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

Toxicity to reproduction

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

dose range finding study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2021-03-03 to 2021-03-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

This 14-day repeated dose toxicitiy study was used as dose-range-finding study for the OECD 421.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OPPTS 870.3050 Repeated Dose 28–Day Oral Toxicity Study in Rodents

Version / remarks:

July 2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 03 October 2008

Deviations:

no

GLP compliance:

no

Remarks:

This study was not performed according to GLP compliances, however the principles of GLP were followed and all data were recorded and retained.

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han:WIST rat of Wistar origin

Details on species / strain selection:

The rat is a commonly used species for toxicological studies in accordance with international recommendations. The Wistar rat was the system of choice because it has been the preferred and most commonly used species for oral toxicity tests and is a well-known laboratory model with sufficient historical data.

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: 50 – 53 days; female animals: 50 – 53 days
- Weight at study initiation: 241 – 266 g for male animals, 152 – 180 g for female animals
- Fasting period before study: no (All animals were food deprived
overnight prior to blood sampling)
- Housing: 5 animals of the same sex/ 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
- Acclimation period: 13 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

Details on route of administration:

The route of application was selected in compliance with international guidelines. The oral route is the anticipated route of human exposure to the test item.

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 40, 100 and 160 mg/mL (calculated by active ingredient). Formulations were prepared in the formulation laboratory of the test facility beforehand, not longer than for three days, and stored at room temperature until use.

VEHICLE:
- Concentration in vehicle: 40, 100 and 160 mg/mL
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The formulation samples were measured within the specified stability range and storage condition. Interference was not detected in control samples. Mean concentrations of the test item in the formulation samples were in the range of 97.0 –109 % of the nominal concentrations. Deviation of the samples taken from different places of the vessels was ≤ 2.37 %, therefore the formulations can be considered homogeneous.

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily (7 days per week)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per sex and per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose. In addition, the study should allow the dose setting for the OECD 421 study.
- Fasting period before blood sampling for clinical biochemistry: overnight

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (at the beginning and end of each working day).
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
On Day 0, animals were observed continuously for 30 minutes then 1, 2, 3 and 5 hours after the administration to estimate the peak period of effects. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
- Time schedule: once a day, after treatment at approximately the same time

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 (prior to study start) and twice weekly – on Days 0, 3, 7, 10 and 13 – with a precision of 1 g.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
-determined with the measurement of given and non-consumed diet with a precision of 1 g once weekly to coincide with body weight measurements (given food on Days 0 and 7; remained food on Days 7 and 13)

OPHTHALMOSCOPIC EXAMINATION: Yes, at necropsy

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of the treatment (Day 14)
- Anaesthetic used for blood collection: Yes, samples were harvested from the retro orbital venous plexus under Isofluran CP® anesthesia
- Animals fasted: Yes, overnight (16 h)
- How many animals: all
- Samples: Three samples were taken from each animal: one for determination of blood clotting times, one for hematology and the third one to obtain serum samples for clinical chemistry.
- Parameters checked in Table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of the treatment (Day 14)
- Animals fasted: Yes, overnight (16 h)
- How many animals: all
- Parameters checked in Table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, during clinical observations (see above)

IMMUNOLOGY: Yes, parameters checked in hematology and clinical biochemistry

OTHER: Blood coagulation (checked in hematology, Table 3)

Sacrifice and pathology:

Gross pathology was performed on every animal: on the day of death (1/5 female at 200 mg/kg bw/day) on Day 13 or one day after the last treatment, on Day 14 (all survivors). Surviving animals were anesthetized with Isofluran CP® and were exsanguinated from the abdominal aorta after verification of deep narcosis. The external appearance (surface of the body, all orifices) was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically for each animal. All observations were recorded with details of the location, color, shape and size.

GROSS PATHOLOGY: Yes (see Table 4)

HISTOPATHOLOGY: Yes
Histopathological examinations were performed on the stomach of all animals in all groups (male and female) based on the necropsy observations at 800 mg/kg bw/day. All organs of dead female animal were processed and are under evaluation histologically.
The fixed and organs were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:
- Body weight
- Hematology
- Blood coagulation
- Clinical chemistry
- Organ weight

The heterogeneity 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 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 was 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.
Frequency of clinical signs, ophthalmoscopy, pathological and histopathological findings by sex and dose was calculated.

