1-Propanaminium, N-(3-aminopropyl)-2-hydroxy-N,N-dimethyl-3-sulfo-, N-(C8-18(even numbered) acyl) derivs., hydroxides, inner salts

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Following daily oral administration (by gavage) of Cocamidopropyl hydroxysultaine at dose levels of 0, 30, 100 or 300 mg/kg/day to Sprague-Dawley rats (10/gender/dose) for at least 5 weeks of dosing in a GLP OECD 422-compliant study, the NOEL for reproductive performance and the NOEL for toxic effects on progeny were both set at 300 mg/kg/day in the absence of any treatment-related effects at this dose-level.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

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. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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: 216 g (females) - 392 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 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) / 10 mL/kg (cyclophosphamide-treated group in micronucleus phase)

Details on mating procedure:

Females were paired with males from the same dose-level group. One female was placed with one male, in the latter's cage, during the night. Sibling pairings were avoided.
Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage.
The day of confirmed mating was designated day 0 post-coitum.
Each female was placed with the same male until mating occurs or 14 days have elapsed. One pair in group 1 with no evidence of mating after 13 days was separated and the female was placed for a further 7 days with a different male from the same dose-level group who has already mated.
The pre-coital time was calculated for each female.

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until the females are mated.

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 week in total, approximately, until study day 41 to day 45).

Frequency of treatment:

Once daily

Details on study schedule:

Age at mating of the mated animals in the study: 11 (females) or 12 (males) weeks

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

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.

Positive control:

No positive control group included in main study.

Parental animals: Observations and examinations:

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..

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.

OTHER:

HAEMATOLOGY:
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:
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.

NEUROBEHAVIOURAL EXAMINATION:
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.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until the females are mated.

Sperm parameters (parental animals):

Testes and epididimydes were weighed in all males at necropsy.
At microscopic examination, special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Litter size:
The total litter size and numbers of pups of each sex was recorded as soon as possible after birth. Any gross malformations in pups were noted.
The litters were observed daily in order to note the number of live, dead and cannibalized pups.
- Clinical signs:
The pups were observed daily for clinical signs or abnormal behavior.
- Body weight:
The body weight of each pup was recorded on days 1 and 5 post-partum.

GROSS EXAMINATION OF DEAD PUPS:
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups found dead. Special attention was paid to whether the pup had fed (e.g. presence of milk in the stomach). No tissues were preserved.

Postmortem examinations (parental animals):

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.

- 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.

GROSS NECROPSY
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.

HISTOPATHOLOGY / ORGAN WEIGHTS
See Tissue Procedure Table below.
- 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.
- 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).
- 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.

Postmortem examinations (offspring):

SACRIFICE
- Pups were sacrificed on day 5 post-partum by an intraperitoneal injection of sodium pentobarbital.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups showing relevant external abnormalities. Special attention was paid to whether the pup had fed (e.g. presence of milk in the stomach). No tissues were preserved.

GROSS NECROPSY
- Gross necropsy consisted of principal thoracic and abdominal organs.

Statistics:

Yes (parametric + non-parametric tests)

Reproductive indices:

The following parameters were calculated:
- pre-implantation loss: Number of corpora lutea - Number of implantation sites / Number of corpora lutea x 100
- post-implantation loss (manually calculated): (Number of implantation sites - Number of live pups) / Number of implantations x 100
- mating index: Number of mated animals / Number of paired animals x 100
- fertility index: Number of pregnant female partners / Number of mated pairs x 100
- gestation index: Number of females with live born pups / Number of pregnant females x 100

Offspring viability indices:

The following parameters were calculated:
- live birth index: Number of live born pups / Number of delivered pups x 100
- viability index on day 4 post-partum: Number of surviving pups on day 4 post-partum / Number of live born pups x 100
- lactation index on day 5 post-partum: Number of surviving pups on day 5 post-partum / Number of surviving pups on day 4 post-partum x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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):

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.

Organ weight findings including organ / body weight ratios:

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.

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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%. Cocamidopropyl hydroxysultaine was not detected in control samples.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 FOOD CONSUMPTION (PARENTAL ANIMALS)
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 p.c., p< 0.05 and -8% on day 5 p.p., p< 0.05) which resulted in a non-statistically significant decrease in mean body weight gain (-29% vs. controls) during the lactation period (days 1-5 p.p.). All these finding were considered to be related to the test item treatment.
There were no effects on mean food consumption during the premating, mating, gestation or lactation period.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no effects on the estrous cycle parameters.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no effects on the sperm parameters, as detected by testes and epididymides weight and microscopic examination.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no treatment-related effects on mating and fertility data.
All animals mated within comparable mean number of days.
There were no relevant differences between control and test item-treated groups (mean duration of gestation, mean number of corpora lutea, mean number of implantations, mean number of pups delivered, mean pre-implantation loss and mean post-implantation loss).

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

HISTOPATHOLOGY (PARENTAL ANIMALS)
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:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: absence of any treatment-related effect on mating and fertility

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
There were no treatment-related effects on the incidences of pups found dead or cannibalized.
There were no treatment-related effects on live birth, viability and lactation indexes.

CLINICAL SIGNS (OFFSPRING)
There were no treatment-related clinical signs.

BODY WEIGHT (OFFSPRING)
There were no significant effects on mean body weight either in male or in female pups during the lactation period (day 1 or day 5 post-partum).

SEXUAL MATURATION (OFFSPRING)
There were no treatment-related effects on sex-ratios (% of male pups).

ORGAN WEIGHTS (OFFSPRING)
Not determined.

GROSS PATHOLOGY (OFFSPRING)
There were no obvious treatment-related findings at necropsy of pups found dead or pups at scheduled sacrifice.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: absence of any treatment-related effect on pups

Reproductive effects observed:

no

-  Mating and fertility data:

 

Dose-level (mg/kg/day)	0	30	100	300
Number of animals paired (M + F)	10 + 10	10 + 10	10 + 10	10 + 10
Number of males mated	10	10	10	10
Number of females mated	10	10	10	10
Mean number of days taken to mate	3.5	2.5	3.6	2.9
Number of pregnant females	9	9	9	10
Male fertility index (%)	100	100	100	100
Female fertility index (%)	90	90	90	100

 

There were no treatment-related effects on mating and fertility data. All animals mated within comparable mean number of days taken to mate. One pair in the control group did not mate within 2 weeks. The female was then paired with a proven male from the same group and they mated in 2 days. All females were pregnant, except one in each of the 0, 30 and 100 mg/kg/day groups.

 

- Delivery data:

 

Dose-level (mg/kg/day)	0	30	100	300
Number of pregnant females	9	9	9	10
Number of females which delivered	9	9	9	10
Mean duration of gestation (days)	21.2	21.0	21.1	21.5
Mean number ofcorpora lutea	16.4	16.6	16.7	17.8
Mean number of implantations	16.3	15.6	15.7	16.5
Mean pre-implantation loss (%)	0.6	5.9	5.2	6.2
Mean number of pups delivered	14.9	14.9	14.7	16.1
Mean post-implantation loss (%)^a	8.7	4.5	6.1	2.4
Viability index on day 4p.p.(%)	97.8	98.5	98.5	98.1

a: manually calculated, no statistics performed.

