the study, the animals were checked daily twice for mortality and at least once for clinical signs. Body weight and food consumption of the dams were recorded repeatedly. Blood collection for determination of thyroid hormone FT3 (free T3) and FT4 (free T4), as well as TSH level was performed on gestation day 20. A Caesarean section and gross pathology were performed on gestation day 20. Organs of the dams were examined macroscopically. Thyroid glands of all sperm positive females were weighed and histological examination was performed on the thyroid glands of all females.

The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and external abnormalities. The ano-genital distance was measured. The placentas were weighed and examined externally. The body of about half of each litter was subjected to visceral examination by means of a dissecting microscope after fixation in Sanomiya mixture. The heads were examined by Wilson's free-hand razor blade method. After double staining, the skeletons of the remaining fetuses (not assigned to visceral examination) were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

There were 22 pregnant females in the control, 100 and 300 mg/kg bw/day group as well as 20 in the 1000 mg/kg bw/day group respectively. After deduction of two females with total post-implantation loss, (100 and 1000 mg/kg bw/day) and one female which died assumably due to a technical reason in the 1000 mg/kg bw/day group, the number of evaluated litters was 22 in the control and 300 mg/kg bw/day, 21 in the 100 and 18 in the 1000 mg/kg bw/day respectively.

Death of one female in the 1000 mg/kg bw/day dose group which was found dead on G.D. 20 after lack of any clinical signs priorly, was assumably attributed to a technical reason (administration of the test item) based on the presence of dried blood around the snout. Considering the stomach irritative properties of the test item, a local effect in the stomach and intestines was also not excluded. The recorded clinical signs (one to six days long in different periods of the gestation) such as reduced activity, piloerection, hunched back and noisy breath occurred in most of the cases in animals where reflux of the test item directly after treatment was observed (or in some cases assumed) in the 100, 300 and 1000 mg/kg. Macroscopic observations in the stomach such as thicker cardia in 14 of 26 females (as well as dark discoloration in the stomach mucosa and thicker cardia mucosa in two different females) observed in the 1000 mg/kg bw/day dose group could be in connection with the local effect of the test item based on the similar findings and histopathological results in the DRF study (non GLP). Treatment related slightly lower body weight on G.D. 8 and 11, weight loss between G.D. 5 and 8 as well as lower body weight gain between G.D. 5 and 20, as well as reduced corrected body weight gain in the 1000 mg/kg bw/day dose group was attributed to the treatment. Reduced food consumption (-13 to -38%) in the 1000 mg/kg bw/day group between G.D. 5 and 14 as well as 5 and 20 was judged as treatment related. Statistical significances in the slightly lower food consumption in the 300 and 100 mg/kg bw/day group for between G.D. 5 and 8 (-10 and -6% respectively) were not considered as adverse based on the small difference and the unaffected body weight parameters. There was no test item effect indicated in the above hormone levels. There were no significant differences in the mean values of thyroid weight in the experimental groups. No lesion of the thyroid tissue in any dam was detected at histological examination. There were no statistically significant differences observed in intrauterine mortality, number of viable fetuses and their sex distribution. Based on lack of dose response, occurrence of one female in the 1000 and one in the 100 mg/kg bw/day dose group with total post-implantation loss was not proved to be test item related.

There were no statistically significant differences observed in the body weights of fetuses, placental weights and ano-genital distances. There was no dose related increase in incidence of malformations or variations in the external, visceral and skeletal examination. The malformations and variations were found with low incidences or/and also present in the current control group or and in the historical control data.

Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity): 300 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

 

Based on available data the test item does not adversely affect reproductive performance. Based on the conducted test, the test substance does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.