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Administrative data

Description of key information

Following daily oral administration (by gavage) of the structural analogue C8-18 Cocamidopropyl hydroxysultaine, at dose levels of 0, 66.7, 200 and 600 mg/kg bw/day, to Wistar rats (10/sex/dose), for 90 days in a GLP OECD 408-compliant study, the NOAEL for systemic toxicity was considered to be 600 mg/kg bw/day (highest dose tested) based on the absence of systemic effects and the NOAEL for local effects was set at 66.7 mg/kg bw/day based on local irritation in the stomach considered to be due to the surfactant properties of the substance. 
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    

Followingdaily oral administration (by gavage)of C8-18 alkylamidopropyl hydroxysultaineat dose levels of 0, 66.7, 200 and 600 mg/kg/day to Crl:WI Wistar Rats(10/gender/dose) for 90 days in a GLP OECD 408-compliant study, the NOAEL for systemic toxicity was considered to be 600 mg/kg bw/day (highest dose tested) based on the absence of systemic effects and the NOAEL for local effects was set at 66.7 mg/kg bw/day based on local irritation in the stomach considered to be due to surfactant properties of the registered substance.

   

Followingdaily oral administration (by gavage)of C8-18 alkylamidopropyl hydroxysultaineat dose levels of 0, 66.7, 200 and 600 mg/kg/day to Crl:WI Wistar Rats(10/gender/dose) for 90 days in a GLP OECD 408-compliant study, the NOAEL for systemic toxicity was considered to be 600 mg/kg bw/day (highest dose tested) based on the absence of systemic effects and the NOAEL for local effects was set at 66.7 mg/kg bw/day based on local irritation in the stomach considered to be due to surfactant properties of the registered substance.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
April - December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Complementary in vivo micronucleus phase added
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation:
. Main study: 9 (females) - 10 (males) weeks old
. Micronucleus phase: 15 weeks old
- Weight at study initiation:
. Main study: 216 g (females) - 392 g (males)
. Micronucleus phase: 277 g (females) - 498 g (males)
- Fasting period before study: No
- Housing: Individual (except during pairing) in polycarbonate 940 cm² cages with stainless stell lids and autoclaved dust
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days (main study) / 7 days (micronucleus phase) before dosing initiation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 50 +/- 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 10 May 2012 To: 20 July 2012
Route of administration:
oral: gavage
Vehicle:
other: drinking water treated by reverse osmosis
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS (main study):
- The test item was administered as a solution in the vehicle, by mixing with the required quantity of vehicle.
- The dose formulations were prepared daily.

VEHICLE
- Concentration in vehicle: The concentration of the test item in samples of each control and test item dose formulation prepared for use in weeks 1, 3, 5 and 7 was determined.
- Administration volume: 5 mL/kg/day (main study)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of the test item in the dose formulations were quantified using a validated analytical method.
The validation of the analytical method was conducted in CiToxLAB France (Study No. 38714 VAA) and precise details concerning the checked parameters, acceptance criteria and obtained results were documented in the corresponding validation report.
Duration of treatment / exposure:
The dose formulations were administered daily according to the following schedule (Day 1 corresponding to the first day of the treatment period):

. In the males:
- 2 weeks before pairing (from study day 1 to 14),
- during the pairing period (3 weeks, from study day 15 until study day 16 to 29),
- until sacrifice (at least 5 weeks in total, from study day 17 to 30 until study day 36).

. In the females:
- 2 weeks before pairing (from study days 1 to 14),
- during the pairing period (3 weeks, from study days 15 to 29),
- during gestation (from study days 16 to 30 until study days 36 to 50),
- during lactation until day 5 post-partum inclusive (from study days 37 to 51 until study days 42 to 56),
- until sacrifice for the non-pregnant females (at least 6 weeks in total, approximately, until study day 41 to day 45).
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0, 30, 100, 300 mg/kg bw/day
Basis:
other: nominal dose levels (main study)
Remarks:
Doses / Concentrations:
0, 6, 20, 60 mg/mL
Basis:
other: nominal concentrations (main study)
No. of animals per sex per dose:
10 (main study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:

In a previous study (CiToxLAB France/Study No. 38719 TSR), Cocamidopropyl hydroxysultaine (batch No. HG04423) was given to rats (three/sex/group), by daily oral administration (gavage) for 2 weeks at 0, 250, 500 or 1000 mg/kg/day.