There were no relevant differences between control and test item-treated groups (mean duration of gestation, mean number ofcorpora lutea, mean number of implantations, mean number of pups delivered and mean post-implantation loss). There was a trend towards a dose-related increase in mean pre-implantation loss but which never reached statistical significance.

- Pup mortality:

 

Dose-level (mg/kg/day)	0	30	100	300
Number of pups found dead	1	3	1	4
Number of pups cannibalized	2	0	1	0
Number of affected litters	3	2	2	2

 

Overall, there were no treatment-related effects on the incidences of pups found dead or cannibalized.

 

- Clinical signs:

At 100 mg/kg/day, thinning appearance and dehydration were noted in one pup on day 5 post-partum. Other incidental clinical signs observed in pups were hematoma on head and/or scab on tail or on hindlimb.

 

- Pup viability:

 

Dose-level (mg/kg/day)	0	30	100	300
Live birth index (%)	100.0	100.0	100.0	100.0
Viability index (day 4 p.p., %)	97.8	98.5	98.5	98.1
Lactation index (day 5 p.p., %)	100.0	99.2	100.0	99.4

Overall, there were no treatment-related effects on live birth, viability and lactation indexes.

 

- Pup body weight:

 

Sex	Male				Female
Dose-level (mg/kg/day)	0	30	100	300	0	30	100	300
Body weight
. Day 1p.p.	6.8	6.9	6.9	6.9	6.5	6.4	6.5	6.4
. Day 5p.p.	10.9	10.9	10.8	10.3	10.4	10.2	10.4	9.8
Body weight change
. Days 1-5p.p.	+4.1	+4.0	+3.9	+3.5	+3.9	+3.8	+3.9	+3.3

 

There were no effects on mean body weight either in male or in female pups during the lactation period (day 1 or day 5 p.p.) with the exception of a trend towards lower mean body weight changes in the 300 mg/kg/day group but which never reached statistical significance.

- Macroscopic post-mortem examination of pups:

 

Dose-level (mg/kg/day)	0	30	100	300
Dead pups:
Number of pup examined (litter)	1 (1)	3 (2)	1 (1)	4 (2)
Autolysis	0	0	1 (1)	3 (2)
Stomach: absence of milk	1 (1)	1 (1)	0	1 (1)
Total dead pup observations	1 (1)	1 (1)	1 (1)	4 (2)
Scheduled sacrifice pups:
Number of pup examined (litter)	0	0	1 (1)	1 (1)
Stomach: absence of milk	0	0	1 (1)	0
Total pup scheduled sacrifice observations	0	0	1 (1)	0

Overall, there were no obvious treatment-related findings at necropsy of pups found dead or in pups at scheduled sacrifice.

Conclusions:

Based on the experimental conditions of this study:
- the No Observed Effect Level (NOEL) for reproductive performance was considered to be 300 mg/kg/ day in the absence of any treatment-related effect on mating and fertility at this dose-level,
- the NOEL for toxic effects on progeny was considered to be 300 mg/kg/day in the absence of any treatment-related effect on pups at this dose-level.

Executive summary:

The potential effects of Cocamidopropyl hydroxysultaine, as a 36.2% aqueous solution, on reproductive and developmental parameters were assessed 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. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5p.p.. The total litter sizes and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs of toxicity and pup body weights were recorded on days 1 and 5p.p..

 

The males were sacrificed after completion of the mating period and dams were sacrificed on day 6p.p.. Body weights and selected organs weights were recorded and a complete macroscopic post-mortem examination performed, with particular attention paid to the reproductive organs. The femur of the first five principal animals in groups 1 to 4 and all group 5 animals were sampled for bone marrow micronucleus analysis. 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 reproductive and developmental parameters, the following observations were made:

 

Mating and fertility data

There were no treatment-related effects on mating and fertility data. All animals mated within comparable mean number of days.

 

Delivery data

There were no relevant differences between control and test item-treated groups (mean duration of gestation, mean number of corpora lutea, mean number of implantations, mean number of pups delivered,mean pre-implantation lossand mean post-implantation loss).

 

Pups mortality

There were no treatment-related effects on the incidences of pups found dead or cannibalized.

 

Pups clinical signs

There were no treatment-related clinical signs.

 

Pups viability

There were no treatment-related effects on live birth, viability and lactation indexes.

 

Pup body weight

There were no significant effects on mean body weight either in male or in female pups during the lactation period (day 1 or day 5p.p.).

Sex ratio

There were no treatment-related effects on sex-ratios (% of male pups).

Pup pathology

There were no treatment-related findings at necropsy of pups found dead or pups at scheduled sacrifice.

 

In conclusion, based on the experimental conditions of this study:

- the No Observed Effect Level (NOEL) for reproductive performance was considered to be 300 mg/kg/ day in the absence of any treatment-related effect on mating and fertility at this dose-level,

- the NOEL for toxic effects on progeny was considered to be 300 mg/kg/day in the absence of any treatment-related effect on pups at this dose-level.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and OECD 422 compliant study (Klimisch 1)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The potential effects of Cocamidopropyl hydroxysultaine, as a 36.2% aqueous solution, on reproductive and developmental parameters were assessed 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.

 

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. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5p.p.. The total litter sizes and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs of toxicity and pup body weights were recorded on days 1 and 5p.p..

 

The males were sacrificed after completion of the mating period and dams were sacrificed on day 6p.p.. Body weights and selected organs weights were recorded and a complete macroscopicpost-mortemexamination performed, with particular attention paid to the reproductive organs. The femur of the first five principal animals in groups 1 to 4 and all group 5 animals were sampled for bone marrow micronucleus analysis. 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 macroscopicpost-mortemexamination.

With regards to reproductive and developmental parameters, there were no treatment-related effects in any of the parameters studied, regarding mating, fertility, delivery and pup growth or development.

 

In conclusion, the No Observed Effect Level (NOEL) for reproductive performance was considered to be 300 mg/kg/ day in the absence of any treatment-related effect on mating and fertility at this dose-level, and the NOEL for toxic effects on progeny was considered to be 300 mg/kg/day in the absence of any treatment-related effect on pups at this dose-level.

Short description of key information:
Following daily oral administration (by gavage) of Cocamidopropyl hydroxysultaine at dose levels of 0, 30, 100 or 300 mg/kg/day to Sprague-Dawley rats (10/gender/dose) for at least 5 weeks of dosing in a GLP OECD 422-compliant study, the NOEL for reproductive performance and the NOEL for toxic effects on progeny were both set at 300 mg/kg/day in the absence of any treatment-related effects at this dose-level.

