Reaction mass of Amines, coco alkyl and β-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and β-Alanine, N-coco alkyl derivs.

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 July 2012 - 03 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Amines, coco alkyl and ß-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and ß-Alanine, N-coco alkyl derivs.

EC Number:

915-790-0

Molecular formula:

n/a

IUPAC Name:

Reaction mass of Amines, coco alkyl and ß-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and ß-Alanine, N-coco alkyl derivs.

Test material form:

other: paste

Details on test material:

- Lot/batch No.: EL 0085
- Name of test material: Reaction mass of Amines, coco alkyl and ß-Alanine, N-(2-carboxyethyl)-, N coco alkyl derives. and ß-Alanine, N coco alkyl derivs.
- Physical state: solid paste
- Expiry date: 15 May 2015
- Storage condition: in a cool place.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Charles River Laboratories France, l’Arbresle, France
- Age and weight at study initiation: on the first day of treatment, the males were approximately 10 weeks old and had a mean body weight of 354 g (range: 327 g to 385 g) and the females were approximately 9 weeks old had a mean body weight of 215 g (range: 191 g to 238 g). The males and the females were sexually mature and were not siblings. The females were previously unmated.
- Fasting period before study: no
- Housing: The animals were individually housed, except during pairing and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France).
Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material.
Each cage contained an object (rat hut) for the enrichment of the environment of the rats. The cages were placed in numerical order on the racks.
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 6 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C (the temperature recorded in the animal room was sometimes outside the target range specified in the study plan)
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 08 August 2012 to 23 September 2012.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was mixed with the required quantity of vehicle, using a mortar and a pestle and then homogenized using a magnetic stirring. The test item dose formulations were prepared daily and stored at room temperature.

VEHICLE
- Concentration in vehicle: 4.4, 13.4 and 40 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurs
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: HPLC/MS-MS,
Test item concentrations: -remained within an acceptable range of variation compared to nominal values.
Homogeneity: satisfactory (0.2 to 200 mg/mL)
Stability: not assessed, dose formulation prepared daily

Duration of treatment / exposure:

In the males:
- 2 weeks before mating,
- during the mating period (3 weeks),
- until sacrifice (i.e. at least 5 weeks in total),

In the females:
- 2 weeks before mating,
- during the mating period (up to 3 weeks),
- during pregnancy,
- during lactation until day 5 post-partum inclusive,
- until sacrifice for females which had not delivered.

Frequency of treatment:

Once daily

Details on study schedule:

- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 11-12 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
200 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
67 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
22 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of a previous study. In this study three groups of Sprague-Dawley rats (three/sex/group) received the test item, daily for 2 weeks, by oral (gavage) administration at 100, 300 or 1000 mg/kg/day. A constant dosage volume of 5 mL/kg/day was used. Another group of three males and three females received the vehicle only (corn oil) for 2 weeks under the same experimental conditions and served as a control group. The following findings were recorded:
-100 mg/kg/day: ptyalism, dyspnea, abdominal breathing and loud breathing,
- 300 mg/kg/day: ptyalism, loud breathing, thin appearance, abdominal breathing and dyspnea. Males had reduced mean body weight gain during the first and last 4 days of the study (-50% vs. controls),
- 1000 mg/kg/day: 1/3 found dead males (on study day 4 with dyspnea, thin appearance, abnormal color/urogenital region, hunched posture, pallor of extremities and loud breathing the days preceding death). At necropsy, the digestive tract had areas of discolorations in the stomach. Surviving animals had ptyalism, dyspnea, abdominal breathing, loud breathing, thin appearance, hunched posture, pallor of eyes and/or extremities, piloerection, soiled mouth and/or anus. Males and females exhibited body weight losses and marked reductions in food consumption.

Therefore, 200 mg/kg/day was selected as the high dose-level. The low-dose and mid-dose were selected using a ratio representing a three-fold interval (i.e. 22 and 67 mg/kg/day).

- Rationale for animal assignment: computerized stratification procedure

Positive control:

no (not required)

Examinations

Parental animals: Observations and examinations:

MORTALITY:
Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

GENERAL CLINICAL EXAMINATIONS:
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 once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL EXAMINATIONS:
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.

BODY WEIGHT (GAIN):
- Time schedule: 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 and on days 0, 7, 14 and 20 post-coitum (p.c.) and days 1 and 5 p.p.

FOOD CONSUMPTION:
- Time schedule: 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 p.c. and during lactation for interval days 1 5 p.p..

NEUROBEHAVIOURAL EXAMINATION:
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 p.p. after sacrifice of the pups.
This included a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity.