At 1000 mg/kg/day, 2/3 males and 2/3 females were found dead on study day 4 and 7, respectively. Abdominal breathing, hunched back, loud breathing, soiled anal/urogenital area, dyspnea, emaciated appearance and/or piloerection preceded the deaths. The surviving male had a marked decrease in body weight (-22.2% vs. controls) on study day 14. In females, there was no evidence of any effect on body weight. Food consumption was severely reduced during the first week of the treatment period (-61.4% in males and -43.2% in females vs. controls, respectively).
At 500 mg/kg/day, there were no unscheduled deaths. Marked clinical signs (hunched back, emaciated appearance or loud breathing) were recorded in males only. All animals (males and females) had ptyalism. A moderate decrease in body weight was observed in males only on study day 14 (-13.2% vs. controls). Food consumption was markedly reduced during all the treatment period in males and during the first week of treatment in females (down to -24.2% in males and 16.8% in females vs. controls, respectively).
At 250 mg/kg/day, there was ptyalism in 1/3 males and in 3/3 females (from study day 12 in males and from study day 4 in females). There were minimal effects on mean body weight (down to -8.3% vs. controls on study day 14 in males) or mean food consumption (down to -9.9% vs.; controls, on period of days 1-8 in males).
At necropsy, black or red discolorations and/or thickened appearance were observed in the forestomach of animals found dead at 1000 mg/kg/day. There were brown or white discolorations and/or thickened forestomach in the animals sacrificed at 500 mg/kg/day. There were no obvious treatment related macroscopic findings at 250 mg/kg/day.

Overall, 500 and 1000 mg/kg/day were considered to be excessive dose-levels, based on in-life and pathology data. Therefore, 300 mg/kg/day was selected as the high dose-level. The low-dose and mid-dose were selected using a ratio representing a 3-fold interval (i.e. 30 and 100 mg/kg/day).

- Rationale for animal assignment (if not random): The animals were allocated to groups (by sex) using a computerized stratification procedure based on body weight, so that the average body weight of each group was similar.
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Each animal was checked for mortality or signs of morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays. From arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed at least once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical examinations were performed on all animals outside the home cage, in a standard arena, once before the beginning of the treatment period and then once a week until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
Main study:
The body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum (p.c.) and days 1 and 5 p.p..
Micronucleus phase:
The body weight of each animal was recorded once before group allocation and on the day of administration of CPA (day 1), in order to adjust the quantity of dose formulation to be given the most appropriately.

FOOD CONSUMPTION:
The quantity of food consumed by each male was measured once a week, over a 7 day period, from the first day of treatment until the start of the pairing period.
The quantity of food consumed by each female was measured once a week, over a 7 day period, from the first day of treatment until the start of the pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 post-coitum and during lactation for interval days 1 5 post-partum.
During the pairing period, the food consumption was measured for neither males nor females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.
Food consumption was not recorded for CPA-treated animals.

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: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours.
Blood samples were taken from the orbital sinus of the animals under light isoflurane anesthesia, into tubes containing the appropriate anticoagulant.
The parameters listed in Table 1 below were determined from the first five males and the first five females to deliver from each group on the day of sacrifice.
A blood smear for possible determination of the differential white cell count (with cell morphology) was prepared for each animal and stained with May Grünwald Giemsa. As all the blood samples were successfully analyzed by the ADVIA 120 the blood smears were archived without further investigation.
A blood smear (stained with blue cresyl) for possible determination of the reticulocyte count was prepared for each animal. As all the blood samples were successfully analyzed by the ADVIA 120 the blood smears were archived without further investigation.