Justification for selection of Effect on fertility via oral route:
Only reproductive and developmental screening toxicity study specific to the test substance available (key study)

Effects on developmental toxicity

Description of key information

Following daily oral administration (by gavage) of Cocamidopropyl hydroxysultaine at dose levels of 0, 66.7, 200 or 600 mg/kg/day to Wistar rats from gestation day 6 to up and including gestation day 19 in a GLP OECD 414-compliant study, the NOAEL for embryotoxicity, foetotoxicity and teratogenecity was set at 600 mg/kg/day in the absence of any treatment-related effects at this dose-level.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 November 2017 to 07 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 (15-25°C, 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 test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony.
- Age at study initiation: at least 12 weeks old at mating.
- Weight at study initiation: 200-260 g at Gestation Day 0
- Fasting period before study: no
- Housing: Standard laboratory conditions; individual housing
- Diet (e.g. ad libitum): The animals were provided with ssniff® SM Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany), ad libitum
- Water (e.g. ad libitum): The animals were provided tap water (in water bottles), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4-24.8°C (target: 22 ± 3°C)
- Humidity (%): 30-56% (target: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 14 November 2017 To: 14 December 2017

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (distilled water) at the following concentrations: 13.3, 40 and 120 mg/mL for the Low, Mid, and High dose groupe, respectively. Formulations were prepared prior to administration to the animals at the appropriate frequency (not more than 10 days before use). A constant volume of 5 mL/kg body weight was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weight of the animals.
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 was filled, and to ensure that the cannula used was the correct length to ensure the test item is dispensed in the stomach and not into the oesophagus.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration was performed at the Test Site using a validated LC-MS (Liquid Chromatography – Mass Spectrometry) method. Representative samples were taken from the test item formulations two times during the study (during the first week and just before the last week of treatment). Samples were taken in duplicate (5 mL/each), one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male : 1 female
- Length of cohabitation: approximately 2-3 hours
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as day 0 of pregnancy
- Any other deviations from standard protocol:

Duration of treatment / exposure:

from gestation day 6 (GD 6) to gestation day 19 (GD 19)

Frequency of treatment:

Daily

Duration of test:

21 days

Dose / conc.:

66.7 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

number of mated females/group:
Control: n= 24
Low dose: n = 26
Mid dose: n = 26
High dose: n = 27

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses were selected by the Study Director and the Sponsor based on available information of the chemical nature and characteristics of the test item and available results of a dose range finding study (DRF) [9], and a developmental toxicity screening test [10]. In the DRF study no signs of systemic toxicity were observed up to and including the 600 mg/kg bw/day dose level. However, in the developmental toxicity screening test, severe maternal toxicity (reduced body weight and food consumption) was observed at 800 mg/kg bw/day, and a similar, less expressed effect was seen in the 600 mg/kg bw/day dose group. Animal deaths occurred in the preliminary teratology study, they are considered to be due to treatment, and not related to the systemic toxicity of the test item; there was no observable effect on the pregnancy of the animals. In a previous DRF study (information supplied by the Sponsor) there was mortality at 1000 mg/kg bw/day so it was clear that the High dose should not be too close to this level.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
Only one general clinical observation was made on the first day (in the afternoon), on the afternoon on those days when detailed clinical observation was made in the morning. Additional general clinical observations were made for confirmatory reason on November 30, post dose, when one high dose animal was found dead. Furthermore, clinical observation (detailed) was made only once on necropsy days (in the morning).

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations were made on all animals at the onset of treatment (GD 6) then weekly.
The animals were monitored for any changes including pertinent behavioural changes and signs of toxicity including mortality, changes in 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, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20, or on the day of death (for found dead animals).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food was measured with precision of ± 1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was also calculated for each interval, including GD 0-6, GD 6-20 and GD 0-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes. The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses.

OTHER:
On GD 13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [half per litter ]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: Yes: [half per litter ]

Statistics:

The statistical evaluation of data was performed with the program package SAS v9.2 in case of Provantis v.9, or SPSS PC+4.0 (SPSS Hungary Kft, Budapest) in the case of data tabulated in Excel, by an appropriate statistical method.
In case of the SAS v9.2 software package (within the validated Provantis system) the following decision tree was applied automatically 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 showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (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 were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then a non-parametric analysis was used. 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.

Indices:

Caesarean Section and Necropsy Data:
- Number of corpora lutea: mean ± S.D.
- Number of implantations: mean ± S.D.
- Number and percentage of live foetuses: mean ± S.D.
- Number and percentage of intrauterine mortality: mean ± S.D. - Classified according to time of death: preimplantation loss, postimplantation loss, early and late embryonic loss, as well as foetal death
- Preimplantation loss: %, group mean
-Postimplantation loss: %, group mean

Foetal Data:
- Sex distribution: %, group mean
- Foetal body weight (accuracy 0.01 g): mean ± S.D.
- External abnormalities*/litter: %, group mean
- Visceral abnormalities*/litter: %, group mean
- Skeletal abnormalities*/litter: %, group mean

Historical control data:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Evaluated animals:
Animals in the Control group were symptom free.
Noisy respiration was observed in 3 out of 23 animals in the Low, 12 out of 24 animals in the Mid, and 20 out of 25 animals in the High dose group. Piloerection was present in 10 out of 24 animals in the Mid, and in all animals of the High dose group.
One animal (number 4505) in the High dose showed the clinical signs of noisy or slight to moderate laboured respiration, hunched back, slightly decreased activity, red discharge around the vulva and the nose.
The increased presence of noisy respiration and piloerection indicates a test item related effect similar to reported signs from other gavage studies with this test item. This surfactant substance, in very small amounts, can cause local effects if there is a slight reflux from the stomach, or if test item on the gavage tube contaminates the upper oesophageal area, with some substance contaminating the upper respiratory tract. All the other symptoms were attributed as secondary to the respiratory effects. The clinical signs were considered to be caused by a local effect, not a systemic toxicity effect.

