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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 16 November 2017 to 28 february 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 01 August 2017 to 07 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: As a preliminary study, this study did not follow a specific guideline and it was designed to allow selection of appropriate dose levels for use in the upcoming 90-day study.
Deviations:
no
Principles of method if other than guideline:
- Principle of test: Subacute repeated dose to xicity by the oral route
- Short description of test conditions: Test Item was administered to Wistar rats at three dose levels after daily oral gavage administration for 14 consecutive days.
- Parameters analysed / observed:
- Clinical signs and mortality
- Body weight
- Food consumption
- Gross macroscopic examination
observations were performed twice daily, and detailed clinical observation was performed
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: UP6C10X10
- Expiration date of the lot/batch: 11 March 2018
- Manufacture date: 11 March 2016
- Purity (sultaine content): 35.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25oC, below 70 RH%), protected from light
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle: During the method validation, 21-day stability of the Test Item in the selected vehicle was obtained. The Test Item proved to be stable on 20±5°C in the concentration range of 1.6-220
mg/L (when adjusted for purity).
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI Wistar rats
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks old
- Weight at study initiation: Males: 240–269g; Females: 181–213g
- Fasting period before study: no
- Housing: Cage type: Type II polypropylene / polycarbonate; Bedding: Lignocel® 3/4 –S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle
1, D-73494 Rosenberg, Germany suitable for the purposes of the study.
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice - breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest,
Germany, ad libitum.
- Water (e.g. ad libitum): tap water from municipal supply, ad libitum
- Acclimation period: 7-8 days

DETAILS OF FOOD AND WATER QUALITY: The supplier of the diet provided analytical certificate for the batches used, which are archived with the study raw data. Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3–24.9°C
- Humidity (%): 31-49%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): Artificial lighting 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 16 November 2017 To: 15 February 2018
Route of administration:
oral: gavage
Details on route of administration:
The animals were treated by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe at a constant dose volume of 5 mL/kg bw. As the Test Item is a surfactant with foaming/irritation potential, in order to avoid as much as possible aspiration into the respiratory tract susceptible to cause unspecific toxicity, attention was paid to very carefully clean the outside of the gavage tube each time the syringe is filled, and to ensure that the cannula used is just the right length to ensure the Test Item is dispensed in the stomach and not into the oesophagus. The Control group was treated concurrently with the vehicle only.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The Test Item was formulated in distilled water at the following concentrations: 13.3, 40 and 120 mg/mL for the Low, mid, and High dose group, respectively. Formulations were prepared according to the obtained stability results, practically for the maximum period of 7-days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
To verify the concentration of the Test Item in formulations, representative samples were taken and analysed from each concentration at one occasion during the study.
The concentration of Test Item in the dose formulations were determined using a validated analytical method (HPLC-MS/M).
The Test Item formulations had measured concentrations of 96-97% of the nominal concentrations; concentrations and stability of the aqueous solutions were fully acceptable. These results were considered suitable for the study purposes.
Duration of treatment / exposure:
14 consecutive days
Frequency of treatment:
daily
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
in terms of active (sultaine) content
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
in terms of active (sultaine) content
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
in terms of active (sultaine) content
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses were selected by the Sponsor based on available information from previous studies.
- Rationale for animal assignment: During the acclimation period, the animals were assigned to their respective dose groups by randomisation based on body weights.
- Fasting period before blood sampling for clinical biochemistry: Not applicable
- Rationale for selecting satellite groups: no satellite groups
- Post-exposure recovery period in satellite groups: no
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). General clinical observations were made at least daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations was made on all animals outside the home cage in a standard arena prior to the first treatment (to allow for within-subject comparisons) and weekly thereafter, in the morning hours (am).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was recorded with a precision of 1g at randomisation (pre-treatment period), on the first day of treatment (Day 1, prior to start of treatment), then on Day 4, 8, 11, on Day 14 (last treatment day) and prior to necropsy (fasted, on Day 15).

FOOD CONSUMPTION:
- Fanimal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Days 3 and 7.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Scheduled gross pathology examination was performed on each animal. On Day 15, animals were euthanised under pentobarbital anaesthesia by exsanguination. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Abnormalities were recorded with details of the location, colour, shape and size, as appropriate. The stomach was retained from all animals and fixed in 10% buffered formalin solution.

