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

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 June 2016 - 22 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

21 September 1998

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: solid paste

Details on test material:

- Name: Reaction mass of Amines, coco alkyl and ß-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and ß-Alanine, N-coco alkyl derivs.
- Synonyms: Amphoram CP1 without solvent ; Amphoram CP 1 ; Amphoram CP1
- Batch No.: EL1699 (also seen as EL 1699)
- Description: yellow to brown paste
- Storage condition: at room temperature
- Expiry date: 13 January 2019.

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 Italia, Calco, Italy
- Age on the first day of treatment: approximately 6 weeks old on the day of treatment
- Mean body weight on the first day of treatment: the males had a mean body weight of 233 g (range: 210 g to 259 g) and the females had a mean body weight of 170 g (range: 139 g to 196 g)
- Fasting period before study: no
- Housing: the animals were housed by two for principal animals and by two or three for recovery animals, by sex and group, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²)
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 12 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 28 June 2016 to 22 November 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING FORMULATIONS:
- Suspension in the vehicle
- According to an homogeneity testing for a range of concentrations covering the lowest and highest used in this study: the test item was weighed, ground using a mortar and pestle, then mixed with the required amount of vehicle and stirred for at least 30 minutes.
- Concentration in vehicle: 2, 6 and 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with tandem Mass Spectrometry detection (LC/MS-MS)
Test item concentrations: remained within an acceptable range of of -12.7% to +14.4%, except for group 2 in Week 8 when compared to the nominal values (± 15% of the nominal concentrations) which was found to be at -24.3% compared to the nominal value. Taking into account the fact that the five other formulations analyzed were conform, this result was considered to have a minor impact on the results of this study.
No test item was observed in the control dose formulation.

Homogeneity/Stability:The dose formulations containing the test item in corn oil at 0.2 mg/mL and 200 mg/mL were found to be homogeneous after preparation. They are therefore considered to be suitable for routine administration in GLP Toxicological studies and should be prepared each dosing day.

Duration of treatment / exposure:

13 weeks followed by an 8-week treatment-free period.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

15 animals per sex (for control and high dose groups)
10 animals per sex (for low and intermediate dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose level selection:
The dose-levels were selected in agreement with the Sponsor, based on the results of a previous study:
- an OECD 422 study where groups of ten male and ten female Sprague-Dawley rats received the test item daily by gavage before mating, during mating and for the females during gestation and up to Day 5 post-partum, at 22, 67 or 200 mg/kg/day in corn oil. The test item treatment effects were essentially seen in the 200 mg/kg/day group with:
- clinical signs such as hypoactivity, loud breathing, half-closed eyes, reflux at dosing and/or dyspnea. Ptyalism was also observed from 22 mg/kg/day but was considered to be of minor toxicological importance,
- decreases in body weight or body weight gain and food consumption with a moderate toxicological significance, in both sexes, throughout the study,
- high neutrophils count (> 70%),
- high liver weights (also seen in the 67 mg/kg/day group),
- acanthosis/hyperkeratosis with ulcer and inflammation in the forestomach, macrophages in the medullary sinuses of mesenteric lymph nodes and hepatocellular hypertrophy. These findings were also seen at 67 mg/kg/day with lower incidence and severity,
- 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).

Therefore, 100 mg/kg/day was selected as the high dose-level. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 30 and 10 mg/kg/day).

- Rationale for animal assignment: computerized randomization procedure.

Positive control:

no (not required)

Examinations

Observations and examinations performed and frequency:

MORTALITY / MORBIDITY:
- Time schedule: once a day during the acclimation period and at least twice a day during the treatment and recovery periods.

CLINICAL OBSERVATIONS:
- Time schedule: each animal was observed once a day, at approximately the same time of day.

DETAILED CLINICAL EXAMINATION:
- Time schedule: detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then at least once a week until the end of the study.

BODY WEIGHT:
- Time schedule: once before the beginning of the treatment period, on the first day of treatment and at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: at least once a week during the study.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule: on all animals (including recovery animals), before the beginning of the treatment period (groups 1, 2, 3 and 4) and on all control and high-dose animals (including recovery animals) on one occasion at the end of the treatment period (groups 1 and 4).

HAEMATOLOGY:
- Time schedule for collection of blood: blood samples were taken from the orbital sinus of animals before the daily treatment
- Time schedule for peripheral blood: at the end of the treatment period
- Time schedule for bone marrow: on completion of the treatment period
- Anaesthetic used for blood collection: Yes - isoflurane
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY:
- Time schedule for blood collection: at the end of the treatment period
- Anaesthetic used for blood collection: Yes - isoflurane
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table [No. 2] were examined.