Non-pregnant animals:
Noisy respiration was observed in 1 out of 3 Low dose, and 1 out of 2 Mid dose animals. Piloerection was observed in 1 out of 2 Mid dose animals. These clinical signs were considered to be related to the test item as described above.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two High dose animals were found dead in the study. One Animal showed hunched back, piloerection from the day before death, and piloerection, hunched back, moderately noisy, gasping respiration on the morning of the death. The other animal showed abnormal skin colour (white) on the limbs and pinna, as well as extremely decreased activity, prostration, and tonic convulsion after treatment. Considering the strong surfactant properties of the Test Item, and the rapid progress of the symptoms, it is considered likely that a small amount of Test Item entered the upper respiratory tract. The deaths were not attributed to systemic toxicity of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant body weight reduction was observed in the High dose group (600 mg/kg bw/day) from GD 8 up to the end of the treatment. These changes resulted in a statistically significant reduction of body weight gain, corrected body weight gain and net body weight gain values during the treatment period (GD 6-20) and the entire study (GD 0-20) when compared to the control values. The GD 20 mean body weight was 13% below the control mean value, confirming an adverse maternal effect of test item.
In the Mid dose group there was a slight but statistically significantly lower body weight gain (p<0.05) for the treatment period (GD 6-20) but no relevant statistical difference for the entire study period or for the individual weighing days. The differences seen in the Mid dose were not considered to be a clearly adverse effect of treatment. No test item related effect on body weight was observed in the Low dose group (66.7 mg/kg bw/day) when compared to control. Similarly, no statistically significant or biologically relevant differences were seen in the body weight gain or corrected body weight gain or net body weight gain values during the treatment period (GD 6-20) or entire study (GD 0-20) compared to the control value for this group. (see details in Table 1)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean daily food consumption in the High dose group (600 mg/kg bw/day) was statistically lower during the whole dosing and treatment period (GD 6-20 and GD 0- 20; 30% and 22.4% below controls, respectively). In the Mid dose group, food consumption was statistically significantly lower between GD 12 and GD 14, and when calculated for the whole treatment period (GD 6-20) but was a relatively small (< 10%) difference). No differences in the food consumption values were found in the Low (66.7 mg/kg bw/day) dose group when compared to the control group. These lower food intake values in the High and Mid dose groups were considered to be test item related effects although the difference in the Mid dose was not enough to be considered a clearly adverse effect of the test item.

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:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed for any of the test item treated groups in the gravid uterine weight parameter when compared to the control value. The gravid uterine weights in the dose groups were in line with the body weights for these groups (see details in table 1).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Evaluated animals:
No macroscopic findings were observed in the Control, Low and Mid dose groups. Diffuse or multifocal thickening of the non-glandular mucosal region of the stomach was observed in 12 out of 25 High dose (600 mg/kg bw/day) animals. These findings are considered to be test item related, local adverse effects, probably an irritant effect in the stomach.

Non-pregnant animals:
No macroscopic findings were observed for non-pregnant animals in any dose groups.

Found dead animals:
Two High dose animals were found dead during the study. Non-collapsed lungs were the common finding in these animals. In one animal, dark red discoloration was observed in all lung lobes (multifocal), in the stomach (in the glandular mucosa), and in the thymus. These findings are considered to be agonal changes. In one animal, multifocal thickness of the non-glandular mucosal region of the stomach was observed, which is in correlation with the macroscopic findings in the evaluated High dose animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No significant differences were noted in preimplantation loss or number of implantations of the test item treated groups when compared to the control.
The early and the late embryonic loss values of the test item treated groups were comparable with control. One dead foetus was present in one litter of the Mid dose group, and no dead foetus was found in any of the other (Control, Low, and High) dose groups. There was no statistically significant difference in the postimplantation loss or total intrauterine mortality between the test item treated and control groups (see details in table 2).

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

One dead foetus was present in one litter of the Mid dose group, and no dead foetus was found in any of the other (Control, Low, and High) dose groups.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Hundred and three females (24 for the Control, 26 for the Low and Mid, 27 for the High dose group, respectively) were mated in the study. The number of confirmed pregnant, evaluated dams was 23 in the Control group, 23 in the Low dose group (66.7 mg/kg bw/day), 24 in the Mid dose group (200 mg/kg bw/day) and 25 in the High dose group (600 mg/kg bw/day). Summary of pregnancy data is seen in Table 3.

Other effects:

no effects observed

Description (incidence and severity):

Corpora lutea: The mean number of corpora lutea was comparable with the control in all test item treated groups.
Evaluation of placentas: No abnormalities were observed on the placentas of any animals in the Control, Low (66.7 mg/kg bw/day), Mid (200 mg/kg bw/day) or High (600 mg/kg bw/day) dose groups.

Key result

Dose descriptor:

NOAEL

Effect level:

66.7 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

mortality

other: The maternal effects were all attributed to local effects due to the surfactants properties of the test substance.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean foetal weight per litter in the test item did not differ significantly from the control mean value (see details in Table 4).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of viable foetuses was comparable with the control mean in all test item treated groups (see details in Table 4).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups (see details in Table 4).

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

There were no external variations or malformations in this study (see details in Table 5).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The skeletal findings in the Low and Mid dose groups correspond with the current historical control (HC) or the concurrent study control data, or were considered to be incidental findings without dose response. The increased skeletal variations in the High dose group are considered to be a secondary effect of the low food intake and reduced body weight of the dams (see details in Tables 6 and 7).
Malformations such as Malformed Vertebrae (Split, Fused) in the Mid (1/115 foetus) dose group; Fused Rib in the High (1/122 foetus) dose group and Lumbar-Sacral Transverse process, fused, Pelvic girdle, malpositioned in the Control (2/123 foetuses in 2 litters) dose group were observed.

These findings correspond with the current historical control or occur only in study Control group data and have isolated occurrence that was considered incidental, ascribed to individual variability and not related to the test item.

In the case of most skeletal variations the foetal or litter based incidence in the test item treated groups was comparable with the current study control or historical control values. Therefore, they were considered as biologically not relevant findings and not related to the test item treatment.

The incidence of statistically significantly higher occurrence of unossified or incomplete ossification (sternum, ribs vertebrae, pubis) in the High dose group litter is in correlation with the reduced foetal body weight. This is compatible with maternal toxicity with reduced body weight in the High dose. The only other statistically different finding in the High dose was Wavy Ribs, which had an incidence fully in line with Historical incidence and hence is not considered to be an effect of test item.

When maternal toxicity affects food intake, causing a body weight reduction in the dams, the otherwise normal development of the foetuses can be delayed – some foetuses may grow more slowly; hence in this study the significantly higher number of retarded foetuses in the High dose group, see 9.6.3, often with delayed ossification. It must be noted that this type of secondary toxicity effect does not result in increased number of malformation of the foetuses [12-14].

In conclusion, all of the foetal findings for the Low and Mid dose groups, in incidence and nature, corresponded to the concurrent study control or current historical control data, were incidental findings and/or were not part of a dose response; hence these were considered as being ascribed to individual variability and not related to treatment. In the High dose group, small increases in the incidence of retarded ossification were attributed as secondary to the maternal body weight and not a direct foetal effect of the test item. Based on these results the test item did not affect adversely the intrauterine development.

Visceral malformations:

no effects observed

Description (incidence and severity):

All of the visceral findings (variations) were consistent in general nature and incidence with the concurrent study control data / existing historical control data or showed incidental occurrence (in the case of convoluted ureter, which was present only in the Mid dose group, with a greater occurrence than in the historical control), therefore considered as incidental findings (see details in Tables 8 and 9).

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The total number of retarded foetuses (runts) were significantly higher in the High dose group. The number of affected litters was similar in all test item treated groups as in the Control group, although the statistical difference correlates with an increased incidence of retarded ossification in this group, attributed to low maternal body weight (see details in Table 4).