HISTOPATHOLOGY: No
Other examinations:

Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.
Statistics:
Group mean and standard deviation were calculated for numerical data. Statistical analysis was performed using SAS 9.2 (built in Provantis System).
The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro- Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests show no significant heterogeneity, an Anova / Ancova (one- way analysis of variance) test was carried out.
If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs recorded during the study. All animals were clinically normal.
Mortality:
no mortality observed
Description (incidence):
There was no mortality in the study, all animals survived until scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no Test Item related effects observed on the animal body weights or body weight gain values during the study.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
When compared to controls, slightly lower food consumption was noted in Mid and High dose on Day 14. As there were no effects on body weight or body weight gain values, this was considered to be non-adverse effect.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no macroscopic findings observed in Control, Low and Mid dose groups at necropsy.
Multifocal thickness at the non-glandular stomach mucosa were noted for 3/4 High dose females and red, single focus was described in the glandular mucosa of the stomach for 1/4 High dose females. These were considered as local irritation effects of the Test Item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
200 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
600 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Conclusions:
In conclusion, under the conditions of this study, after 14 days of oral gavage treatment at 600 mg/kg bw/day (High dose) multifocal thickness at the non-glandular stomach mucosa were noted for 3/4 High dose females and red, single focus was described in the mucosa of the glandular stomach for 1/4 High dose females. These were considered as local irritation effects of the Test Item. There were no adverse effects of treatment in the Mid or Low dose group therefore, the systemic NOAEL (no observed adverse effect level) was considered to be 600 mg/kg bw/day (High dose).
Executive summary:

The objective of the study was to obtain preliminary information on the toxic potential of the Test Item administered to Wistar rats at three dose levels (150, 300 and 600 mg/kg bw/day) after daily oral gavage administration for 14 consecutive days, in preparation of a main 90-day study to be conducted.

The Test Item formulations had concentrations of 96-97% of the nominal concentrations. These results and stability of the aqueous solutions were fully

acceptable. General clinical and mortality observations were performed twice daily, and detailed clinical observation was performed weekly during the study. Body weight was measured on Day 4, 8, 11, on Day 14, fasted body weight was recorded prior to scheduled necropsy (Day 15). Food consumption was recorded weekly. Following repeated dose administration daily for 14 consecutive days, gross macroscopic examination was performed at necropsy. The stomach was retained from all animals. No histopathology was required.

There was no mortality in the study. There were no clinical signs recorded during the study. There were no Test Item related effects observed on the animal body weights or body weight gain values. Slightly lower food consumption was noted in Mid and High dose on Day 14. As there were no effects on body weight or body weight gain values, this was considered to be a non-adverse effect.

At necropsy, multifocal thickness at the non-glandular stomach mucosa were noted for 3/4 High dose females and red, single focus was described in the glandular mucosa of the stomach for 1/4 High dose females. These were considered as local irritation effects of the Test Item.

In conclusion, under the conditions of this study, 14-days oral gavage treatment at 600 mg/kg bw/day (High dose) was associated with local irritation effects of the Test Item in the mucosa of non-glandular and glandular stomach. There were no adverse effects of treatment in the Mid or Low dose group and therefore, the systemic NOAEL (no observed adverse effect level) was considered to be 600 mg/kg bw/day (High dose). It was considered that suitable dose levels for the 90-day study are 66.6, 200 and 600 mg/kg/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: UP6C10X10
- Expiration date of the lot/batch: 11 March 2018
- Manufacture date: 11 March 2016
- Purity (sultaine content): 35.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25oC, below 70 RH%), protected from light
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle: During the method validation, 21-day stability of the Test Item in the selected vehicle was obtained. The Test Item proved to be stable on 20±5°C in the concentration range of 1.6-220
mg/L (when adjusted for purity).

