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Toxicological information

Toxicity to reproduction

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

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

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

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

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

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

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

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

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

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

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

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

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

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

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

URINALYSIS: No

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

See Tissue Procedure Table below.

- Sacrifice:

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

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

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

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

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

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

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

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

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

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

HAEMATOLOGY
There were no effects on the hematological parameters.

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

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

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

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

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

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

 

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

 

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

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

 

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

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

Mortality

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

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

 

Clinical signs

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

 

Functional Observation Battery

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

 

Motor activity

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

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

 

Body weight and body weight change

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

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

 

Food consumption

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

 

Hematology

There were no effects on the hematological parameters.

 

Blood biochemistry

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

Pathology

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

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

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

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

Reason / purpose:
reference to same study
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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:
reference to same study
Reason / purpose:
reference to same study
Principles of method if other than guideline:
Test performed based on the methods described by Schmid (Schmid W. The micronucleus test. Mutation Research 31: 9-15, 1975) and modified by Salamone et al. (Salamone M, Heddle J, Stuart E and Katz M. Toward an improved micronucleus test. Studies on 3 model agents, mitomycin C, CPA and dimethylbenzanthracene. Mutation Research 74: 347-356, 1980).
Apart from detecting chromosome breakage events (clastogenesis), the micronucleus test is capable of detecting chemicals which induce whole chromosome loss (aneuploidy) in the absence of clastogenic activity. In the bone marrow of rats exposed to a chemical which induces cytogenetic damage, chromosomal fragments or entire chromosomes which are left behind at cell division were not incorporated into the nuclei of daughter cells. Most of these fragments condense and form one or more micronuclei in the cytoplasm. The visualization of micronuclei is facilitated in erythrocytes because their nucleus is extruded during erythropoiesis. Accordingly, the basis of this test is an evaluation of the increase in the number of Micronucleated Polychromatic Erythrocytes (MPE).
Substances which inhibit either proliferation or maturation of erythroblasts and those which are toxic for nucleated cells, decrease the proportion of immature erythrocytes (polychromatic, PE) when compared to mature erythrocytes (normochromatic, NE). Thus, the cytotoxicity of a substance can be evaluated by a decrease in the PE/NE ratio.
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation:
. Main study: 9 (females) - 10 (males) weeks old
. Micronucleus phase: 15 weeks old
- Weight at study initiation:
. Main study: 216 g (females) - 392 g (males)
. Micronucleus phase: 277 g (females) - 498 g (males)
- Fasting period before study: No
- Housing: Individual (except during pairing) in polycarbonate 940 cm² cages with stainless stell lids and autoclaved dust
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days (main study) / 7 days (micronucleus phase) before dosing initiation

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

IN-LIFE DATES: From: 10 May 2012 To: 20 July 2012
Route of administration:
oral: gavage
Vehicle:
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)
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):

MAIN STUDY:
. In the males:
- 2 weeks before pairing (from study day 1 to 14),
- during the pairing period (3 weeks, from study day 15 until study day 16 to 29),
- until sacrifice (at least 5 weeks in total, from study day 17 to 30 until study day 36).
. In the females:
- 2 weeks before pairing (from study days 1 to 14),
- during the pairing period (3 weeks, from study days 15 to 29),
- during gestation (from study days 16 to 30 until study days 36 to 50),
- during lactation until day 5 post-partum inclusive (from study days 37 to 51 until study days 42 to 56),
- until sacrifice for the non-pregnant females (at least 6 weeks in total, approximately, until study day 41 to day 45).

MICRONUCLEUS PHASE:
The positive control dose formulation was administered as a single dose on the day preceding the scheduled sacrifice (i.e. on completion of the treatment period for groups 1 to 4).
Frequency of treatment:
Once daily
Post exposure period:
None
Remarks:
Doses / Concentrations:
0, 30, 100, 300 mg/kg bw/day
Basis:
other: nominal dose levels (main study)
No. of animals per sex per dose:
10 (main study) / 5 (micronucleus phase)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Nature: cyclophosphamide (CPA, CAS No. 6055-19-2, Sigma, Saint-Quentin-Fallavier, France)
- Justification for choice of positive control: In the absence of any specific recommendation in the OECD guideline No. 474, the dose level was selected based on scientific literature, in order to insure a clear positive response.
- Route of administration: oral (gavage)
- Doses / concentrations: 30 mg/kg bw as a single dose 24 hours prior to euthanasia at the end of the treatment period
Tissues and cell types examined:
Bone marrow cells (increase of the frequency of micronucleated cells) were examined in the first five animals in sultaine-treated groups and all CPA-treated animals.
Details of tissue and slide preparation:
- Preparation of the smears:
At the end of the treatment period, all animals (first five principal animals in sultaine-treated groups, all CPA-treated animals) were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination. The femurs were removed and bone marrows were flushed and suspended in fetal calf serum. The separation of anucleated erythrocytic cells from other myeloic cells was carried using a cellulose column. This elution step enables the production of slides containing only polychromatic and normochromatic erythrocytes without any nucleated cells or mast cell granules. After centrifugation of the eluate containing the cells, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air dried and stained with Giemsa.
The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring). Thereafter, CPA-treated animals were discarded without any further investigations.