The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately.

Clinical signs:

no effects observed

Description (incidence and severity):

Test item relates clinical signs were not detected at any dose level (male or female) during the 14-day observation period.
The behavior and physical condition of all animals (male and female) were normal in the control, 200, 500 and 800 mg/kg bw/day during the course of 14-day administration.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no test item related mortality in the 200, 500 or 800 mg/kg bw/day groups during the 14-day treatment period (male and female).
At 200 mg/kg bw/day, one female animal (1/5) was found dead before the actual daily treatment on Day 13. Piloerection, decreased activity, dyspnea, narrow eye aperture and side position were noted for this animal on Days 11-12. Based on the body weight loss from Day 3 and necropsy findings (pulmonary hemorrhages, foamy liquid content in the lungs, thin wall of stomach, dilated and brownish-red mucous filled intestines), the cause of the death was probably an individual disease. There were no similar observations or mortality at the higher doses, therefore a test item related effect was excluded.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development was undisturbed in male and female animals in all test item treated groups (200, 500 or 800 mg/kg bw/day) throughout the entire observation period.
In the dead female animal at 200 mg/kg bw/day, the body weight decreased after Day 3. Thus, the terminal body weight was lower than at the beginning of treatment (Day 0). This observation was considered to be due to individual disease and not test item related.
The mean body weight and body weight gain were similar – there were no statistical or biological significances with respect to their control – in male or female animals in the control, 200, 500 and 800 mg/kg bw/day treated groups during the entire observation period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

Description (incidence and severity):

The mean daily food consumption was comparable in the control, 200, 500 and 800 mg/kg bw/day groups (male and female) during the 14-day observation period.

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

not examined

Ophthalmological findings:

not examined

Description (incidence and severity):

Examination in necropsy did not reveal any effects.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological investigations revealed changes in white blood cell parameters (neutrophil granulocytes, lymphocytes in male animals and white blood cell count in female animals) at 800 mg/kg bw/day and in platelet count and percentage of reticulocytes (in female animals) at 800 mg/kg bw/day presumably in correlation with gastric lesions. Nevertheless, most of individual values of platelet count and reticulocytes remained within the historical control range.
In the male animals, statistical significance with respect to the control was detected at the higher mean percentage of neutrophil granulocytes (NEU) and lower mean percentage of lymphocytes (LYM) at 800 mg/kg bw/day as well as at the shorter mean prothrombin time (PT) at 200, 500 and 800 mg/kg bw/day.
In the female animals, the following statistically significant differences with respect to the control were detected:
- higher mean white blood cell count at 800 mg/kg bw/day,
- higher mean platelet count (PLT) at 200, 500 and 800 mg/kg bw/day;
- lower mean percentage of reticulocytes (RET) at 200 mg/kg bw/day;
- higher mean percentage of reticulocytes at 500 and 800 mg/kg bw/day;
Changes in WBC, NEU, LYM, PLT and RET may be the consequences of the local effect of the test item in the stomach at 800 mg/kg bw/day.
The PLT and RET values remained within or were marginal to the historical control range in female animals at 200 and 500 mg/kg bw/day. Moreover, the actual control value of PLT was below the historical control. Therefore, the toxicological significance of these findings in female low and mid dose groups is questionable.
PT values also remained within the historical control ranges and tendency in changes in PT has no clinical meaning. Therefore, this minor finding was judged to be of little or no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no pathological changes in the investigated clinical chemistry parameters at 200, 500 or 800 mg/kg bw/day (male and female). Slight elevation in alanine aminotransferase activity may be indicative of enhanced hepatic function (adaptation) at 500 mg/kg bw/day (male) and at 800 mg/kg bw/day (male and female).
The examined clinical chemistry parameters were comparable in the male animals in control and 200 mg/kg bw/day groups.
The mean activity of alanine aminotransferase (ALT) slightly exceeded the control in male animals at 500 and 800 mg/kg bw/day.
Statistical significance with respect to the control was also observed at the slightly higher mean activity of aspartate aminotransferase (AST) and at the lower mean concentration of inorganic phosphorous (Pi) in male animals at 500 mg/kg bw/day.
In the female animals, elevated mean concentration of glucose (GLUC), cholesterol (CHOL) at 200 mg/kg bw/day, lowered mean concentration of chloride (Cl-) at 500 mg/kg bw/day, as well as higher mean activity of alanine aminotransferase and higher mean concentrations of cholesterol and inorganic phosphorous at 800 mg/kg bw/day were detected when compared to their control.