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI Wistar rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks old
- Weight at study initiation: Males: 240–269g; Females: 181–213g
- Fasting period before study: no
- Housing: Cage type: Type II polypropylene / polycarbonate; Bedding: Lignocel® 3/4 –S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle
1, D-73494 Rosenberg, Germany suitable for the purposes of the study.
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice - breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest,
Germany, ad libitum.
- Water (e.g. ad libitum): tap water from municipal supply, ad libitum
- Acclimation period: 7-8 days

DETAILS OF FOOD AND WATER QUALITY: The supplier of the diet provided analytical certificate for the batches used, which are archived with the study raw data. Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3–24.9°C
- Humidity (%): 31-49%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): Artificial lighting 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 16 November 2017 To: 15 February 2018

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The animals were treated by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe at a constant dose volume of 5 mL/kg bw. As the Test Item is a surfactant with foaming/irritation potential, in order to avoid as much as possible aspiration into the respiratory tract susceptible to cause unspecific toxicity, attention was paid to very carefully clean the outside of the gavage tube each time the syringe is filled, and to ensure that the cannula used is just the right length to ensure the Test Item is dispensed in the stomach and not into the oesophagus. The Control group was treated concurrently with the vehicle only.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The Test Item was formulated in distilled water at the following concentrations: 13.3, 40 and 120 mg/mL for the Low, mid, and High dose group, respectively. Formulations were prepared according to the obtained stability results, practically for the maximum period of 7-days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
To verify the concentration of the Test Item in formulations, representative samples were taken and analysed from each concentration at 4 times during the study (at Week 1, 5, 9 and Week 12), Set 1 (approximately 5 mL) and Set 2 (approximately 5 mL). Similarly, one sample was taken in duplicate from the Group 1 (Control).
The concentration of Test Item in the dose formulations were determined using a validated analytical method (HPLC-MS/M).
The Test Item formulations had measured concentrations of 95.1-107.6% of the nominal concentrations; concentrations and stability of the aqueous solutions were fully acceptable. These results were considered suitable for the study purposes.
Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
66.7 mg/kg bw/day (nominal)
Remarks:
in terms of active (sultaine) content
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
in terms of active (sultaine) content
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
in terms of active (sultaine) content
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses were selected by the Sponsor based on available information and results of 14-day study by oral gavage in rats performed at Citoxlab Hungary Ltd. (Study code: 17/133-101PE; see section 7.5.1 subacute study 2019). In that study, after 14 days of oral gavage treatment at 600 mg/kg bw/day (High dose) multifocal thickness at the non-glandular stomach mucosa were noted for 3/4 High dose females and red, single focus was described in the mucosa of the glandular stomach for 1/4 High dose females. Based on these results, the 600 mg/kg bw/day dose was considered acceptable as top dose in the 90-day repeated dose study.
- Rationale for animal assignment: During the acclimation period, the animals were assigned to their respective dose groups by randomisation based on body weights.
- Fasting period before blood sampling for clinical biochemistry: yes
- Rationale for selecting satellite groups: no satellite groups
- Post-exposure recovery period in satellite groups: no
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). General clinical observations were made at least daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena at randomization, prior to the first treatment (to allow for within-subject comparisons) and weekly thereafter, in the morning hours (am).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was recorded with a precision of 1g at randomisation (pre-treatment period), on the first day of treatment (Day 1, prior to start of treatment), then weekly, including on Day 90 (last treatment day) and prior to necropsy or at death.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The determination of food consumption was performed for all groups once a week. The remaining, non-consumed food was weighed at least weekly from Day 8 with a precision of 1 g.

WATER CONSUMPTION: YNo

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: Ophthalmoscopic examination was conducted in all animals before treatment and in the Control (Group 1) and High dose (Group 4) animals on Week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91.
- Anaesthetic used for blood collection: Yes: blood samples were collected by heart puncture under pentobarbital anaesthesia.
- Animals fasted: Yes (overnight prior the blood sampling)
- How many animals: all animals
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91.
- Anaesthetic used for blood collection: Yes: blood samples were collected by heart puncture under pentobarbital anaesthesia.
- Animals fasted: Yes (overnight prior the blood sampling)
- How many animals: all animals
- Parameters checked in table [2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine collection was conducted over approximately 16 hours, during an overnight period of food deprivation of animals, which were placed in metabolic cages
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the treatment period on Day 82-84.
- Dose groups that were examined: all animmals in all groups.
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Each animal was subjected to the functional observation battery (FOB), including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment (SMART).
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
A detailed assessment for neurotoxicity effects were made on the basis of these measurements (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
Parameters such as body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated.
To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured.
Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of Test Item. The rats were held appropriately such that the fore limbs are allowed to grip the support bar and pulled back until they release the bar; the device measures
the maximum grip strength. This was performed 3 times for each animal on the test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data.
Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 30 min, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Data from all groups were evaluated for distance travelled in 5 minute segments. The data from the 5 minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.