HAEMATOLOGY:
Time schedule: on the day of sacrifice.

CLINICAL CHEMISTRY:
Time schedule: on the day of sacrifice.

URINALYSIS:
Time schedule: on the day of sacrifice.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded

Oestrous cyclicity (parental animals):

fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females are mated.

Sperm parameters (parental animals):

Parameters examined in males of parental generation:
- weighing and microscopic examination: see Tissue Procedure Table below
- microscopic evaluation of stages of the spermatogenic cycle and testicular interstitial cells (group 4)

Litter observations:

STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
- external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all animals after the end of the mating period
- Female animals: all surviving animals = day 6 post-partum or, for females which had not delivered yet, day 25 post-coitum (mothers with litter dying entirely were sacrificed as appropriate).

ORGAN WEIGHTS: see table below

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination

HISTOPATHOLOGY:
- on all tissues listed in the table below for the first five control and high dose animals (groups 1 and 4) sacrificed as scheduled,
- on all macroscopic lesions,
- all females sacrificed because of non-delivery to investigate possible causes

Postmortem examinations (offspring):

SACRIFICE: on day 5 post-partum

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No

Reproductive indices:

Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live concepti) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)

Offspring viability indices:

Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

no in-vivo observation of sperm

Reproductive performance:

no effects observed

Details on results (P0)

MORTALITY:
One male treated at 22 mg/kg/day and one male treated at 200 mg/kg/day were prematurely sacrificed on day 26 or 23, respectively.
Prior to death, abdominal and loud breathing, dyspnea and blood in mouth were noted in both males.
In addition, the male treated at 22 mg/kg/day had hypoactivity, half-closed eyes and reflux at dosing and the male treated at 200 mg/kg/day had ptyalism.
At necropsy and for both males, there were lesions indicative of gavage errors.
There were no unscheduled deaths in females during the study.

CLINICAL SIGNS:
Hypoactivity, loud breathing, half-closed eyes, reflux at dosing and/or dyspnea recorded in animals treated at 200 mg/kg/day, were considered to be treatment-related and of toxicological significance.
Ptyalism was observed from 22 mg/kg/day and considered to be of minor toxicological importance.
Other clinical sign (cutaneous lesion, area of hairloss and abnormal growth of teeth) are commonly observed in this species and strain.

BODY WEIGHT (GAIN):
In males:
When compared with controls, there was a statistically significant lower mean body weight in the 200 mg/kg/day group from study day 15 (up to -12.4%) which resulted in a statistically significant lower mean body weight change over the whole study period (-43.6%).

In females:
During the premating period, there were no effects on mean body weight or mean body weight changes.
During the gestation period and when compared with controls, there was a statistically significant lower mean body weight in the 200 mg/kg/day group on day 20 p.c. (-8.5%) which resulted in a statistically significant lower mean body weight change (-18.7%) during gestation.
During the lactation period and when compared with controls, there was a statistically significant lower mean body weight in the 200 mg/kg/day group from day 1 p.p. (up to -10.8%).
Taking into account, the amplitudes of the changes, the decreases observed in both sexes at 200 mg/kg/day were considered of moderate toxicological significance.

FOOD CONSUMPTION:
In males and when compared with controls, there was a statistically significant lower mean food consumption during the period of days 1-8 (-10.0%) in the 67 mg/kg/day group and during the periods of days 1-8 (-16.7%) and 8-15 (-17.2) in the 200 mg/kg/day group.
In females and when compared with controls, there were no effects on mean food consumption, except during the lactation period with a statistically significant lower mean food consumption ( 16.2%) in the 200 mg/kg/day group.
Taking into account, the amplitudes of the changes, the decreases observed in both sexes at 200 mg/kg/day were considered of moderate toxicological significance.

NEUROBEHAVIOURAL EXAMINATION:
Motor activity
When compared with controls, there were trends towards an increase in the number of horizontal movements in males at 22 mg/kg/day and a decrease in the number of horizontal movements in females at 67 mg/kg/day.
In the absence of similar finding in both sexes and of any dose-relationships, a test item treatment related effect was considered unlikely.

FUNCTIONAL OBSERVATION BATTERY:
Hypoactivity and slightly modified breathing were in line with clinical signs already observed (see above) at 200 mg/kg/day in males and considered to be related to treatment with the test item.
Others finding are commonly observed in this species and strain and therefore considered to be fortuitous.