CLINICAL CHEMISTRY: Yes
The parameters listed in Table 2 below were determined from the first five males and the first five females to deliver from each group on the day of sacrifice.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional Observation Battery:
The first five males and the first five females to deliver from each group were evaluated once at the end of the treatment period. For females, this was performed on day 5 post partum after sacrifice of the pups.
This included a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity.
The animals were not randomized in order to ensure "blind" evaluation.
All animals were observed in the cage, in the hand and in the standard arena.
The following parameters were assessed and graded:
- "touch escape" or ease of removal from the cage,
- in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.
Reactivity to manipulation or to different stimuli:
The following parameter measurements, reflexes and responses were recorded: touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, at the end of observation: rectal temperature.
Motor activity:
Finally, motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period.
Sacrifice and pathology:
ORGAN WEIGHT: Yes
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

See Tissue Procedure Table below.

- Sacrifice:

On completion of the treatment period, after at least 14 hours fasting, all males and females were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.
Males: after the end of the pairing period (at least 5 weeks of treatment in total),
Females: on day 6 post partum.
The following females were sacrificed by the same way without overnight fasting:
Females which did not deliver: on day 25 post coitum (after a body weight recording to check for a possible un-noticed delivery) except for female Y23122 (300 mg/kg) which was sacrificed on day 23 post-coitum.
Pups were sacrificed by an intraperitoneal injection of sodium pentobarbital.
Surviving pups: on day 5 post partum.

- Animals prematurely sacrificed or found dead:
. Males
The male found dead Y23056 (300 mg/kg) was submitted to a macroscopic post-mortem examination of the principal thoracic and abdominal organs.
No other male was prematurely sacrificed or found dead during the study period.
. Females
No females were prematurely sacrificed or found dead during the study period.
. Pups
Any pup was prematurely sacrificed. A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all found dead pups. Special attention was paid to whether the pup has fed (e.g. presence of milk in the stomach). No tissues were preserved.

- Organ weights (parental animals):
The body weight of each animal sacrificed as scheduled was recorded before sacrifice, and the organs specified in the Tissue Procedure Table below were weighed (wet) as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

- Macroscopic post-mortem examination:
. Parent animals
A complete macroscopic post-mortem examination was performed on all parent animals including the male Y23056 (300 mg/kg) found dead during the study. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. The numbers of corpora lutea and implantation sites were also recorded for females sacrificed as scheduled on day 6 post-partum.
The numbers of corpora lutea and implantation sites were recorded for females sacrificed on day 25 post-coitum or day 23 post-coitum for female Y23122 (300 mg/kg) due to the absence of delivery. For apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.
. Pup examinations
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups showing relevant external abnormalities. No tissues were preserved.

- Preservation of tissues:
The tissues specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except for the testes and epididymides which were fixed in Davidson's fixative).
Two bone marrow smears for micronucleus analysis (see § Other examinations) were prepared from the left femur of each animal sacrificed on completion of the treatment period (first five principal animals in sultaine-treated groups, all CPA-treated animals).

- Preparation of histological slides:
All tissues required for microscopic examination were trimmed based on the RITA guidelines, embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS). This tissue processing was performed at CiToxLAB France.

- Microscopic examination:
A microscopic examination was performed on:
. all tissues listed in the Tissue Procedure Table below from the first five sacrificed as scheduled males and the first five females to deliver and be sacrificed on day 6 post-partum of the control and high-dose groups (0 and 300 mg/kg) and for the male that died,
. stomach, forestomach, kidneys, lungs and trachea from the first five sacrificed as scheduled males and the first five females to deliver and be sacrificed on day 6 post-partum of the low- and mid dose groups (30 and 100 mg/kg),
. all macroscopic lesions of all the animals of the low- and intermediate-dose groups (30 and 100 mg/kg) sacrificed on completion of the treatment period,
. all females sacrificed because of no delivery to investigate possible causes.
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Statistics:
Yes (parametric + non-parametric tests)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Loud breathing and ptyalism at 300 mg/kg
Mortality:
mortality observed, treatment-related
Description (incidence):
Loud breathing and ptyalism at 300 mg/kg
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decrease in bodyweight gain of females at 300 mg/kg during premating period. Statistically significant decrease in bodyweight of females at 300 mg/kg during gestation and lactation periods.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg: forestomach squamous cell hyperplasia, pulmonary bronchioalveolar inflammation and tracheal epithelial alteration, minimal to slight degeneration/hypertrophy of tubular epithelium or minimal tubular vacuolation in kidneys.
Histopathological findings: neoplastic:
no effects observed
Details on results:
The test item concentrations in the administered dose formulations analyzed in weeks 1, 3, 5 and 7 remained within an acceptable range of -7.0% to +4.5% when compared to the nominal values, except for the 100 mg/kg group analyzed in week 5 found at -16.0% and 100 and 300 mg/kg groups analyzed in week 7 found respectively at -11.7% and -12.3%. Cocamidopropyl hydroxysultaine was not detected in control samples.