OTHER: Examination of vaginal smears
Prior to necropsy, the oestrus cycle of all females were determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution.
The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see list of tissues and organs table 4)
Necropsy and macroscopic examination were performed on all surviving animals, at the end of treatment period, on Day 91 (after the sample collection for clinical pathology evaluation). The animals were euthanized by exsanguination under pentobarbital anaesthesia.
Macroscopic evaluation:
After exsanguination the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically.
In addition, bone marrow smears from the femur of each animal were prepared at necropsy (but not examined).

HISTOPATHOLOGY: Yes (see list of tissues and organs table 4)
The eyes with the optic nerve and the testes with epididymides were preserved in modified Davidson’s fixative; all other organs in 10% buffered formalin solution.
The tissues and organs to be examined were embedded in paraffin wax, sections were cut at 4-6μ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Full histopathology was performed in Groups 1 (Control) and 4 (High dose) and any animals found dead during the study. In addition, any organs or tissues with macroscopic abnormalities (except
common background findings, unless the incidence is affected by treatment) were subjected to histological examination from all groups.
As Test Item related microscopic findings were identified in the kidney (tubular cell vacuolation) and stomach (hyperkeratosis of the non-glandular stomach mucosa, erosion/ulcer) of the High dose animals, these organs from the Low and Mid dose were processed to slides. The Mid and Low dose slides were also examined microscopically.
Other examinations:
ORGAN WEIGHTS:
The following organs were trimmed of fat and weighed in all animals:
With precision of 0.01g : Brain, Seminal vesicles with coagulating glands, Epididymides, Heart, Spleen, Kidneys, Testes, Liver, Thymus, Prostate, Uterus including cervix.
With precision of 0.001g: Adrenals, Ovaries, Thyroids with parathyroids, Pituitary

Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.
Statistics:
Data were collected using the software PROVANTIS v.9 or recorded on the appropriate forms as per relevant SOPs, then tabulated using PROVANTIS v.9, Microsoft Office Word and/or Excel, as appropriate.
Group mean and standard deviation were calculated for numerical data. Statistical analysis was performed using SAS 9.2 (built in Provantis System).
The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests show no significant heterogeneity, an Anova / Ancova (oneway analysis of variance) test was carried out.
If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there wa a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001 as appropriate.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were Test Item related non-adverse clinical signs recorded during the study in Low, Mid and High doses.
Low dose, slight noisy respiration was recorded in one male and in one females. Whole body tonic convulsion was recorded for another female rat in the Low dose on four occasions during the study.
In Mid dose, slight noisy respiration was recorded on a few occasions in males (2/10) and female (1/10). Whole body tonic convulsion was recorded for the same female rat in on five occasions during the study.
In High dose, slight noisy respiration was recorded for 6/10 males and for 6/10 females, piloerection was recorded for 1/10 male and 3/10 female rats (1 female had hunched back as well). Red discharge from the nose or/and the eyes were observed for one male and one female.
Noisy respiration was not considered to reflect a systemic toxicity of the Test Item, but a local effect related to the strong surfactant properties of the substance (even a very small amount entering the respiratory tract by reflux would be expected to be very irritating). Hence the finding is not considered as adverse in the context of a systemic toxicity study. Some other minor findings were considered to be secondary to the respiration effect.
Noisy respiration is a common clinical sign in animals which have minor local pulmonary effects, which in known to occur when very small amounts of surfactant
enters the upper respiratory tract. This can occur by reflux from the stomach.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two High dose animals (one male on Day 32 and one female on Day 57) died during the study. Severe broncho-alveolar pneumonia was considered to be cause of death for male. The macroscopic and microscopic findings did not indicate a specific cause of death for the female.
In previous studies with this Test Item (a strong surfactant) there were many mortalities which are ascribed to small amounts of the Test Item entering the upper respiratory tract (probably from outside of the gavage tube). This information is based on discussions with the Sponsor. In this study great care was taken to clean the gavage tubes, so mortality was restricted to these 2 animals, and was probably caused by reflux of small amounts of Test Item. In the male, there was histological evidence of local pulmonary damage, indicating exposure was several days before the death. In the female it is likely that an acute respiratory tract exposure caused the death (reddish foamy material was present in the trachea) but there is no other clinical or histological evidence to confirm this. However, with a lack of any other potential caused of mortality, the 2 deaths were ascribing to local respiratory tract effects; there was no evidence for any systemic adverse effects that could cause death.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant Test Item related effects observed on the animal body weights or body weight gain values during the study. All treated groups were comparable with the Control (see graph in attachement).
On one occasion during the study, the body weight gain of the High dose males were statistically significantly lower, but the overall conclusion was that there was no significant effect of the Test Item on body weight.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no Test Item related adverse effects noted on animal food consumption under the conditions of this study.
There were no consistent changes measured in the food consumption during the treatment period between the dose groups. However, there were sporadic differences in food consumption measurements in both sexes compared to the Control group with statistical significance in males only, but these were considered not to be clearly related to the Test Item.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No Test Item related changes compared to pre-treatment or/and the Control were noted at ophthalmoscopy examination at any of the dose levels. All examined animals were found to be normal.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse effects observed on haematology parameters.
Statistical variations in High dose males were minor (<6% difference from the Control) and similar (MCH) or slightly out (MCV) of the historical control range for these parameters. There were no statistical differences see in females (see Table 2).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no Test Item related effects on the clinical chemistry parameters evaluated at the completion of the 90-day treatment period.
In High dose males, urea (approximately -21%), albumin (approximately -5%), in High dose females, creatinine (-15%), Albumin/Globulin Ratio (approximately -14%) reached statistical difference at the terminal evaluation of these parameters. These difference were minor small of magnitude and similar to the historical control range for these parameters, except the creatinine for High and Mid dose females where these values were slightly out of the historical range (see Table 3).
Urinalysis findings:
no effects observed
Description (incidence and severity):
No effects were noted on the urinalysis parameters evaluated, which were considered to be related to the Test Item.
The urinalysis parameters were comparable to the controls in all dose groups. Variations occurred and consisted of minor differences to controls, these were unrelated to treatment and within the normal range.
Urine sediment analysis in the groups showed similar results, no treatment related effect was noted.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In female High dose, the grip strengths of the hind limb were statistically significantly lower (-23.4%), this was considered to Test Item related non-adverse finding.
There was no Test Item related findings in the animal behaviour, general physical condition, in the reactions to different type of stimuli. The quantitative and semiquantitative measures for neurotoxicity (FOB, LMA (locomotor activity, SMART), landing foot splay) were considered to be unaffected by treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative to body weights and brain weight of the kidneys increased statistically significantly both in male and female High dose animals and was considered as Test Item-related. Based on the histopathology examination, the increase of female’s kidney weights was supported by a non-adverse Test Item-related increased vacuolation of tubular cells although there was no histopathologic change in males. The kidney weight differences were considered as not being adverse.
The relative to body organ weight of the pituitary in the High dose males and the relative to body organ weight of the ovary in the High dose females increased and they were statistically significant, but without microscopic changes. They were not considered to be related to treatment (see Table 4).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No systemic effect Test Item-related macroscopic findings were seen in the found dead experimental rats. In the both animals the lungs were dark red/ mottled and noncollapsed.
In the female, reddish foamy material was present in the trachea. The thymus was seen with dark red foci at necropsy.
Test item-related focal/multifocal/diffuse thickness of the non-glandular gastric mucosa was observed in both the High dose males (6/9) and females (9/9) and in 2/10 Mid dose males. Additionally, dark red depressed area was seen in 1/10 High dose female. A few white raised area of the non-glandular gastric mucosa seen in 1/10 Low dose female were not considered to be Test Item-related, since no treatment effects were seen by
light microscopy in Low dose females.
The gastric changes indicate a local irritation-type effect of the Test Item, clearly
observed at the High dose, with some evidence of affected males at the Mid dose. The
gastric observations at the Low dose were not considered to be treatment related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No clearly Test Item-related systemic microscopic effects were noted in the found dead rats. In the male, severe broncho-alveolar pneumonia seen in the lungs was considered as cause of death (congestion was also present) this correlated with a finding of noisy respiration. Agonal pulmonary congestion was present as well. In the liver slight multifocal hepatocellular vacuolation was present, but this may not reflect a systemic effect of the Test Item in this dead animal. In the female the lungs showed agonal congestion, in the thymus moderate congestion/haemorrhage could be seen as incidental.