The values of ALT activity exceeded the control value in female animals at 800 mg/kg bw/day.
Most of the above-mentioned differences (Pi, AST in males; GLUC, CHOL, Cl- and Pi in females) with respect to control were small and were considered to be independent from the treatment because values were within or marginal to the historical control ranges .
Elevated activity of AST in male animals at 500 mg/kg bw/day exceeded the actual control and also historical control range. However, a similar finding was not detected at the higher dose.
Therefore, these findings were not considered to be toxicologically relevant.

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

see 'clinical signs'

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Elevated weights of liver (female at 200 and 500 mg/kg bw/day, male and female at 800 mg/kg bw/day) may reflect a possible test item influence on the hepatic function as an adaptive reaction. Histopathological examinations would be necessary to reveal the nature of changes in these organs.
In the male animals at 200 and 500 mg/kg bw/day, the weights of all examined organs were comparable with their control.
At 800 mg/kg bw/day, the mean liver and kidney weights, both relative to body weight, exceeded the control in male animals. Minor change (8 %) in the kidney weight relative to body weight was considered to be not toxicologically relevant.
In the female animals statistically significant difference with respect to the control was observed at the higher mean weights of liver as follows:
- higher mean liver weight relative to brain weigh at 200 mg/kg bw/day;
- higher mean liver weights (absolute and relative to body and brain weights) at 500 and 800 mg/kg bw/day

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross necropsy revealed alterations in the stomach (thickened wall, hemorrhages at cardia) probably related to the local effect of the test item in some male and especially in female animals at 800 mg/kg bw/day.
Dead animal
In dead female animal (1/5) at 200 mg/kg bw/day, hemorrhage and foamy liquid content in the lungs, thin wall of the stomach and dilated, brownish-red mucus filled intestines were observed at the necropsy on day of death (Day 13).
Surviving animals
In the male animals, right side pyelectasia were detected in each group: 1/5 control, 1/5 at 200 mg/kg bw/day, 1/5 at 500 mg/kg bw/day and 2/5 at 800 mg/kg bw/day.
At 800 mg/kg bw/day, thickened wall and hemorrhages at cardia of the stomach (1/5) as well as thickened wall of urinary bladder and mucous cyst around the right lobe of prostate (1/5) were seen in single male animal.
In the female animals, right or both sided pyelectasia (1/5 control, 1/5 at 800 mg/kg bw/day) and moderate hydrometra (1/5 control, 2/4 at 200 mg/kg bw/day, 1/5 at 500 mg/kg bw/day) were observed with the highest incidence at the necropsy.
In single female animals at 500 mg/kg bw/day, thymic hemorrhages (1/5) and Hernia diaphragmatica (1/5) were also detected.
At 800 mg/kg bw/day, acute pulmonary hemorrhage (1/5), thickened wall (2/5) and hemorrhage (3/5) in the stomach occurred in female animals beside the above mentioned pyelectasia.
Macroscopic findings in one dead female animal of the 200 mg/kg bw/day dose group were probably caused by individual disease as death or similar findings were not detected in the higher dose groups.
Changes in the stomach were only observed in high dose treated animals and these presumably were related to the local effect of the test item at the cardiac part.
Pyelectasia is a common finding in experimental rats of this strain. This renal change occurs commonly in experimental rats of this strain, therefore it was considered to be toxicologically not relevant in this study.
Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats.
In the lack of related inflammatory or other pathological signs, pyelectasia and hydrometra were judged to be toxicologically not relevant and not test item related.
Acute pulmonary and thymic hemorrhages were probably due to the exsanguination procedure.
Some macroscopic findings were only detected in single animals and were considered to be individual one: thickened wall of urinary bladder and mucous cyst around the right lobe of prostate, Hernia diaphragmatica.