HAEMATOLOGY:
There were increases in neutrophils count at 200 mg/kg/day in males and from 22 mg/kg/day in females. When compared with controls, the differences were statistically significant in neutrophils count in males (+71.3%) and females (+70.6%) at 200 mg/kg/day group. These increases were considered to be treatment-related but of minor toxicological significance taking into account the amplitude of the changes.
A test tem treatment-related effect was considered unlikely for the other differences in the absence of dose-relationship or significance in the opposite sex.

CLINICAL CHEMISTRY:
A test tem treatment-related effect was considered unlikely for these differences in the absence of dose-relationship or significance in the opposite sex.

URINALYSIS:
There were no effects on mean urinalysis parameters.

REPRODUCTIVE PERFORMANCE:
Mating and fertility data
There were no treatment-related effects on mating and fertility data. All females were pregnant.
All animals mated within comparable mean number of days.

Delivery data
There were no effects on mean duration of gestation, mean pre-implantation loss, mean number of pups delivered or viability index.
In the 200 mg/kg/day group, there was a higher mean percentage of post-implantation loss which remained within the range of our historical control data [5.4%; 15.9%, Historical Control Data 2010]. Therefore and in the absence of any dose-relationship, a test item treatment-related effect was considered unlikely.

ORGAN WEIGHTS:
When compared with controls, the mean absolute and relative liver weights were significantly higher in males and females given 200 mg/kg/day (p<0.01 or p<0.05). The mean relative liver weights were also significantly higher in females given 67 mg/kg/day (p<0.05).
Other organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates and/or were not consistent for the sexes.

GROSS PATHOLOGY:
Unscheduled deaths
One male given 200 mg/kg/day prematurely sacrificed on day 23 had red discoloration of the external surface of the trachea and of the base of the tongue. One male given 22 mg/kg/day prematurely sacrificed on day 26 had a gelatinous thymus. These lesions suggested that the moribundity was caused by a gavage related trauma.

Terminal sacrifice
The white discoloration of the forestomach in one female treated at 200 mg/kg/day and the thickening of the stomach in one female treated at 67 mg/kg/day were related to test item administration. These findings correlated respectively to acanthosis/hyperkeratosis/ulcer and submucosal inflammation at microscopic examination.
The other macroscopic findings had no histological correlates or correlated with common histological findings in control rats, and were considered to be incidental.

HISTOPATHOLOGY:
Unscheduled deaths
One male had acute granulocytic and hemorrhagic inflammation of the trachea and of the esophagus, together with an hematoma adjacent to the tongue and lung hemorrhages. The slight increase in macrophages seen in the mesenteric lymph node and the minimal acanthosis/hyperkeratosis in the forestomach were related to the test item administration.
One male had a subacute edematous interstitial inflammation in the thymus, associated with pulmonary hemorrhages and subacute to acute inflammation in the lungs, trachea and esophagus that displayed also a necrotic focus. The minimal acanthosis/hyperkeratosis in the forestomach was related to the test item.

Terminal sacrifice
Microscopic findings were seen in forestomach, mesenteric lymph nodes and liver.

. Forestomach (and stomach)
Minimal to marked acanthosis associated with hyperkeratosis was seen in two females treated at 67 mg/kg/day and in males and females treated at 200 mg/kg/day. There were also occasionally an erosion/ulcer and/or submucosal inflammation (with edema or fibrosis) in mid- or high-dose animals. In view of the marked severity of the acanthosis/hyperkeratosis, this finding was considered as adverse at 200 mg/kg/day. These findings were considered to be the consequences of the irritant properties of the compound.
There was a slight subacute inflammation in the submucosa of the stomach from a female treated at 67 mg/kg/day that correlated to the thickening seen macroscopically. Although this was an isolate finding not dose-related, it was considered as the result of a contiguous extension of the forestomach submucosal edematous inflammation in this animal.

. Mesenteric lymph nodes
Minimal to moderate, non adverse increase in macrophages was seen in males and females treated at 67 or 200 mg/kg/day in the medullary sinuses. The macrophages had a foamy cytoplasm.

. Liver
Minimal to slight non adverse hepatocellular hypertrophy was seen in one female treated at 67 mg/kg/day and in males and females treated at 200 mg/kg/day.
There were no associated degenerative changes at any of the dose-levels.
This finding correlated to the increase in liver weights seen in females at 67 mg/kg/day and in males and females at 200 mg/kg/day.

. Prostate
All examined males treated at 200 mg/kg/day presented a minimal infiltrate of mononuclear cells in the prostate versus one out of five controls, low- and mid-dose males.
In CiToxLAB historical control data, the mean percentage of affected animals in control groups is 45% (13-14 week-studies, 105 animals, range: 0-70%). In view of the low severity of this finding and the low incidence in the control group, the relationship to test item administration of this minimal increase in incidence of mononuclear inflammatory cell infiltrate at 200 mg/kg/day remained questionable.

Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, and/or are common background findings for the Sprague-Dawley rat.

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive performance

2

Dose descriptor:

NOAEL

Effect level:

67 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on microscopic examination findings

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

MORTALITY/VIABILITY:
The number of pups found dead, the number of pups cannibalized and the number of litters affected are presented in the table below:

Dose-level (mg/kg/day) 0 22 67 200
Number of pups found dead 4 9 7 6
Number of pups cannibalized 0 2 0 4
Number of affected litters 3 4 5 4

Overall, there were no evidences of a test item treatment-related effect on the repartition of found dead pups, pups cannibalized or affected litters.

CLINICAL SIGNS:
The findings recorded were the following:
- Control group: hematoma on the back (one pup from one dam and another one from one dam),
- 22 and 67 mg/kg/day groups: there were no clinical signs,
- 200 mg/kg/day: thin appearance and cold to the touch were recorded in all pups from one dam on day 1 p.p. Dehydration was also noted in one of these pups from day 1 p.p. Thin appearance and cold to the touch were also recorded in one pup from two dams from day 1 p.p. Desquamation was recorded in all pups from one dam on day 1 p.p.

Thin appearance, cold to the touch and desquamation were considered to be related to treatment with the test item and of toxicological importance. All these findings were considered to be the consequence of altered/reduced maternal care.

BODY WEIGHT (GAIN):
There were no effects on mean pup body weight and body weight change.

GROSS PATHOLOGY:
There were no test item treatment-related findings.

ORGAN WEIGHTS:
When compared with controls, the mean absolute and relative liver weights were significantly higher in males and females given 200 mg/kg/day (p<0.01 or p<0.05). The mean relative liver weights were also significantly higher in females given 67 mg/kg/day (p<0.05).
Other organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates and/or were not consistent for the sexes.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

67 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: toxic effects on progeny

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and for females throughout gestation and until day 5 post-partum, at dose-levels of 22, 67 or 200 mg/kg/day.
Based on the experimental conditions of this study:
- the No Observed Effect Level (NOEL) for reproductive performance was considered to be 200 mg/kg/ day in the absence of any treatment-related effect on mating and fertility at this dose-level,
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 67 mg/kg/day based on microscopic examination findings (acanthosis/hyperkeratosis with ulcer and inflammation in the forestomach in both sexes which were considered as adverse at 200 mg/kg/day),
- the No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was considered to be 67 mg/kg/day based on clinical signs recorded in pups at 200 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (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.

This study provides information on the possible health hazards (including neurological and immunological effects) likely to arise from repeated exposure over a limited period of time. It can also indicate effects on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

Methods

Three groups of 10 male and 10 female Sprague-Dawley rats received the test item, daily, by oral administration (gavage), before mating, during mating and, for the females, throughout gestation until day 5 p.p., at dose-levels of 22, 67 or 200 mg/kg/day. An additional group of 10 males and 10 females received the vehicle control, corn oil, 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 5 p.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 5 p.p.

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 and urine for urinalysis.

 

The males were sacrificed after completion of the mating period. Body weights and selected organs weights were recorded and a complete macroscopic post-mortem examination performed, with particular attention paid to the reproductive organs. A microscopic examination was conducted on selected organs from the first five males in the control group and the high-dose group and also on liver, mesenteric lymph nodes, prostate and forestomach from the first five animals of the low- and mid-dose groups (groups 2 and 3). Microscopic examination was conducted on all macroscopic lesions from all groups.

 

Dams were sacrificed on day 6p.p. Body weights and selected organs weights were recorded and a complete macroscopic examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was then conducted on selected organs from the first five females to deliver in the control group and the high-dose group and on any macroscopic lesions from all groups.

 

Pups, including those found dead before study termination, were also submitted for a macroscopic post-mortem examination.

Results

The test item concentrations in the administered dose formulations analyzed in weeks 1, 3 and 7 remained within an acceptable when compared to the nominal values. The test itemwas not detected in control samples.

 

Mortality

One male treated at 22 mg/kg/day and one male treated at 200 mg/kg/day were prematurely sacrificed on day 26 or 23, respectively, these deaths were related to the gavage procedure. There were no unscheduled deaths in females during the study.

 

Clinical signs

Hypoactivity, loud breathing, half-closed eyes, reflux at dosing and/or dyspnea recorded in animals treated at 200 mg/kg/day, were considered to be treatment-related and of toxicological significance.

