Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
April - December 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and OECD test guideline compliant study performed on an analogue substance (for justification of read-across between Lauramidopropylhydroxysultaine and cocamidopropylhydroxysultaine, please refer to corresponding assessment report in Section 13).
Cross-reference
Reason / purpose:
reference to same study

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
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Cocamidopropyl hydroxysultaine
- Physical state: Yellow liquid (aqueous solution)
- Analytical purity: 36.2% Alkylamidopropyl hydroxysultaine
- Impurities (identity and concentrations): Glycerol 2.65%, Laurylamidopropyl dimethylamine (C12) < 0.05%
- Purity test date: 31 January 2012
- Lot/batch No.: HG04423
- Manufacturing date of the lot/batch: 27 September 2011
- Expiry date of the lot/batch: 26 September 2013
- Storage condition of test material: At room temperature and protected from light
- Other: Date of receipt at test facility 08 February 2012

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:
. 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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)

Results and discussion

Results of examinations

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.

Effect levels

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:
other: histopathology (forestomach, lungs, kidneys)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

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.