Key result

Dose descriptor:

NOAEL

Remarks:

embryotoxicity

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects reported

Remarks on result:

other: highest dose tested

Key result

Dose descriptor:

NOAEC

Remarks:

foetotoxicity

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects reported

Remarks on result:

other:

Remarks:

highest dose tested

Key result

Dose descriptor:

NOAEC

Remarks:

teratogenecity

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects reported

Remarks on result:

other:

Remarks:

highest dose tested

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Summary of  maternal body weight parameters

Parameters	Dose (mg/kg bw/day)
	0	66.7	200	600
Number of evaluated dams	23	23	24	25
Body weight on GD20 (g)	334.6	328.5	321.3	290.8**	D
Body weight gain GD6-20 (g)	92.0	86.7	77.1*	50.2**	D
Body weight gain GD0-20 (g)	113.0	107.1	98.5	68.7**	U
Gravid Uterus (g)	63.4	59.5	58.9	53.8	NS
Corrected body weight on GD20 (g)	271.2	269.0	262.4	237.0**	U
Corrected body weight gain GD0-20 (g)	49.6	47.6	39.6	14.9**	U
Net body weight gain GD6-20 (g)	28.6	27.1	18.2*	-3.6**	U

Notes: Body weight data were rounded to one decimal place. Corrected and net weight / weight gains refer to body weight values minus the weight of the gravid uterus.

*= p<0.05; **= p<0.01; D: Dunnett two sided test.; U: Dunn two sided test

Table 2: Summary of the intrauterine evaluation

Parameters	Dose (mg/kg bw/day)
	0	66.7	200	600
Number of evaluated dams	23	23	24	25
Mean number of corpora lutea	12.04	11.78	11.67	11.24	NS
Preimplantation loss, mean	1.04	1.70	1.75	1.24	NS
Preimplantation loss (%), mean	8.79	14.09	15.25	10.60	NS
Mean number of implantations	11.00	10.09	9.92	10.00	NS
Early embryonic loss, mean	0.17	0.26	0.17	0.20	NS
Early embryonic loss (%), mean	1.48	2.55	2.25	2.15	NS
Late embryonic loss, mean	0.22	0.00	0.13	0.08	NS
Late embryonic loss (%), mean	1.80	0.00	1.50	0.73	NS
Dead foetuses, mean	0.00	0.00	0.04	0.00	NS
Dead foetuses (%), mean	0.00	0.00	0.38	0.00	NS
Postimplantation loss, mean	0.39	0.26	0.33	0.28	NS
Postimplantation loss (%), mean	3.28	2.55	4.14	2.89	NS
Total intrauterine mortality, mean	1.43	1.96	2.08	1.52	NS
Total intrauterine mortality (%), mean	11.97	16.34	18.36	13.35	NS
Viable foetuses, mean	10.61	9.83	9.58	9.72	NS

Notes: Most important parameters are shown in bold.

NS: Statistically not significant when compared to the vehicle control.

Table 3: Summary of pregnancy data

 

Parameters	Dose (mg/kg bw/day)
	0	66.7	200	600
Number of mated females	24	26	26	27
Pre-terminal death or euthanasia	0	0	0	2
Number of non-pregnant females	1	3	2	0
Number of females with ≤ 5 implantation sites	0	0	1	0
Number of evaluated females on GD20 (Caesarean section)	23	23	24	25

 

Table 4: Examination of viable foetuses

 

Parameters	Dose (mg/kg bw/day)
	0	66.7	200	600
Number of examined litters	23	23	24	25
Viable foetuses, mean	10.61	9.83	9.58	9.72	NS
Male foetuses, mean	5.13	4.91	4.71	5.24	NS
Female foetuses, mean	5.48	4.91	4.88	4.48	NS
Total number of foetuses	244	226	230	243	NS
Total number of male foetuses	118	113	113	131	NS
Total number of female foetuses	126	113	117	112	NS
Sex distribution (% of males / females)	49/51	50/50	50/50	53/47	NS
Mean foetal weight / litter (g)	3.43	3.49	3.51	3.45	NS
Number of foetuses with retarded body weight	10	17	12	33**	CH2
Number of affected litters (with runts)	8	8	6	11	NS

Notes: Most important parameters are shown in bold.

NS: Statistically not significant when compared to the vehicle control.

CH2: Chi Square test; **= p<0.01

Table 5: Summary table of the external abnormalities

 

Parameter	Dose (mg/kg bw/day)				HC data
	0	66.7	200	600
Total number of examined litters	23	23	24	25	670
Total number of examined foetuses	244	226	230	243	6889
Total number of intact (normal) foetuses	244	226	230	243	--

Note: HC: historical control

Table 6: Summary table of the skeletal abnormalities

 

Parameter	Dose (mg/kg bw/day)				HC data
	0	66.7	200	600
Total number of examined litters	23	23	24	25	669
Total number of examined foetuses	123	112	115	122	3435
Total number of intact (normal) foetuses	111	93	98	90CH**	--
Total number of foetuses / litters with malformation	2 / 2	0 / 0	1 / 1	1 / 1	--
Total number of foetuses / litters with variation	10 / 7	19CH*/ 11	16 / 9	31CH**/ 15	--

Notes: Numbers represent the number of abnormalities / number of affected litters. HC: historical control

CH: Chi2test; * = p < 0.05 ** = p < 0.01

 

Table 7: Details of the skeletal abnormalities

 

Parameter			Dose (mg/kg bw/day)				HC data
			0	66.7	200	600
Total number of examined litters			23	23	24	25	669
Total number of examined foetuses			123	112	115	122	3435
Skeletalmalformations
Malformed Vertebrae (Split, Fused)	Litter incidence	n	0	0	1	0	3
		%	0.0	0.0	4.2	0.0	0.448
	Foetal incidence	n	0	0	1	0	3
		%	0.000	0.000	0.870	0.000	0.087
Rib, Fused	Litter incidence	n	0	0	0	1	--
		%	0.000	0.000	0.000	4.000	--
	Foetal incidence	n	0	0	0	1	--
		%	0.000	0.000	0.000	0.820	--
Transverse process fused; Pelvic girdle, malpositioned#	Litter incidence	n	2	0	0	0	6
		%	8.696	0.000	0.000	0.000	0.897
	Foetal incidence	n	2	0	0	0	6
		%	1.626	0.000	0.000	0.000	0.175

Notes: Numbers represent the number (n) or ratio (%) of abnormalities. HC: historical control (data provided where considered useful)

No statistically significant differences were noted compared to the control group.