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths in control, 30 and 100 mg/kg/day groups.
In the 300 mg/kg/day group, on male was found dead on study day 34. At macroscopic post mortem examination, there were enlargment of lungs (with presence of red discoloration) and white discoloration and irregular surface of the wall of stomach. At microscopic examination, the cause of death was moderate subacute bronchioalveolar inflammation, most likely secondary to aspiration of the test item after regurgitation at dosing.
At 300 mg/kg/day, loud breathing was recorded during the period of days 17 to 19 in one male, during all the pregnancy period in one female and at the end of the lactation period in another one. This clinical sign was considered to be related to the treatment with the test item and of toxicological significance. Ptyalism was considered to be related to the test item but of minor toxicological importance.

BODY WEIGHT AND WEIGHT GAIN
In males, there were no effects on mean body weight or mean body weight gain.
In females, at 300 mg/kg/day, there was a dose-related decrease in mean body weight gain (-37% vs. controls, p< 0.05) during the premating period and decreases in mean body weight during the pregnancy and lactation periods (-7% vs. controls on day 0 post-coitum, p< 0.05 and -8% on day 5 post-partum, p< 0.05) related with a non-statistically significant decrease in mean body weight gain ( 29% vs. controls) during the lactation period (days 1-5 post-partum). All these finding were considered to be treatment-related.

FOOD CONSUMPTION
There were no effects on mean food consumption during the premating, mating, gestation or lactation period.

HAEMATOLOGY
There were no effects on the hematological parameters.

CLINICAL CHEMISTRY
A few isolated findings were considered to be of no toxicological significance.

NEUROBEHAVIOUR
In the Functional Observation Battery, there were no findings considered to have obvious biological or toxicological significance. During the motor activity assessments, there were no relevant differences in rearing in treated group animals when compared with control animals. There was a trend towards a decrease in the number of horizontal movements in females. However, in the absence of associated clinical signs, in the absence of effects in males and taking into account variability across groups, a treatment-related effect was considered unlikely.

ORGAN WEIGHTS AND GROSS PATHOLOGY
There were no organ weight or macroscopic changes attributed to the test item.

HISTOPATHOLOGY
The test item administration at the highest dose-level induced microscopic changes in the stomach, lungs, trachea and kidneys. In the forestomach, squamous cell hyperplasia was most likely due to irritant properties of the test item. Pulmonary bronchioalveolar inflammation and tracheal epithelial alteration were thought to be related to aspiration of compound after regurgitation at dosing. In the kidneys, there were minimal to slight degeneration/hypertrophy of the tubular epithelium, principally in males, and minimal tubular vacuolation in some females.
At 100 mg/kg/day, there was only minimal epithelial alteration in the trachea from a single male, which was not considered as adverse in view of its low incidence and magnitude. There were no microscopic findings in the stomach, forestomach, kidneys or lungs.
There were no pathological findings at 30 mg/kg/day.
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 100 mg/kg/day based on microscopic findings in the forestomach, lungs and kidneys of animals given 300 mg/kg/day.
Executive summary:

The subacute toxicity of Cocamidopropyl hydroxysultaine was tested in an OECD 422 compliant study following daily oral administration (by gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until day 5 post‑partum (p.p.) inclusive.