Neuropathological findings:

no effects observed

Description (incidence and severity):

see 'clinical signs'

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histological examination revealed squamous cell hyperplasia in the forestomach in the middle (500 mg/kg bw/day) and high (800 mg/kg bw/day) dose treated male and female animals accompanied with erosion and inflammatory cell infiltrates in some animals only at 800 mg/kg bw/day.
These finding are commonly seen following administration of a chemical by gavage but may occur by feed-dosed or by drinking water routes as well.
Hyperplasia is characterized by thickening of the epithelial layer that often forms a broad-based lesion with marked hyperkeratosis and infiltration of inflammatory cells in the lamina propria. Hyperplastic lesions are often crateriform with a central focus of inflammatory cells. When hyperplasia consists of a diffuse thickening of the squamous epithelium, hyperkeratosis is often a component of the lesion. Maturation of cells is orderly; mitotic activity may be increased but cellular atypia and invasion are not seen. More marked hyperplasia may show prominent rete pegs that will appear as islands of cells in the lamina propria.
There were no histological findings in the stomach of male or female animals in the control and 200 mg/kg bw/day groups.
Minimal squamous cell hyperplasia was observed in the stomach of male (2/5) and female (3/5) animals at 500 mg/kg bw/day.
At 800 mg/kg bw/day, mild or moderate squamous cell hyperplasia (5/5 male and 5/5 female), erosion (1/5 male and 2/5 female) and minimal (2/5 female) or mild (2/5 male) inflammation were detected in the stomach.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

other: no NOAEL determination in DRF study

Remarks on result:

not determinable

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Conclusions:

Under the conditions of the present study, Fatty acids, C18 (linear and branched, unsaturated) and fatty acids C18 (linear and branched, unsaturated) -N,N-dibutylamide, monosulphated, sodium salts induced changes in hematological parameters (WBC, NEU, LYM, PLT, RET), in the forestomach (thickened wall, hemorrhages, squamous cell hyperplasia, erosion, inflammatory cell infiltration) and in the liver function (ALT, liver weight) in male or female Han:WIST rats after the consecutive 14-day oral (by gavage) administration of 800 mg/kg bw/day .
At 500 mg/kg bw/day, slight changes were detected in the platelet count, in percentage of reticulocytes, in the forestomach (thickened wall, hemorrhages, squamous cell hyperplasia) and in the liver function (ALT, liver weight) in male or female animals.
At 200 mg/kg bw/day, the PLT count, liver weight (relative to brain weight) of female animals slightly exceeded the actual control. However individual values met well the historical control values.
Detected findings in the stomach – and subsequently in some hematological parameters – and liver function were attributed to the test item.
Based on the observations made in this 14-day repeated dose toxicity study, the dose levels for a Reproduction/Developmental Toxicity Screening Test in the Rat (main study according to test guideline OECD 421) were determined as follows:

Group 1: Vehicle control
Group 2: 100 mg/kg bw/day
Group 3: 300 mg/kg bw/day
Group 4: 700 mg/kg bw/day

Executive summary:

The purpose of this study was to obtain first information on the toxic potential of Fatty acids, C18 (linear and branched, unsaturated) and fatty acids C18 (linear and branched, unsaturated) -N,N-dibutylamide, monosulphated, sodium salts in rats at three dose levels to allow a dose-setting for a Reproduction/Developmental Toxicity Screening Test in the Rat (main study according to test guideline OECD 421).
The dose setting with 0, 200, 500 and 800 mg/kg bw/day (calculated by active ingredient) based on the results of a 28-day repeated dose toxicity study provided by the sponsor has been chosen in agreement with the Sponsor.
Doses of 0 (vehicle only), 200, 500 and 800 mg/kg bw/day were orally administered (by gavage) to four groups of Han:WIST rats consisting of five animals per group and sex at a dosing volume of 5 mL/kg in concentrations of 40, 100 and 160 mg/mL. A group of vehicle (distilled water) treated animals (n= 5/sex) served as a control. The suitability of the chosen vehicle for the test item was analytically verified. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. Measured concentrations of formulations applied in the study varied in the acceptable range (between 97 % and 109 % of the nominal concentrations) and all formulations were homogenous, thereby confirming proper dosing.