#: Historical control database contains Transverse processes, fused

 

Table 7: Details of the skeletal abnormalities (continued)

Parameter			Dose (mg/kg bw/day)				HC data
			0	66.7	200	600
Total number of examined litters			23	23	24	25	669
Total number of examined foetuses			123	112	115	122	3435
Skeletalvariations
Skull: 3 or More Bones, Incomplete Ossification	Litter incidence	n	1	3	1	4	123
		%	4.3	13.0	4.2	16.0	18.385
	Foetal incidence	n	1	5	1	5	153
		%	0.813	4.464	0.870	4.098	4.454
Skull: Hyoid, body, unossified	Litter incidence	n	0	1	0	1	--
		%	0.000	4.348	0.000	4.000	--
	Foetal incidence	n	0	1	0	3	--
		%	0.000	0.893	0.000	2.459	--
Sternum: Ossified Sternebra (4 or less) #	Litter incidence	n	5	5	5	9	269
		%	21.7	21.7	20.8	36.0	40.2
	Foetal incidence	n	5	7	10	18CH**	385
		%	4.065	6.250	8.696	14.754	11.208
Ribs: Wavy	Litter incidence	n	2	6	2	7	198
		%	8.7	26.1	8.3	28.0	29.596
	Foetal incidence	n	3	10CH*	3	11CH*	311
		%	2.439	8.929	2.609	9.016	9.054
Ribs: Rib or Cartilage Interrupted	Litter incidence	n	1	0	0	2	4
		%	4.348	0.000	0.000	8.000	0.598
	Foetal incidence	n	1	0	0	2	6
		%	0.813	0.000	0.000	1.639	0.175
Vertebrae: 2 or More Dumbbell or Asymmetric Ossification	Litter incidence	n	0	1	1	1	85
		%	0.000	4.348	4.167	4.000	12.706
	Foetal incidence	n	0	1	2	1	92
		%	0.000	0.893	1.739	0.820	2.697
Vertebrae: Sacral Unossified	Litter incidence	n	0	0	1	0	--
		%	0.000	0.000	4.167	0.000	--
	Foetal incidence	n	0	0	1	0	--
		%	0.000	0.000	0.870	0.000	--
Bipartite Ossification	Litter incidence	n	0	0	2	0	27
		%	0.000	0.000	8.333	0.000	4.036
	Foetal incidence	n	0	0	2	0	27
		%	0.000	0.000	1.739	0.000	0.786
Pubis unossified	Litter incidence	n	0	0	1	1	6
		%	0.000	0.000	4.167	4.000	0.897
	Foetal incidence	n	0	0	1	1	6
		%	0.000	0.000	0.870	0.820	0.175

Notes: Numbers represent the number (n) or ratio (%) of abnormalities. HC: historical control (data provided where considered useful)

#: Ossified sternebra (3 or less) is recorded in the historical control database CH: Chi2test; * = p < 0.05; ** = p < 0.01

Table 7: Details of the skeletal abnormalities (continued)

Parameter			Dose (mg/kg bw/day)				HC data
			0	66.7	200	600
Total number of examined litters			23	23	24	25	669
Total number of examined foetuses			123	112	115	122	3435
Skeletalvariations
Limbs (C/T): Tarsal ≤3	Litter incidence	n	1	1	1	1	34
		%	4.348	4.348	4.167	4.000	5.082
	Foetal incidence	n	1	1	2	1	49
		%	0.813	0.893	1.739	0.820	1.426

Notes: Numbers represent the number (n) or ratio (%) of abnormalities. HC: historical control (data provided where considered useful)

 

Table 8: Summary table of the visceral abnormalities

Parameter	Dose (mg/kg bw/day)				HC data
	0	66.7	200	600
Total number of examined litters	23	23	24	25	670
Total number of examined foetuses	121	114	115	121	3450
Total number of intact (normal) foetuses	116	108	109	121*	--
Total number of foetuses / litters with malformation	1 / 1	0 / 0	2 / 2	0 / 0	--
Total number of foetuses / litters with variation	4 / 4	6 / 6	4 / 3	0 / 0*	--

Notes: Numbers represent the number of abnormalities / number of affected litters. HC: historical control

*= p<0.05; Chi Square test

Table 9: Details of the visceral abnormalities

Parameter			Dose (mg/kg bw/day)				HC data
			0	66.7	200	600
Total number of examined litters			23	23	24	25	670
Total number of examined foetuses			121	114	115	121	3450
Visceral malformations
Kidney, misshapen (Small)	Litter incidence	n	1	0	0	0	1
		%	4.3	0.0	0.0	0.0	0.149
	Foetal incidence	n	1	0	0	0	1
		%	0.826	0.000	0.000	0.000	0.029
Abnormal Lung Lobes	Litter incidence	n	0	0	1	0	1
		%	0.0	0.0	4.2	0.0	0.149
	Foetal incidence	n	0	0	1	0	1
		%	0.000	0.000	0.870	0.000	0.029
Testis, malpositioned	Litter incidence	n	0	0	1	0	--
		%	0.0	0.0	4.2	0.0	--
	Foetal incidence	n	0	0	1	0	--
		%	0.000	0.000	0.870	0.000	--
Visceral variations
Brachiocephalic trunk, short	Litterincidence	n	2	1	0	0	45
		%	8.7	4.3	0.0	0.0	6.7
	Foetalincidence	n	2	1	0	0	53
		%	1.653	0.877	0.000	0.000	1.536
Renal papilla, absent	Litterincidence	n	0	0	1	0	--
		%	0.0	0.0	4.2	0.0	--
	Foetalincidence	n	0	0	1	0	--
		%	0.000	0.000	0.870	0.000	--
Renal pelvis, dilated	Litterincidence	n	0	0	1	0	--
		%	0.0	0.0	4.2	0.0	--
	Foetalincidence	n	0	0	1	0	--
		%	0.000	0.000	0.870	0.000	--
Carotid artery, malpositioned	Litterincidence	n	0	0	1	0	1
		%	0.0	0.0	4.2	0.0	0.149
	Foetalincidence	n	0	0	1	0	1
		%	0.000	0.000	0.870	0.000	0.029

Table 9: Details of the visceral abnormalities (Continued)

Parameter			Dose (mg/kg bw/day)				HC data
			0	66.7	200	600
Total number of examined litters			23	23	24	25	670
Total number of examined foetuses			121	114	115	121	3450
Visceral variations
Kidney, malpositioned	Litterincidence	n	0	1	0	0	1
		%	0.0	4.3	0.0	0.0	0.149
	Foetalincidence	n	0	1	0	0	1
		%	0.000	0.877	0.000	0.000	0.029
Adrenal gland, malpositioned	Litterincidence	n	0	1	0	0	1
		%	0.0	4.3	0.0	0.0	0.149
	Foetalincidence	n	0	1	0	0	1
		%	0.000	0.877	0.000	0.000	0.029
Ureter convoluted	Litterincidence	n	0	0	1	0	7
		%	0.000	0.000	4.167	0.000	1.045
	Foetalincidence	n	0	0	1	0	7
		%	0.000	0.000	0.870	0.000	0.203
Thymic cord	Litterincidence	n	0	3	1	0	69
		%	0.000	13.043	4.167	0.000	10.299
	Foetalincidence	n	0	3	2	0	82
		%	0.000	2.632	1.739	0.000	2.377
Renal papilla, small	Litterincidence	n	2	1	0	0	20
		%	8.7	4.3	0.0	0.0	2.985
	Foetalincidence	n	2	1	0	0	22
		%	1.653	0.877	0.000	0.000	0.638

Notes: Numbers represent the number (n) or ratio (%) of abnormalities. HC: historical control (data provided where considered useful)

No statistically significant differences were noted compared to the control group.