Test item-related microscopic findings were noted in the stomach (non-glandular) from the High dose females, and High and Mid dose males. In the kidneys, Test Item-related change was seen in High dose females.

Stomach:
Minimal or slight multifocal hyperkeratosis (without epithelial hyperplasia) of the nonglandular stomach mucosa occurred in 7/9 High dose and 2/10 Mid dose males. Minimal to moderate (mainly moderate) hyperkeratosis was seen in 7/9 High dose females. In 2/7 of these High dose females, focal/multifocal erosion/ulcers were observed. These changes were correlated with necropsy findings in affected animals.
Kidney:
In the High dose females, there was Test Item-related increase in severity of the tubular cell vacuolation comparing to Controls (7/10 minimal in the controls; 2/9 slight and 7/9 moderate in the High dose). In the Mid dose females, minimal tubular cell vacuolation was seen in 3/10 cases, hence no Test Item effect was considered. No vacuolation was observed at the Low dose level. In the High dose males, there was no meaningful difference between observed minimal tubular cell vacuolation in 2/9 High dose and 1/10 Control male rats, hence no Test Item-related change was concluded.
Alterations such as tubular basophilia and casts in the kidneys, minimal/slight multifocal, hepatocellular vacuolation, inflammatory cell infiltrate in the prostate,
dilatation tubule in the testes, congestion/haemorrhage in the thymus, broncho-alveolar pneumonia and increased cellularity in the lung-associated lymph nodes (in one High dose female), based on low incidence, the occurrence and/or distribution in control and dosed groups, were considered as incidental or background.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
66.7 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
systemic effetcs
Effect level:
600 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Dose formulation analysis

Samples for Test Item concentration determination of the dosing formulations were collected four times during the treatment period. Formulation stability was established before this study. The Test Item concentration was analysed at the Test Site using an LC-MS method.

 The mean concentration of all formulations were found to be in the range of 95.1-107.6%of their nominal concentrations (13.3, 40 and 120 mg/mL). No Test Item was detected in the vehicle control formulations.Based on these results, Test Item formulations were considered suitable for the study purposes.

 

Table 1 - Summary of the dose formulation analysis:

 

Analytical occasion

(week of sampling)

Nominal concentration(mg/mL)

Measured concentrations with the 95% confidence intervals(mg/mL)

Measured concentration in percentage of the nominal

08 Dec 2017

(week 1)

0

<LOQ

N/A

13.3

12.64

±

0.26

95.1

40

40.37

±

0.74

100.9

120

111.9

±

0.89

93.9

22 Dec 2017

(week 5)

0

<LOQ

N/A

13.3

14.01

±

0.08

105.3

40

43.02

±

0.84

107.5

120

126.1

±

1.08

105.1

15 Jan 2018

(week 9)

0

<LOQ

N/A

13.3

13.40

±

0.42

100.8

40

43.05

±

0.75

107.6

120

128.3

±

2.69

106.9

07 Febr 2018

(week 12)

0

<LOQ

N/A

13.3

13.17

±

0.08

99.0

40

40.04

±

1.52

100.1

120

120.3

±

3.57

100.2

 

Table 2 - Hematology:

 There were no adverse effects observed on haematology parameters.

 Statisticalvariations in High dose males were minor (<6% difference from the Control) and similar (MCH) or slightly out (MCV) of the historical control range for these parameters. There were no statistical differences see in females.