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Cocamidopropyl hydroxysultaine, as a 36.2% aqueous solution, daily, by oral administration (gavage), over the administration period, at dose‑levels of 30, 100 or 300 mg/kg/day.An additional group of 10 males and 10 females received the vehicle control, drinking water, under the same experimental conditions. The dosing volume was 5 mL/kg/day.

 

Animals were checked daily for clinical signs, mortality, and detailed clinical observations were conducted weekly. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. A Functional Observation Battery including touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature and motor activity was performed on five males and females per group at the end of the study. Prior to sacrifice, blood samples were also taken from these animals for analysis of hematology and blood biochemistry parameters.

 

The males were sacrificed after completion of the mating period and dams were sacrificed on day 6 p.p.. Body weights and selected organs weights were recorded and a complete macroscopicpost-mortemexamination performed, with particular attention paid to the reproductive organs. A microscopic examination was also conducted on selected organs from the first five animals in the control groups and the high-dose groups. Microscopic examination was conducted on all macroscopic lesions from all groups.

Based upon the microscopic results of the high-dose group, stomach, forestomach, kidneys, lungs and trachea of the first five animals of the low- and intermediate-dose groups were also examined. Pups, including those found dead before study termination, were also submitted for a macroscopic post-mortem examination.

 

The test item concentrations in the administered dose formulations analyzed in weeks 1, 3, 5 and 7 remained within an acceptable range of -7.0% to +4.5% when compared to the nominal values, except for group 3 analyzed in week 5 found at -16.0% and groups 3 and 4 analyzed in week 7 found respectively at -11.7% and -12.3%. When compared with the nominal values (±10%), these variations were of small amplitude and therefore to considered to have no impact on the integrity of the study. Cocamidopropyl hydroxysultaine was not detected in control samples.

With regards to repeated-dose toxicity parameters, the following observations were made:

Mortality

There were no unscheduled deaths in control, 30 and 100 mg/kg/day groups.

In the 300 mg/kg/day group, one male was found dead on study day 34. At macroscopic post‑mortem examination, there were enlargment of lungs (with presence of red discoloration) and white discoloration and irregular surface of the wall of stomach. At microscopic examination, the cause of death was moderate subacute bronchioalveolar inflammation, most likely secondary to aspiration of the test item after regurgitation at dosing. This mortality was not considered incidental but attributed to the test item.

 

Clinical signs

At 300 mg/kg/day, loud breathing was recorded during the period of days 17 to19 in one male, during all the pregnancy period in one female and at the end of the lactation period in another one. This clinical sign was considered to be related to the treatment with the test item and of toxicological significance. Ptyalism, frequently observed in most animals given 300 mg/kg/day, was considered to be related to the test item but of minor toxicological importance.

 

Functional Observation Battery

There were no findings considered to have obvious biological or toxicological significance.

 

Motor activity

There were no relevant differences in rearing in treated group animals when compared with control animals.

There was a trend towards a decrease in the number of horizontal movements in females. However, in the absence of associated clinical signs, in the absence of effects in males and taking into account variability across groups, a treatment-related effect was excluded.

 

Body weight and body weight change

In males, there were no effects on mean body weight or mean body weight gain.

In females, there was a dose-related decrease in mean body weight gain (-37% at 300 mg/kg/day vs. controls, p< 0.05) during the premating period and decreases in mean body weight during the pregnancy and lactation periods (-7%vs.controls on day 0p.c., p<0.05and -8% on day 5 p.p., p< 0.05, at 300 mg/kg/day) which was associated with a non-statistically significant decrease in mean body weight gain (‑29%vs. controls at 300 mg/kg/day) during the lactation period (days 1-5p.p.). All these finding were considered to be treatment-related.

 

Food consumption

There were no effects on mean food consumption during the premating, mating, gestation or lactation period.

 

Hematology

There were no effects on the hematological parameters.

 

Blood biochemistry

A few isolated findings were considered to be of non toxicological significance.

Pathology

There were no organ weight or macroscopic changes attributed to the test item.