Conclusions:

In conclusion, C8-18 alkylamidopropyl hydroxysultaine, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 600 mg/kg bw/day induced maternal toxicity, with clinical signs associated with a local respiratory effect, reduced food intake, reduced body weight and body weight gain, and thickening of the non-glandular mucosal region of the stomach. All the maternal changes in the High dose group were compatible with being related to the local irritancy of the test item, there was no evidence of direct systemic toxicity.

The Mid dose of 200 mg/kg bw/day had lesser signs with no adverse effect on body weight, but there was evidence of a slight maternal toxicity. A few animals in the Low dose had respiratory effects with no consequences, this was not considered to reflect a significant maternal toxicity.

In the High dose group, the incidence of runts was higher, and the incidence of retarded ossification were statistically higher compared to the control group; these observations were attributed to maternal toxicity. There were no such effects in the Mid or Low dose groups. There were no malformations or developmental effects attributed to test item at any dose level.
The following no-observed-adverse-effect (NOAEL) levels were derived: NOAELmaternal toxicity: 66.7 mg/kg bw/day; based on significant maternal effects at 600 mg/kg bw/day and clinical signs of local effects at 200 mg/kg bw/day. The maternal effects were all attributed to local effects, there was no evidence of direct systemic toxicity in any group.
NOAELembryotoxicity: 600 mg/kg bw/day; based on the lack of any test-item related intrauterine effect in any treatment group.
NOAELfoetotoxicity: 600 mg/kg bw/day; based on the lack of any adverse developmental effects in any treatment group.
NOAELteratogenecity: 600 mg/kg bw/day; bBased on the lack of any developmental effects in any treatment group.

Executive summary:

This developmental toxicity study was performed according to OECD Test Guideline 414 to assess the effects of the test item C8-18 alkylamidopropyl hydroxysultaine on the embryonic and foetal development (including the organogenesis period) of HannoverWistar rats in their first pregnancy. The dams (one control and three test item treated groups) were treated daily by oral (gavage) administration, from gestation day 6 (GD 6) up to and including gestation day 19 (GD 19), where sperm positive day was counted as day 0 of pregnancy (GD 0). Control dams were treated with the vehicle (distilled water) only. Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

The doses were selected based on available information of the chemical nature and characteristics of the test item and available results of a dose range finding study,and a developmental toxicity screening test. Based on this information, the doses of 66.7, 200 and 600 mg/kg bw/day were deemed suitable for the purpose of the study.

Test item formulations were analysed for concentration two times during the treatment period using a validated LC-MS method. Simultaneously, vehicle control formulations were analysed for concentration.

Parameters monitored during the study included mortality and clinical observations, bodyweight,body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically.

 The number of confirmed pregnant, evaluated dams was 23 in the Control, 23 in the Low (66.7 mg/kg bw/day), 24 in the Mid (200 mg/kg bw/day) and 25 in the High  (600 mg/kg bw/day) dose groups, respectively.

 

Results:

All test item formulations were within the range of 94.8-104.1% of nominal concentration. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.

Two animals were found dead in the High dose groups, with respiratory symptoms suggesting that their death was caused by the strong surfactant activity (a local effect, not attributed to systemic toxicity) of the Test Item.

Apart from incidental findings, noisy respiration and piloerection were present in almost half of the animals in the Mid dose group (in 12 and 10 animals from the 24 evaluated dams,respectively) and dominantly present (in 20 and 25 animals from the 25 evaluated dams, respectively) in the High dose group. A few Low dose animals were similarly affected. These adverse effects were considered related to a local irritant type effect of the test item. There were no signs of systemic toxicity.

Statistically significant lower body weight was observed in the High dose group from GD 8 up to the end of the treatment, with significantly reduced body weight gain, corrected body weight gain and net body weight gain values. These changes were considered to be test item related adverse effects, probably secondary to the local respiratory and stomach effects of the test item as observed in the clinical signs and at necropsy.

There were no test item related adverse effect on maternal body weight / body weight gain and maternal corrected body weight / net body weight gain in the Low and Mid dose groups (66.7 and 200 mg/kg bw/day, respectively).

A relatively large, statistically significant reduction of the food consumption was observed in the High dose animals after the start of the treatment, this reduction contributed to the lower body weight in this group. A minor difference in the Mid dose group (<10%) may have been related to treatment but had no significant consequences.

At necropsy of the dams, no remarkable external observations related to test item treatment were recorded for any pregnant animals during necropsy. Diffuse or multifocal thickening of the non-glandular mucosal region of the stomach was observed in the High dose animals, indicating a test item related adverse local effect. No abnormalities were observed on the placentas in any examined groups.

The maternal findings showed a clear maternal toxicity at the High dose, with clinical signs associated with a local respiratory effect, signs of stomach irritation, and lower food intake and bodyweight than controls (Average body weight on GD 20 was approximately 13% below control). In the Mid dose group noisy respiration and piloerection was observed in almost half of the animals, the group had a transient lower food intake. It is concluded that there was a slight maternal toxicity at the Mid dose,but there was no adverse effect on body weight. A few animals in the Low dose had respiratory effects with no consequences,this was not considered to reflect a significant maternal toxicity for the group as awhole.

There were no toxicologically significant differences,or test item related-changes in the examined parameters of the intrauterine development up to and including 600 mg/kg bw/day.

The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean.

The total number of retarded foetuses was statistical significantly higher in the High dose animals, associated with maternal toxicity.

There were no test item related effects on external or visceral development of foetuses inthestudy.AsmallincreasedincidenceofretardedossificationintheHighdosegroup was considered to be compatible with the maternal toxicity in this group.There were no other differences observed between the groups at foetal examination.

Foetal malformations observed in the study were all considered to be incidental, they showed no dose dependency, thus not regarded as a test item related effect.

In conclusion, C8-18 alkylamidopropyl hydroxysultaine, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 600mg/kg bw/day induced maternal toxicity, with clinical signs associated with a local respiratory effect, reduced food intake, reduced bodyweight and body weight gain, and thickening of the non-glandular mucosal region of the stomach. All the maternal changes in the High dose group were compatible with being related to the local irritancy of the test item, there was no evidence of direct systemic toxicity.

 

The Mid dose of 200mg/kg bw/day had lesser signs with no adverse effect on body weight, but there was evidence of a slight maternal toxicity. A few animals in the Low dose had respiratory effects with no consequences, this was not considered to reflect a significant maternal toxicity.