THYROID HORMONES (principal animals):
- Time schedule: an additional blood samplewas taken on the day of scheduled sacrifice from all principal animals
- Animals fasted: Yes

Sacrifice and pathology:

ORGAN WEIGHTS: see table 3
The body weight of each animal was recorded before sacrifice at the end of the treatment or recovery period. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all animals.

PRESERVATION OF TISSUES:
For all study animals, the tissues specified in the Tissue Procedures Table were preserved in 10% buffered formalin (except for the eyes and optic nerves and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's Fixative).
Two bone marrow smears for potential determination of the bone marrow differential cell count (see § Bone marrow) were prepared from the femur of each animal sacrificed on completion of the treatment period.

PREPARATION OF HISTOLOGICAL SLIDES
All tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin.

HISTOPATHOLOGY:
A microscopic examination was performed in principal animals on:
- all tissues listed in the Tissue Procedure Table, for the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period and for all animals found dead,
- for all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period.

Following the microscopic results of the high dose group, a microscopic examination was performed in all principal animals in groups 2 and 3 and in recovery animals in groups 1 and 4:
- forestomach,
- mesenteric lymph nodes,
- liver,
- thyroids.

Statistics:

Citox software was used to perform the statistical analyses of body weight, food consumption, hematology, and blood biochemistry data.
PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In group 4 (100 mg/kg/day), hypoactivity and loud breathing were noted in one female on Day 35.
During the recovery period, in group 4, one treated male presented hunched posture, loud breathing and dyspnea from Day 123 to Day 125 or 133. In another male, hunched posture was recorded on Day 93.
These clinical signs were considered incidental, since they were observed 1 day or for a short period, and in very few number of animals.
Other clinical signs (piloerection, alopecia, ptyalism, head or neck soiled, scabs, thinning of hair and reflux at dosing) were considered incidental since they reflected the normal inter-animal variation in this species.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the control group, one male and one female were found dead on Day 20. At necropsy, foamy content and red diffuse discoloration were observed in lungs correlating histologically with slight or moderate congestion/edema.
These changes are highly suggestive of mis-dosing.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In all groups, in male and female rats, no relevant changes in body weight and body weight gain were noted during the study period.

Other changes in body weight gain (in particular: decrease in all treated females between Days 15-22; in group 4 males between Days 43/50 and in group 4 females between Days 91/98) were considered incidental and not related to the test item administration, due to their low magnitude, the absence of changes in food consumption, and the lack of associated clinical signs.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No relevant changes in mean food consumption in both genders were noted at all doses, during the study period.
In female groups including control group, although the presence of wasting was noted in some cages, the food consumption was not considered affected by the test item.

Food efficiency:

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant changes in ophthalmological examination, in both genders, were noted at all doses, during the study period.
Chorioretinopathy was noted in two females in group 3 on Day 84, and was considered incidental since they reflected the normal inter-animal variation in this species.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant changes in hematology parameters, in both genders, were noted at all doses, during the study period.
Changes in hematology parameters (including red blood cells, white blood cells, fibrinogen, thrombocytes and lymphocytes, large unstained cells) were considered incidental since they reflected the normal inter animal variation in this species, were of minimal magnitude and/or lacked dose-relationship.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant changes in biochemistry parameters, in both genders, were noted at all doses, during the study period.
Changes in biochemistry parameters (including sodium, cholesterol, triglycerides, phosphate, bilirubin concentrations and ASAT activity) were considered incidental since they reflected the normal inter-animal variation in this species, were of minimal magnitude and/or lacked dose-relationship.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See Table 1.
Terminal sacrificed animals
When compared to controls, the mean absolute and relative liver weights were marginally higher in females treated at 30 mg/kg/day and in both sexes at 100 mg/kg/day, reaching a statistical significance for the relative weight in both sexes at 100 mg/kg/day (p<0.01 for males, p<0.05 for females).
These changes correlated with hepatocellular hypertrophy and were considered to be test item-related.
Other changes, including the statistically significant higher absolute ovaries weights were considered to be part of the normal intergroup variations and without any relationship to the test item.

Animals sacrificed at the end of the treatment-free period
There were no test item-related changes in the mean organ weights.
When compared to controls, the thymus weights were higher in females previously treated at 100 mg/kg/day (up to 50% for the relative weight) reaching a statistically significant level for the absolute weight. In the absence of similar trend at the end of the treatment period and as this was mainly due to a single animal, any relationship to the test item was excluded.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Unscheduled deaths
Foamy content and red diffuse discoloration were observed in lungs of both decedents.