The test item administration at the highest dose-level induced microscopic changes in the stomach, lungs, trachea and kidneys.In the forestomach, squamous cell hyperplasia was most likely due to irritant properties of the test item. Pulmonary bronchioalveolar inflammation and tracheal epithelial alteration were thought to be related to aspiration of compound after regurgitation at dosing. In the kidneys, there were minimal to slight degeneration/hypertrophy of the tubular epithelium, principally in males, and minimal tubular vacuolation in some females.

At 100 mg/kg/day, there was only minimal epithelial alteration in the trachea from a single male, which was not considered as adverse in view of its low incidence and magnitude. There were no microscopic findings in the stomach, forestomach, kidneys or lungs.

In conclusion, based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 100 mg/kg/day based on microscopic findings in the forestomach, lungs, trachea and kidneys of animals given 300 mg/kg/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
C8-C18 AAPHS and C12-18 AAPHS have the same functional groups, and general composition. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3)
Target chemical = C12-18 cocamidopropyl hydroxysultaine (EC 939-457-4)

3. ANALOGUE APPROACH JUSTIFICATION
The alkyl C-chain distribution of the source chemical significantly overlaps with the one of the target chemical, the C12-alkyl derivative being the major constituent in both chemicals. The structural differences in side C-chains is not expected to lead to significant differences on phys-chem properties.

4. DATA MATRIX: see "Documentation and scientific justification of the read-across approach" in section 13.2
Reason / purpose:
read-across source
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Conclusions:
Under the experimental conditions of the OECD 422 study performed on C8-18 Cocoamidopropyl hydroxysultaine, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 100 mg/kg/day based on microscopic findings in the forestomach, lungs and kidneys of animals given 300 mg/kg/day. By analogy, similar effects are expected for C12-18 Cocoamidopropyl hydroxysultaine.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
C8-C18 AAPHS and C12-18 AAPHS have the same functional groups, and general composition. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3)
Target chemical = C12-18 cocamidopropyl hydroxysultaine (EC 939-457-4)

3. ANALOGUE APPROACH JUSTIFICATION
The alkyl C-chain distribution of the source chemical significantly overlaps with the one of the target chemical, the C12-alkyl derivative being the major constituent in both chemicals. The structural differences in side C-chains is not expected to lead to significant differences on phys-chem properties.

4. DATA MATRIX: see "Documentation and scientific justification of the read-across approach" in section 13.2
Reason / purpose:
read-across source
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:
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
Conclusions:
In conclusion, under the conditions of this study, after 14 days of oral gavage treatment at 600 mg/kg bw/day (High dose) with C8-18 Cocoamidopropyl hydroxysultaine, 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). By analogy similar effects are expected with C12-18 Cocoamidopropyl hydroxysultaine.
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
C8-C18 AAPHS and C12-18 AAPHS have the same functional groups, and general composition. The main variable resides in the alkyl chain distribution.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical = C8-18 cocamidopropyl hydroxysultaine (EC 939-455-3)
Target chemical = C12-18 cocamidopropyl hydroxysultaine (EC 939-457-4)

3. ANALOGUE APPROACH JUSTIFICATION
The alkyl C-chain distribution of the source chemical significantly overlaps with the one of the target chemical, the C12-alkyl derivative being the major constituent in both chemicals. The structural differences in side C-chains is not expected to lead to significant differences on phys-chem properties.

4. DATA MATRIX: see "Documentation and scientific justification of the read-across approach" in section 13.2
Reason / purpose:
read-across source
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:
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:
no
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Conclusions:
In the 90-day study performed on C8-18 Cocoamidopropyl hydroxysultaine, 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). By analogy, similar effects are expected with C12-18 Cocoamidopropyl hydroxysultaine.
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.

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
Reason / purpose:
reference to other study
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
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:
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
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
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
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.
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
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
Critical effects observed:
not specified

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

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).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP and OECD 408 compliant study (Klimisch 1)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

No systemic toxicity was reported up to the 600 mg/kg bw/day (highest dose tested) in the 90 -day repeated dose toxicity study. Only local irritation in the stomach of the rats was reported from the dose of 200 mg/kg bw/day. This effect is not expected to occur in human as the registered substance is not expected to be intentionnaly swallowed.