Detailed clinical observations were performed daily after the treatment. On Day 0, animals were observed continuously for 30 minutes then 1, 2, 3 and 5 hours after the administration to estimate the peak period of effects. Body weights were recorded twice weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted one day after the last treatment (on Day 14). Selected organs were weighed. Histopathological evaluation was performed on the stomach of each animal in all groups based on macroscopic (necropsy) observations at 800 mg/kg bw/day.

 

There was no test item related mortality in 200, 500 or 800 mg/kg bw/day groups. One female animal was found dead at 200 mg/kg bw/day on Day 13. Clinical signs (piloerection, decreased activity, dyspnea, narrow eye aperture and side position on Days 11-12), body weight loss from Day 3 and on necropsy findings (pulmonary hemorrhages, foamy liquid content in the lungs, thin wall of stomach, dilated and brownish-red mucous filled intestines) were detected. Histological examination revealed serous-purulent bronchopneumonia – presumably related to miss-swallowing in connection with the treatment procedure – as the cause of death. Test item related clinical signs were not detected in male or female animals at any dose level (200, 500 or 800 mg/kg bw/day). The body weight development was unaffected at 200, 500 or 800 mg/kg bw/day during the two weeks of the study. The mean daily food consumption was similar in male and female in the control and test item administered groups. Hematological investigations revealed changes in white blood cell parameters (neutrophil granulocytes, lymphocytes in male animals and total white blood cell count in female animals) 800 mg/kg bw/day and in platelet count and percentage of reticulocytes (female animals) at 500 and 800 mg/kg bw/day presumably in correlation with gastric lesions. Nevertheless, most of individual values of platelet count and reticulocytes remained within the historical control range. Slight elevation in alanine aminotransferase activity at 500 mg/kg bw/day (male) and at 800 mg/kg bw/day (male and female) may be indicative of enhanced hepatic function in accordance with organ weight changes. Gross necropsy revealed changes in the stomach (thickened wall, hemorrhages) in some male and female animals at 800 mg/kg bw/day. Elevated weights of liver (female at 200 mg/kg bw/day, male and female at 500 and 800 mg/kg bw/day) may reflect a possible test item influence on the hepatic function as an adaptive reaction. Histopathological examinations could reveal the nature of changes in these organs. Gastric lesions – squamous cell hyperplasia, erosions, inflammatory cell infiltration – in the forestomach refer to local effect of the test item in male and female animals at 800 mg/kg bw/day. Squamous cell hyperplasia was also detected in some animals at 500 mg/kg bw/day without erosion and inflammation.

Under the conditions of the present study, the test item induced changes in hematological parameters (WBC, NEU, LYM, PLT, RET), in the forestomach (thickened wall, hemorrhages, squamous cell hyperplasia, erosion, inflammatory cell infiltration) and in the liver function (ALT, liver weight) in male or female Han:WIST rats after the consecutive 14-day oral (by gavage) administration of 800 mg/kg bw/day. At 500 mg/kg bw/day, slight changes were detected in the platelet count, in percentage of reticulocytes, in the forestomach (thickened wall, hemorrhages, squamous cell hyperplasia) and in the liver function (ALT, liver weight) in male or female animals. At 200 mg/kg bw/day, the PLT count, liver weight (relative to brain weight) of female animals slightly exceeded the actual control. However individual values remained within the historical control values. Detected findings in the stomach – and subsequently in some hematological parameters – and liver function were attributed to the test item.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Version / remarks:

July 2000

Deviations:

no

Test guideline 2

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.

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dibutyloleamide and oleic acid, sulphated, sodium salts

EC Number:

915-926-9

Molecular formula:

C18H34Na2O6S + C26H52NNaO5S

IUPAC Name:

N,N-dibutyloleamide and oleic acid, sulphated, sodium salts

Test material form:

liquid

Details on test material:

The water content of the actual test item was 57.6% (w/w); the water content of the REACH registration substance was analytically determined to be 3.8% (w/w).

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

no

Applicant's summary and conclusion

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