 

In the High dose group, the incidence of runts was higher, and the incidence of retarded ossification were statistically higher compared to the control group; these observations were attributed to maternal toxicity. There were no such effects in the Mid or Low dose groups. There were no malformations or developmental effects attributed to test item at any dose level.

The following no-observed-adverse-effect (NOAEL) levels were derived:

- NOAELmaternal toxicity: 66.7 mg/kg bw/day; based on significant maternal effects at 600 mg/kg bw/day and clinical signs of local effects at 200 mg/kg bw/day. The maternal effects were all attributed to local effects, there was no evidence of direct systemic toxicity in any group.

- NOAELembryotoxicity: 600 mg/kg bw/day; based on the lack of any test-item related intrauterine effect in any treatment group.

- NOAELfoetotoxicity: 600 mg/kg bw/day; based on the lack of any adverse developmental effects in any treatment group.

- NOAELteratogenecity: 600 mg/kg bw/day; based on the lack of any developmental effects in any treatment group.

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and OECD 414 compliant study (Klimisch 1)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The potential effects of Cocamidopropyl hydroxysultaine, as a 35.4% aqueous solution, on the embryonic and foetal development (including the organogenesis period) were assessed in an OECD 414 compliant study following daily oral administration (by gavage) to Hannover Wistar rats from gestation day 6 up to and including gestation day (GD) 19.

The dams were treated at the doses of 66.7, 200 and 600 mg/kg bw/day. Control dams were treated with the vehicle (distilled water) only. Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD 20.

Parameters monitored during the study included mortality and clinical observations, bodyweight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically.

 The number of confirmed pregnant, evaluated dams was 23, 23, 24 and 25 in the Control, Low, Mid and High dose groups, respectively.

 

Two animals were found dead in the High dose groups, with respiratory symptoms suggesting that their death was caused by the strong surfactant activity (a local effect, not attributed to systemic toxicity) of the Test Item.

Apart from incidental findings, noisy respiration and piloerection were present in almost half of the animals in the Mid dose group (in 12 and 10 animals from the 24 evaluated dams, respectively) and dominantly present (in 20 and 25 animals from the 25 evaluated dams, respectively) in the High dose group. A few Low dose animals were similarly affected. These adverse effects were considered related to a local irritant type effect of the test item. There were no signs of systemic toxicity.

Statistically significant lower body weight was observed in the High dose group from GD 8 up to the end of the treatment, with significantly reduced body weight gain, corrected body weight gain and net body weight gain values. These changes were considered to be test item related adverse effects, probably secondary to the local respiratory and stomach effects of the test item as observed in the clinical signs and at necropsy.

There were no test item related adverse effect on maternal body weight / body weight gain and maternal corrected body weight / net body weight gain in the Low and Mid dose groups.

A relatively large, statistically significant reduction of the food consumption was observed in the High dose animals after the start of the treatment, this reduction contributed to the lower body weight in this group. A minor difference in the Mid dose group (<10%) may have been related to treatment but had no significant consequences.

At necropsy of the dams, no remarkable external observations related to test item treatment were recorded for any pregnant animals during necropsy. Diffuse or multifocal thickening of the non-glandular mucosal region of the stomach was observed in the High dose animals, indicating a test item related adverse local effect. No abnormalities were observed on the placentas in any examined groups.

The maternal findings showed a clear maternal toxicity at the High dose, with clinical signs associated with a local respiratory effect, signs of stomach irritation, and lower food intake and bodyweight than controls. In the Mid dose group noisy respiration and piloerection was observed in almost half of the animals, the group had a transient lower food intake. It is concluded that there was a slight maternal toxicity at the Mid dose, but there was no adverse effect on body weight. A few animals in the Low dose had respiratory effects with no consequences, this was not considered to reflect a significant maternal toxicity for the group as a whole.

There were no toxicologically significant differences, or test item related-changes in the examined parameters of the intrauterine development up to and including 600 mg/kg bw/day.

The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean.

The total number of retarded foetuses was statistical significantly higher in the High dose animals, associated with maternal toxicity.

There were no test item related effects on external or visceral development of fetuses in the study.A small increased incidence of retarded ossification in the High dose group was considered to be compatible with the maternal toxicity in this group. There were no other differences observed between the groups at foetal examination.

Foetal malformations observed in the study were all considered to be incidental, they showed no dose dependency, thus not regarded as a test item related effect.

In conclusion, C8-18 alkylamidopropyl hydroxysultaine, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 600mg/kg bw/day induced maternal toxicity, with clinical signs associated with a local respiratory effect, reduced food intake, reduced bodyweight and body weight gain, and thickening of the non-glandular mucosal region of the stomach. All the maternal changes in the High dose group were compatible with being related to the local irritancy of the test item, there was no evidence of direct systemic toxicity.

 

The Mid dose of 200 mg/kg bw/day had lesser signs with no adverse effect on body weight, but there was evidence of a slight maternal toxicity. A few animals in the Low dose had respiratory effects with no consequences, this was not considered to reflect a significant maternal toxicity.

 

In the High dose group, the incidence of runts was higher, and the incidence of retarded ossification were statistically higher compared to the control group; these observations were attributed to maternal toxicity. There were no such effects in the Mid or Low dose groups. There were no malformations or developmental effects attributed to test item at any dose level.

 

The following no-observed-adverse-effect (NOAEL) levels were derived:

- NOAELmaternal toxicity: 66.7 mg/kg bw/day; based on significant maternal effects at 600 mg/kg bw/day and clinical signs of local effects at 200 mg/kg bw/day. The maternal effects were all attributed to local effects, there was no evidence of direct systemic toxicity in any group.

- NOAELembryotoxicity: 600 mg/kg bw/day; based on the lack of any test-item related intrauterine effect in any treatment group.

- NOAELfoetotoxicity: 600 mg/kg bw/day; based on the lack of any adverse developmental effects in any treatment group.

- NOAELteratogenecity: 600 mg/kg bw/day; based on the lack of any developmental effects in any treatment group.

Short description of key information:
Following daily oral administration (by gavage) of Cocamidopropyl hydroxysultaine at dose levels of 0, 66.7, 200 or 600 mg/kg/day to Wistar rats from gestation day 6 to up and including gestation day 19 in a GLP OECD 414-compliant study, the NOAEL for embryotoxicity, foetotoxicity and teratogenecity was set at 600 mg/kg/day in the absence of any treatment-related effects at this dose-level.

Justification for selection of Effect on developmental toxicity via oral route:

Only one pre-natal developmental toxicity study specific to the test substance available (key study). The oral route was selected since it is a route of administration recommended by the OECD guideline No. 414 and it was considered suitable to provide the exposure required for this developmental toxicology study.

Justification for classification or non-classification

In the absence of any treatment-related effects on reproductive and developmental parameters in an OECD 422 compliant study and on the embryonic and foetal development in an OECD 414 compliant study, no classification for this endpoint is warranted.