Additional information

As the repeated dose toxicity of the substance EC 939-457-4 has not been investigated experimentally, a read-across approach was followed to fill in the endpoint based on studies conducted on the structural analogue substance C8 -18 cocamidopropyl hydroxysultaine (= source substance EC 939-455-3).

In a GLP OECD 408-compliant study (Key study), Cocamidopropyl hydroxysultaine was administered daily by oral gavage for 90 consecutive days to Wistar rats (10/sex/dose) at the dose levels of 66.7, 100 or 600 mg/kw bw/day. Control animals (10/sex) received the vehicle only (distilled water).

Two High dose animals died during the study. Severe broncho-alveolar pneumonia was considered to be cause of death one animal. The macroscopic an d microscopic findings did not indicate a specific cause of death for the other dead animal.

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 Highdose 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 benon-adverse.

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

 

These results were confirmed by the results of the Dose Range Finding study (selected as supporting study) aiming at determining dose levels for the above 90-day study. In this study the Cocamidopropyl hydroxusultaine was administered daily by oral gavage to Wistar rats at three dose levels (150, 300 and 600 mg/kg bw/day) for 14 consecutive days.

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.

It was concluded that, 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).

 

Prior to the above studies, the subacute toxicity of Cocamidopropyl hydroxysultaine, as a 36.2% aqueous solution, was tested at doselevels of 30, 100 or 300 mg/kg/day (5 mL/kg) in an OECD 422 compliant study following daily oral administration (by gavage) to male and female Sprague-Dawley rats (10 per gender and per dose) from before mating, during mating and, for the females, throughout gestation until day 5 postpartum (p.p.) inclusive (selected as supporting study)

In this study, one male given 300 mg/kg/day was found dead on day 34. At macroscopic post-mortem examination, enlargment of lungs (with red discoloration) and white discoloration and irregular surface of the stomach wall were observed. Histopathology showed that death was associated with moderate subacute bronchioalveolar inflammation, most likely secondary to aspiration of the test item after regurgitation at dosing. At 300 mg/kg/day, loud breathing was recorded over days 17 to 19 in one male, during all the pregnancy period in one female and at the end of the lactation period in another female. Ptyalism was also frequently observed in most animals given 300 mg/kg/day. In females given 300 mg/kg/day, there was a dose-related decrease in mean body weight gain during the premating period and decreases in mean body weight during the pregnancy and lactation periods which were associated with a non-statistically significant decrease in mean body weight gain during the lactation period, with no correlates in food intake. At scheduled euthanasia, no organ weight or macroscopic changes were noted. Histopathology evidenced microscopic changes in the stomach, lungs, trachea and kidneys. In the forestomach, squamous cell hyperplasia was most likely due to irritant properties of the test item. Pulmonary bronchioalveolar inflammation and tracheal epithelial alteration were thought to be related to aspiration of compound after regurgitation at dosing. In the kidneys, there were minimal to slight degeneration/hypertrophy of the tubular epithelium, principally in males, and minimal tubular vacuolation in some females. Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 100 mg/kg/day based on microscopic findings in the forestomach, lungs, trachea and kidneys of animals given 300 mg/kg/day.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The 90-day study was selected as it was the study with the longuest exposure period among the 3 available repeated-dose toxicity studies and no study by other routes of exposure was performed.

Justification for classification or non-classification

In accordance with Regulation (EC) No. 1272/2008 (CLP) criteria, no classification is warranted with regards to Specific Target Organ Toxicity following Repeated Exposure (STOT-RE) because no systemic toxicity was reported up to the highest dose tested (NOAEL = 600 mg/kg bw/day) in a 90 -day repeated dose toxicity study in rats conducted in the structural analogue Cocamidopropyl hydroxysultaine. Only local irritation in the stomach was reported from the dose of 200 mg/kg bw/day due to the surfactant properties of the substance. This local effect is not expected to occur in human as the registed substance is not expected to be swallowed.