 

 

Dose group (mg/kg bw/day)

Control

66.7

200

600

Males

Mean Cell Volume (flitre)3

52.21

52.01

52.16

54.69dd

differences %

-0.4

-0.1

4.7

Mean Cell Haemoglobin (pg)4

16.13

16.06

16.13

17.04dd

differences %

-0.4

0.0

5.7

 

dd= Dunnett 2 Sided p < 0.01

Table 3- Clinical chemistry:

There were no Test Item related effects on the clinical chemistry parameters evaluated at the completion of the 90-day treatment period.

 

 

Dose group (mg/kg bw/day)

Control

66.7

200

600

Males

Urea (mmol/L)5

7.611

7.011

6.581

6.033dd

differences %

-7.9

-13.5

-20.7

Albumin (g/L)6

31.51

32.24

31.57

29.83u

differences %

2.3

0.2

-5.3

Females

Creatinine (µmol/L)7

48.11

47.24

46.90

40.89d

differences %

-1.8

-2.5

-15.0

A/G ratio8

1.53

1.44

1.45

1.32dd

differences %

-5.9

-5.2

-13.6

u= Dunn-Test 2 Sided p < 0.05

d= Dunnett 2 Sided p < 0.05

dd= Dunnett 2 Sided p < 0.01

Table 4 - Organ weight:

There were Test Item related findings in organ weight data.

The absolute and relative to body weights and brain weight of the kidneys increased statistically significantly both in male and female High dose animals and was considered asTest Item-related. Based on the histopathology examination, the increase of female's kidney weights was supported by a non-adverse Test Item-related increased vacuolation of tubular cells although there was no histopathologic change in males.The kidney weight differences were considered as not being adverse.

 

 

Dose group (mg/kg bw/day)

Control

66.7

200

600

Males

Body weight (g) Day 91 (fasted)

574.5

558.7

585.8

540.6

(differences%)

-2.8

2.0

-5.9

Kidney (absolute g)

3.163

3.256

3.377

3.562dd

(differences%)

2.9

6.8

12.6

Kidney (relative to body weight %)

0.551

0.585

0.579

0.662dd

(differences%)

6.2

5.0

20.0

Kidney (relative to brain weight %)

140.25

145.00

148.19

156.45dd

(differences%)

3.4

5.7

11.5

Females

Body weight (g) Day 91 (fasted)

290.8

296.5

289.0

278.6

(differences%)

2.0

-0.6

-4.2

Kidney (absolute g)

1.785

1.753

1.771

1.950u

(differences%)

-1.8

-0.8

9.2

Kidney (relative to body weight %)

0.617

0.594

0.613

0.701dd

(differences%)

-3.8

-0.7

13.6

Kidney (relative to brain weight %)

85.89

85.76

86.43

94.94u

(differences%)

-0.2

0.6

10.5

u= Dunn-Test 2 Sided p < 0.05

d= Dunnett 2 Sided p < 0.05

dd= Dunnett 2 Sided p < 0.01

Applicant's summary and conclusion

Conclusions:
It is concluded that local irritation in the stomach was an adverse effect, with a local effect NOAEL of (66.7 mg/kg bw/day). However, the systemic NOAEL was considered to be the High dose (600 mg/kg bw/day).
Executive summary:

The purpose of this study was to obtain information on the toxicity of the registered substance when administered daily for 90 days by oral gavage to the rat at 3 dose levels selected in agreement with the Sponsor, based on previous data available, including the results of a 14-day DRF study by oral gavage in rats (Citoxlab study code:17/133-101PE (see section 7.5.1).

Male and female Wistar rats were treated once daily for 90 days by oral gavage administration according to the following Experimental Design:

 

Gr.

No.

 

Group Designation

 

Dose Levels

 

Concentration

Dose volume

 

Animal Numbers

 

 

(mg/kgbw/day)

(mg/mL)

(mL/kg bw)

Male

Female

1

Control

0

0

 

10

10

2

Low Dose

66.7

13.3

5

10

10

3

Mid Dose

200

40

10

10

4

High dose

600

120

 

10

10

The first day of dosing of each animal was regarded as Day 1. Control animals received the vehicle only.

Analysis of Test Item formulations for concentrations were performed and no Test Item was detected in the Control solution samples. All formulations were found to be acceptable for the study purposes.

The Test Item is known to have strong surfactant properties, so the gavage treatment was performed very carefully to try to prevent any Test Item being deposited in the upper oesophagus area, from where it can enter the upper respiratory tract.