Terminal sacrificed
No test item-related changes were observed at necropsy. Changes recorded were considered to be part of the normal background commonly seen in rats of this age and without any relationship to the test item.

Animals sacrificed at the end of the treatment-free period
There were no test item-related changes. Changes recorded were considered to be part of the normal background commonly seen in rats of this age and without any relationship to the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Tables 2 to 6.
Unscheduled deaths
Slight or moderate congestion/edema correlating with gross observation was noted in lungs of both decedents. These changes were highly suggestive of mis-dosing. Additional changes in lungs consisted of slight multifocal mixed inflammatory cell infiltrate with a perivascular distribution.
Slight cortical hypertrophy was seen in adrenals of the female. This observation was considered to be non specific, probably stress-related.

Terminal sacrificed
Test item-related changes were observed in the forestomach, liver, mesenteric lymph nodes and thyroids.

. Forestomach
A spectrum of changes including minimal to marked hyperplasia of squamous cells, minimal to moderate hyperkeratosis or inflammation in submucosa and minimal to slight occasional erosion/ulcers was observed in the forestomach of males at 100 mg/kg/day. In females, only minimal hyperplasia of squamous cells with hyperkeratosis was observed in a few animals at the limiting ridge along with a minimal erosion in one of them.
At 30 mg/kg/day, only slight hyperplasia of squamous cells along with minimal hyperkeratosis was observed in a single male.
Considering the severity of the lesions in males at 100 mg/kg/day the changes in this sex were considered to be adverse at this dose-level. However, in the absence of a forestomach in human stomach, changes in this nonglandular structure from rodents were considered to be of questionable toxicological relevance in human.

. Mesenteric lymph nodes
Non adverse macrophages infiltrates and/or aggregates were observed at a slightly higher incidence/severity in females at 30 mg/kg/day and in both sexes at 100 mg/kg/day than in controls. Also, there was a trend towards a higher development of the paracortex in test item-treated animals at 100 mg/kg/day when compared to controls.

. Liver
Minimal to slight hepatocellular hypertrophy was observed, at 100 mg/kg/day, particularly in males, correlating with the higher liver weight.
In females treated at 100 mg/kg/day, the incidence and severity of periportal vacuolation was higher than in controls. Although this observation is commonly seen in controls, any relationship to the test item cannot be excluded.
In the absence of associated degenerative test item-related changes, these liver changes were considered to be non adverse.
Other liver changes were considered to be part of the normal background commonly seen in rats.

. Thyroids
There was a trend towards an increase in incidence of hypertrophy of follicular cells in both sexes occasionally with decreased amount of colloid, particularly at 100 mg/kg/day suggesting a slightly higher activation of the thyroids.
These thyroid changes were considered to be minor and non-adverse and likely secondary to the liver hypertrophy

Animals sacrificed at the end of the treatment-free period
Following an 8-week treatment-free period, full recovery was seen in the forestomach, the liver and the thyroids.
In the mesenteric lymph node, only aggregates of macrophages remained slightly more pronounced in 2/5 females previously treated at 100 mg/kg/day, suggestion ongoing recovery.
Marked focal hepatocellular hyperplasia was observed in the liver of one male previously treated at 100 mg/kg/day. This isolated change in a single test item treated animal was considered to be likely incidental.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1:

 

Sex	Male			Female
Group	2	3	4	2	3	4
Dose-level (mg/kg/day)	10	30	100	10	30	100
Number of animals	10	10	10	10	10	10
- Final body weight	+7	+1	-2	+3	0	-1
- Liver
. absolute	+11	+5	+13	+7	+10	+11
. relative	+4	+4	+15**	+3	+10	+12*

Statistically significant from controls: *: p<0.05, **: p<0.01.

The significance concerned the organ weights values and not the percentages.