- Microscopic examination of slides:
In a first instance, the micronucleus analysis was performed on the slides of the males only. Based on the toxicological results obtained in this study (as differences were not observed between males and females), the slides analysis was not performed for the females.
For each sampled male, the number of Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio were established by scoring a total of 1000 erythrocytes (PE + NE). Scoring was performed "blind".
Evaluation criteria:
All individual data of MPE/2000PE and the PE/NE ratio were presented in tabular form. Mean and standard deviations of MPE/1000PE and PE/NE ratios were given for each experimental group. Because MPE/1000PE is considered as a percentage, this parameter was statistically analyzed after Arcsine transformation. The model was validated by a comparison between the vehicle group and the positive control. Effect of the test item was determined by comparisons of each group to the control one.
For a result to be considered positive, there must be: either a dose-related increase in the frequency of MPE when compared to the vehicle control group, or an increase in the frequency of MPE in a single dose group, of at least 2-fold the frequency of the vehicle control group.
Biological relevance of the results was considered first. Statistical analysis was used as an aid in evaluating the test results.
Statistics:
Normality and homogeneity of variances were tested using a Kolmogorov-Smirnov test and a Bartlett test. If normality and homogeneity of variances are demonstrated, the statistical comparisons are performed using a Student t-test (two groups) or a one-way analysis of variance (three groups) followed by a Dunnett test (if necessary). If normality or homogeneity of variances is not demonstrated, a Mann/Whitney test (two groups) or a Kruskall Wallis test (three groups) will be performed followed by a Dunn test (if necessary). All these analyses were performed using the software SAS Enterprise Guide Version 2.05.89 (SAS Release 8.02 TS Level 02M0, SAS Institute Inc), with a level of significance of 0.05 for all tests.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The positive control cyclophosphamide induced a significant increase (p < 0.001) in the frequency of MPE when compared with the vehicle group, indicating the sensitivity of the test system under our experimental conditions. The test conditions were therefore considered to be valid.
The mean values of the PE/NE ratios in animals treated with the test item at 30, 100 or 300 mg/kg/day were not statistically significantly different from that of the vehicle control animals.
The mean frequencies of MPE in the three test item treated groups were not found significantly different from that in the vehicle group. These results met the criteria of a negative response.
(See Table below)

Table: Results of the cytogenetic test: data summary

Group

Dose(1)

MPE/1000PE

PE/NE ratio

(mg/kg/day)

mean

(sd)

mean

(sd)

Males

Vehicle

0

0.8

(0.4)

0.42

(0.19)

Test item

30

1.3

(0.8)

0.54

(0.09)

100

1.5

(0.9)

0.38

(0.11)

300

0.8

(0.3)

0.48

(0.13)

Cyclophosphamide

30

15.1***

(3.3)

0.22*

(0.06)

(1): expressed as active material

Five animals per group

MPE: Micronucleated Polychromatic Erythrocytes

PE: Polychromatic Erythrocytes

NE: Normochromatic Erythrocytes

sd: standard deviation

*** p<0.001         * p<0.05

The mean values of the polychromatic/normochromatic erythrocytes (PE/NE) ratios in animals treated with the test item at 30, 100 or 300 mg/kg/day were not statistically significantly different from that of the vehicle control animals. However, based on the effects observed in kidneys (distant from the site of administration) in the repeated-dose toxicity phase of the study, the target tissue (bone marrow) was considered to have been exposed to the test substance and/or its metabolites or degradation products.

Conclusions:
Under the experimental conditions of this study, Cocamidopropyl hydroxysultaine, did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells up to 300 mg/kg administered daily for the whole dosing period.
Executive summary:

As part of 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, an evaluation of the potential of the test item to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells (increase in the frequency of micronucleated cells) was performed.

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. Another group of five males and five females received Cyclophosphamide (CPA) as a single dose of 30 mg/kg on the day preceding schedule sacrifice, and acted as a positive control group for micronuclei induction. At necropsy, 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.

 

There were no pathological findings in animals given 30 mg/kg/day of CPA at microscopic examination.

The positive control cyclophosphamide induced a significant increase (p < 0.001) in the frequency of micronucleated polychromatic erythrocytes (MPE) when compared with the vehicle group, indicating the sensitivity of the test system under our experimental conditions. The test conditions were therefore considered to be valid.

The mean values of the polychromatic/normochromatic erythrocytes (PE/NE) ratios in animals treated with the test item at 30, 100 or 300 mg/kg/day were not statistically significantly different from that of the vehicle control animals. However, based on the effects observed in kidneys (distant from the site of administration) in the repeated-dose toxicity phase of the study, the target tissue (bone marrow) was considered to have been exposed to the test substance and/or its metabolites or degradation products.

The mean frequencies of MPE in the three test item treated groups were not found significantly different from that in the vehicle group. These results met the criteria of a negative response.

In conclusion, under the experimental conditions of the study, Cocamidopropyl hydroxysultaine did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells up to 300 mg/kg administered daily for the whole dosing period (i.e., approximately 5 to 6 weeks).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Complementary in vivo micronucleus phase added
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation:
. Main study: 9 (females) - 10 (males) weeks old
. Micronucleus phase: 15 weeks old
- Weight at study initiation: 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg: forestomach squamous cell hyperplasia, pulmonary bronchioalveolar inflammation and tracheal epithelial alteration, minimal to slight degeneration/hypertrophy of tubular epithelium or minimal tubular vacuolation in kidneys.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

-  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.

Applicant's summary and conclusion

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.