During the study, observations for mortality were performed twice a day, clinical signs and detailed clinical observations were also performed at least daily and weekly, respectively. Body weight and food consumption measurements were performed in weekly intervals.

Ophthalmoscopy was conducted before randomisation and in the last week, and neurological assessment including FOB (functional observation battery), locomotor activity (SMART), landing foot splay and grip strength were performed during Week 12 of the treatment.

Clinical pathology examinations (haematology, clinical chemistry, coagulation and urinalysis) were conducted prior to necropsy on all surviving animals on Day 91

followed by necropsy with macroscopic examination and selected organ weight measurements. Full histopathology was performed for all Control and High dose

animals, on lesions from other animals found at necropsy and on the kidney and stomach from the Low and Mid dose groups.

Results:

Two High dose animals (one male on Day 32 and one female on Day 57) died during the study. Severe broncho-alveolar pneumonia was considered to be cause of death for the male.

The macroscopic and microscopic findings did not indicate a specific cause of death for the female. There were Test Item related clinical signs recorded during the study in Low, Mid and High doses (noisy respiration), these were considered to reflect a local irritation effect, and not adverse in the context of a systemic toxicity study. There were no significant adverse effects noted on body weight or body weight gain, food consumption, neurological assessment however, in female High dose, the grip strengths of the hind limb were statistically significantly lower (-23.4%), this was considered to Test Item related non-adverse finding. There were no significant adverse effects noted on ophthalmoscopy or oestrus cycle, in the animal behaviour, general physical condition, in the reactions to different type of stimuli, in haematology, clinical chemistry or urinalysis parameters.

The absolute and relative to body weights and brain weight of the kidneys increased statistically significantly both in male and female High dose animals and was

considered as Test Item-related. Based on the histopathology examination, the increase of female’s kidney weights was supported by a non-adverse Test Item-related increased vacuolation of tubular cells although there was no histopathologic change in males. The kidney weight differences were considered as not being adverse.

Gastric irritation was observed as a local adverse effect in High dose males and females and a few Mid dose males. Test item-related focal/multifocal/diffuse thickness of the non-glandular gastric mucosa (minimal to moderate hyperkeratosis) was observed in both sexes at the High dose and in 2/10 Mid dose males. Additionally, a dark red depressed area was seen in 1/10 High dose females. In 2/7 High dose females, focal/multifocal erosion/ulcers were observed. These changes were considered as local irritation effects of the Test Item.

An increase in severity of the tubular cell vacuolation when compared to controls was observed in High dose females. No Test Item-related increase vacuolation was seen in High dose males or in Mid or Low dose females. The increase of vacuolation in High dose females was not associated with any degenerative or inflammatory responses, hence considered to be non-adverse.

In conclusion, under the conditions of this study, the oral administration of C8-18 alkylamidopropyl hydroxysultaine to Crl:WI Wistar Rats for 90 days caused no effects in body weight, food intake, neurological assessment, ophthalmology, haematology, clinical chemistry or urine parameters. Local irritation effects were observed at all dose levels, with pulmonary symptoms, probably caused by minor reflux into the respiratory tract. In the most severe cases, mortality occurred in 2 High dose rats. The pulmonary changes were considered to reflect a local irritation effect of this surfactant substance, and not adverse in the context of a systemic toxicity study.

The High dose groups of both sexes showed signs of gastric irritation with multifocal hyperkeratosis of the non-glandular mucosa (not accompanied with epithelial

hyperplasia). A similar effect was observed in some Mid dose males. In a few High dose females, the gastric changes included focal/multifocal erosion/ulcers. These changes were considered as local irritation effects of the Test Item.

Increased kidney weights in both sexes at the High dose and an increased incidence and severity of the tubular cell vacuolation in the kidneys in High dose females (not in males or Mid dose females) was observed. The histopathology changes were not associated with any degenerative or inflammatory responses; hence the renal observations were considered to be non-adverse.

It is concluded that local irritation in the stomach was an adverse effect, with a local effect NOAEL of (66.7 mg/kg bw/day). However, the systemic NOAEL was considered to be the High dose (600 mg/kg bw/day).