Table 2:

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	10	30	100	0	10	30	100
Number of animals	9	10	10	10	9	10	10	10
Hyperplasia; squamous cells
Grade 1 (minimal)	-	-	-	3	-	-	-	4
Grade 2 (slight)	-	-	1	2	-	-	-	-
Grade 3 (moderate)	-	-	-	4	-	-	-	-
Grade 4 (marked)	-	-	-	1	-	-	-	-
Total	0	0	1	10	0	0	0	4
Hyperkeratosis
Grade 1 (minimal)	-	-	1	2	-	-	-	4
Grade 2 (slight)	-	-	-	5	-	-	-	-
Grade 3 (moderate)	-	-	-	2	-	-	-	-
Total	0	0	1	9	0	0	0	4
Erosion/ulcer
Grade 1 (minimal)	-	-	-	3	-	-	-	1
Grade 2 (slight)	-	-	-	1	-	-	-	-
Total	0	0	0	4	0	0	0	1
Inflammation; submucosa
Grade 1 (minimal)	-	-	-	3	-	-	-	-
Grade 2 (slight)	-	-	-	3	-	-	-	-
Grade 3 (moderate)	-	-	-	2	-	-	-	-
Total	0	0	0	8	0	0	0	0
Edema
Grade 1 (minimal)	-	-	-	2	-	-	-	-
Grade 2 (slight)	-	-	-	5	-	-	-	-
Total	0	0	0	7	0	0	0	0

-: not observed in this group.

Table 3:

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	10	30	100	0	10	30	100
Number of animals	9	10	10	10	9	10	10	10
Development of paracortex
Grade 1 (minimal)	3	1	2	-	4	1	1	-
Grade 2 (slight)	4	7	6	7	5	6	9	7
Grade 3 (moderate)	2	2	2	3	-	3	-	3
Total	9	10	10	10	9	10	10	10
Infiltrate of macrophages
Grade 1 (minimal)	4	5	3	5	7	7	5	5
Grade 2 (slight)	-	-	-	2	-	-	2	2
Total	4	5	3	7	7	7	7	7
Aggregates of macrophages
Grade 1 (minimal)	5	5	3	7	2	3	6	4
Grade 2 (slight)	-	-	-	1	-	-	-	4
Total	5	5	3	8	2	3	6	8

-: not observed in this group.

Table 4:

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	10	30	100	0	10	30	100
Number of animals	9	10	10	10	9	10	10	10
Hepatocellular hypertrophy
Grade 1 (minimal)	-	1	-	6	1	-	1	3
Grade 2 (slight)	-	-	-	1	-	-	-	-
Total	0	1	0	7	1	0	1	3
Vacuolation; periportal
Grade 1 (minimal)	-	2	1	-	3	-	2	5
Grade 2 (slight)	1	-	-	-	-	-	-	3
Total	1	2	1	0	3	0	2	8

-: not observed in this group.

Table 5:

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	10	30	100	0	10	30	100
Number of animals	9	10	10	10	9	10	10	10
Hypertrophy; follicular cells
Grade 1 (minimal)	3	3	5	9	-	2	3	5
Total	3	3	5	9	0	2	3	5

-: not observed in this group.

Table 6:

 

Sex	Male		Female
Group	1	4	1	4
Dose level (mg/kg/day)	0	100	0	100
Number of animals	5	5	5	5
Aggregates of macrophages
Grade 1 (minimal)	4	3	2	1
Grade 2 (slight)	-	-	-	2
Total	4	3	2	3

-: not observed in this group.

Applicant's summary and conclusion

Conclusions:

The test item was administered by oral route, to male and female rats Sprague Dawleys rats, for 13 weeks at 10, 30 or 100 mg/kg/day, following by an 8-week treatment-free period.

At all doses, the test item was clinically well tolerated and no relevant signs of toxicity in hematology and biochemistry parameters were observed during the study.

At necropsy, in the forestomach, adverse changes including hyperplasia of squamous cells along with hyperkeratosis, submucosal inflammation, edema and erosion/ulcers were observed in males at 100 mg/kg/day. In females, only minor changes were seen.
In the mesenteric lymph nodes, increased macrophages was observed along with a trend towards higher development of the paracortex. In the liver, minimal to slight hepatocellular hypertrophy was seen along with higher incidence of periportal vacuolation in females. In the thyroids, there was a trend towards slightly increased follicular cell hypertrophy with decreased amount of colloid.

At 30 mg/kg/day, only minor changes were seen in the forestomach of one male and in the mesenteric lymph nodes of females.

Consequently under the experimental conditions of the study, adverse changes were observed in the forestomach of male rats treated at 100 mg/kg/day and no adverse changes were noted in female rats at high dose, following daily oral administration of the test item for 13 weeks. The NOAEL (No Observable Adverse Effect Level) was established at 30 mg/kg/day in male rats and at 100 mg/kg/day in female rats.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item, following daily oral administration (gavage) to rats for 13 weeks. On completion of the treatment period, designated animals were held for an 8-week recovery period in order to evaluate the reversibility of any findings.