Ptyalism was observed from 22 mg/kg/day and considered to be of minor toxicological importance.

 

Motor activity

Notest item treatment-related effects were observed.

 

Functional Observation

Hypoactivity and slightly modified breathing were in line with clinical signs already observed (see above) at 200 mg/kg/day in males and considered to be related to treatment with the test item.

 

Body weight and body weight change

In males: when compared with controls, there was a statistically significant lower mean body weight in the 200 mg/kg/day group from study day 15 (up to -12.4%) which resulted in a statistically significant lower mean body weight change over the whole study period (-43.6%).

In females: during the premating period, there were no effects on mean body weight or mean body weight changes. During the gestation period and when compared with controls, there was a statistically significant lower mean body weight in the 200 mg/kg/day group on day 20p.c.(-8.5%)which resulted in a statistically significant lower mean body weight change (-18.7%) during gestation. During the lactation period and when compared with controls, there was a statistically significant lower mean body weight in the 200 mg/kg/day group from day 1 p.p. (up to -10.8%).

Taking into account, the amplitudes of the changes, the decreases observed in both sexes at 200 mg/kg/day were considered of moderate toxicological significance.

 

Food consumption

In males and when compared with controls, there was a statistically significantlower mean food consumption during the period of days 1-8 (-10.0%) in the 67 mg/kg/day group and during the periods of days 1-8 (-16.7%) and 8-15 (-17.2) in the 200 mg/kg/day group.

 

In females and when compared with controls, there were no effects on mean food consumption, except during the lactation period with a statistically significantlower mean food consumption (-16.2%) in the 200 mg/kg/day group.

Taking into account, the amplitudes of the changes, the decreases observed in both sexes at 200 mg/kg/day were considered of moderate toxicological significance.

Hematology

When compared with controls, there were statistically significant increases in neutrophils counts in males (+71.3%) and females (+70.6%) from the 200 mg/kg/day group. These increases were considered to be treatment-related but of minor toxicological significance taking into account the amplitude of the changes.

 

Blood biochemistry

No test tem treatment-related effects were observed.

 

Urinalysis

There were no effects on mean urinalysis parameters.

 

Mating and fertility data

There were no treatment-related effects on mating and fertility data. All females were pregnant.

All animals mated within comparable mean number of days.

 

Delivery data

There were no effects on mean duration of gestation, mean pre-implantation loss, mean number of pups delivered or viability index.

In the 200 mg/kg/day group, there was a higher mean percentage of post-implantation loss which remained within the range of the historical control data. Therefore and in the absence of any dose-relationship, a test item treatment-related effect was considered unlikely.

 

Pup mortality

There were no evidences of a test item treatment-related effect on the repartition of found dead pups, pups cannibalized or affected litters.

 

Pup clinical signs

In the 200 mg/kg/day group, thin appearance, cold to the touch and desquamation were considered to be related to treatment with the test item and of toxicological importance.

 

Pupviability

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

 

Pup body weight

There were no effects on mean pup body weight and body weight change.

 

Sex ratio

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

 

Macroscopic post-mortem examination of pups

There were no test item treatment-related findings.

 

Pathology

Liver weights were higher in females given 67 mg/kg/day and in males and females given 200 mg/kg/day.

At macroscopic examination, there were a thickening of the stomach in one female treated at 67 mg/kg/day and a white discoloration of the forestomach in one female treated at 200 mg/kg/day.

At microscopic examination, there were acanthosis/hyperkeratosis with ulcer and inflammation in the forestomach in females at 67 mg/kg/day and in males and females at 200 mg/kg/day (considered as adverse at 200 mg/kg/day), macrophages in the medullary sinuses of mesenteric lymph nodes from males and females treated at 67 or 200 mg/kg/day, and hepatocellular hypertrophy in one female treated at 67 mg/kg/day and in males and females treated at 200 mg/kg/day.

 

Conclusion

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and for females throughout gestation and until day 5 post-partum, at dose-levels of 22, 67 or 200 mg/kg/day.

Based on the experimental conditions of this study:

.         the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 67 mg/kg/day based on microscopic examination findings (acanthosis/hyperkeratosis with ulcer and inflammation in the forestomach in both sexes which were considered as adverse at 200 mg/kg/day),

.         the No Observed Effect Level (NOEL) for reproductive performance was considered to be 200 mg/kg/ day in the absence of any treatment-related effect on mating and fertility at this dose-level,

.         the No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was considered to be 67 mg/kg/day based on clinical signs recorded in pups at 200 mg/kg/day.