Methods

Three groups of ten principal and five recovery (group 4 only) animals per sex (male and female Sprague-Dawley rats) received the test item daily by oral administration (gavage) for 13 weeks at dose-levels of 10, 30 or 100 mg/kg/day (groups 2, 3 and 4, respectively).

The test item was administered as a suspension in a vehicle (corn oil).

A control group of ten principal and five recovery animals per sex (male and female Sprague-Dawley rats) received the vehicle alone under the same experimental conditions.

At the end of the treatment period, recovery animals (groups 1 and 4) were kept for an 8-week treatment-free period.

Formulation samples were taken on Weeks 1, 2, 4, 8, 9 (group 2 only) and 13 and chemical analysis was performed to determine the concentration of test item in dose formulations.

Each animal was checked daily for mortality and clinical signs.

Body weight was recorded once before the beginning of the treatment period, on the first day of treatment and at least once a week until the end of the study.

Food consumption was recordedat least once a week during the study.

Ophthalmological examinations were performed before the beginning of the treatment and on Day 84.

Hematology and blood biochemistry were performed on all animals at the end of the treatment period.

A blood sample for thyroid hormones was collected at the end of treatment period.

On completion of the treatment period, animals were sacrificed and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on selected tissues for the control and all treated animals.

Results

The test item concentrations in the administered dose formulations analyzed in Weeks 1, 2, 4, 8, 9 and 13 remained within an acceptable range of -12.7% to +14.4%, except for group 2 in Week 8 when compared to the nominal values (± 15% of the nominal concentrations)which was found to be at -24.3% compared to the nominal value. Taking into account the fact that the five other formulations analyzed were conform, this result was considered to have a minor impact on the results of this study.

No test item was observed in the control dose formulation.

There were no test item-related moribund or prematurely sacrificed animals.

No clinical signs, no relevant changes in ophthalmological examination, in mean body weight and in mean food consumption were observed during the study.

No relevant changes were noted in hematology and biochemistry parameters, at all doses.

At microscopic examination adverse changes were noted in the forestomach of males at 100 mg/kg/day. These included hyperplasia of squamous cells along with hyperkeratosis, submucosal inflammation, edema and erosion/ulcers. In females, only minor changes were seen at this dose-level.

In the mesenteric lymph nodes, non adverse increased macrophages (infiltrates and/or aggregates) were observed along with a trend towards higher development of the paracortex.

 

In the liver, non adverse minimal to slight hepatocellular hypertrophy (correlating with higher weight) was seen along with higher incidence of periportal vacuolation in females.

 

In the thyroids, there was a non adverse slight increase in incidence in follicular cell hypertrophy in both sexes occasionally with decreased amount of colloid given 100 mg/kg/day.

 

At 30 mg/kg/day, minimal non adverse changes were seen in the forestomach of a single male. In the mesenteric lymph nodes of females there was a slight increase of macrophages (infiltrates and aggregates).

 

Following an 8-week treatment-free period complete recovery was seen in the forestomach, the liver and the thyroids.

In the mesenteric lymph node, only aggregates of macrophages remained slightly more pronounced in females previously treated at 100 mg/kg/day, suggestion ongoing recovery.

Conclusion

The test item was administered by oral route, to male and female rats Sprague-Dawleys rats, for 13 weeks at 10, 30 or 100 mg/kg/day, following by an 8-week treatment-free period.

At all doses, the test item was clinically well tolerated and no relevant signs of toxicity in hematology and biochemistry parameterswere observed during the study.

At necropsy, in the forestomach, adverse changes including hyperplasia of squamous cells along with hyperkeratosis, submucosal inflammation, edema and erosion/ulcers were observed in males at 100 mg/kg/day. In females, only minor changes were seen.

In the mesenteric lymph nodes, increased macrophages was observed along with a trend towards higher development of the paracortex. In the liver, minimal to slight hepatocellular hypertrophy was seen along with higher incidence of periportal vacuolation in females. In the thyroids, there was a trend towards slightly increased follicular cell hypertrophy with decreased amount of colloid.

 

At 30 mg/kg/day, only minor changes were seen in the forestomach of one male and in the mesenteric lymph nodes of females.

 

Consequently under the experimental conditions of the study, adverse changes were observed in the forestomach of male rats treated at 100 mg/kg/day and no adverse changes were noted in female rats at high-dose, following daily oral administration of the test item for 13 weeks. The NOAEL (No Observable Adverse Effect Level) was established at 30 mg/kg/day in male rats and at 100 mg/kg/day in female rats.