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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November 2017 - 21 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

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

Constituent 1
Chemical structure
Reference substance name:
3-methyl-1,5-pentanediyl diacrylate
EC Number:
264-727-7
EC Name:
3-methyl-1,5-pentanediyl diacrylate
Cas Number:
64194-22-5
Molecular formula:
C12H18O4
IUPAC Name:
3-methyl-5-(prop-2-enoyloxy)pentyl prop-2-enoate
Test material form:
liquid

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, the animals were 6 weeks old.
- Mean body weight: on the first day of treatment, the males had a mean body weight of 217 g (range: 194 g to 247 g) and the females had a mean body weight of 158 g (range: 141 g to 176 g).
- Fasting period before study: no
- Housing: the animals were group-housed in twos, from the same sex and group, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust (SICSA, Alfortville, France).
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for 7 days before the beginning of the study.

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

IN-LIFE DATES: 19 December 2017 to 21 March 2018.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING FORMULATIONS:
- Solution in the vehicle
- Concentration in vehicle: 5, 15 and 60 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: Gas Chromatography with FID detection (GC-FID)
Test item concentrations: remained within an acceptable range of ± 10% when compared to the nominal values (-2.3% to +4.7% of the nominal concentrations).
Homogeneity: homogenous formulations at 2 and 200 mg/mL.
Stability: stable formulation at 2 and 200 mg/mL at room temperature for 12 days.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose selection:
The dose levels were selected in agreement with the Sponsor, following the results of a previous toxicology study performed in the same species. In this study, male and female Sprague-Dawley rats received the test item daily by gavage at dose levels of 100, 300 or 1000 mg/kg/day for 2 weeks.
At 1000 mg/kg/day, in life observations were limited to ptyalism in both sexes, and lower body weight gain at treatment initiation that resulted in a lower body weight in males on Day 8. Histopathology findings consisted of adverse, moderate to marked forestomach squamous cell hyperplasia, correlating with macroscopic thickening and mass, associated with hyperkeratosis and correlating with white discoloration. In addition, there was minimal to marked hepatocellular vacuolation along with increased weights and increased lobular pattern in the liver. Minimal tubular vacuolation was additionally observed in the kidneys in females only.
At 300 mg/kg/day, the test item was clinically well tolerated. Histopathology findings consisted of minimal or slight squamous cell hyperplasia that correlated with macroscopic thickening in the forestomach, together with hyperkeratosis in a single female, and minimal to moderate hepatocellular vacuolation correlating with increased lobular pattern in the liver.
At 100 mg/kg/day, the test item was clinically well tolerated. Histopathology findings consisted of minimal to moderate hepatocellular vacuolation that correlated with increased lobular pattern in the liver.
Following results obtained in the previous toxicology study (Citoxlab France/Study No. 45402 TSR), the high dose-level chosen in the present study was 300 mg/kg/day due to adverse microscopic findings observed in forestomach in animals given 1000 mg/kg/day.

- Rationale for animal assignment: computerized stratification procedure.
Positive control:
no (not required)

Examinations

Observations and examinations performed and frequency:
MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment period, including weekends and public holidays.

CLINICAL SIGNS:
- Time schedule: each animal was observed at least once a day, at approximately the same time on the days of treatment.

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

BODY WEIGHT:
- Time schedule: the body weight of each animal was recorded once before the beginning of the treatment period, on the first day of treatment and then once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by the animals in each cage was recorded once a week during the study. Food consumption was calculated per animal and per day.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule: ophthalmological examinations were performed on all animals before the beginning of the treatment period and on control and high-dose animals on one occasion at the end of the treatment period (i.e. Week 13).

NEUROBEHAVIOURAL EXAMINATION:
Functional Observation Battery (FOB)
Each animal was evaluated once in Week 12 before the daily treatment.
This evaluation included a detailed clinical examination, the assessment of reactivity to manipulation and different stimuli, and motor activity.
The animals were randomized and all animals were observed in the cage, in the hand and in the standard arena.

Detailed clinical observation
The following parameters were assessed and graded:
- in the cage "touch escape",
- in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (two-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.

Reactivity to manipulation or to different stimuli
The following measurements, reflexes and responses were recorded:
- touch response,
- forelimb grip strength,
- pupil reflex,
- visual stimulus,
- auditory startle reflex,
- tail pinch response,
- righting reflex,
- landing foot splay,
- at the end of observation: rectal temperature.

Motor activity
For each animal, motor activity was measured by automated infra-red sensor equipment over a 60-minute period.

HEMATOLOGY:
- Time schedule for collection of blood: the parameters were determined for all animals at the end of the treatment period.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: the parameters were determined for all animals at the end of the treatment period.
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [2] were examined.

URINALYSIS:
- Time schedule for collection of urine: the parameters were determined for all animals at the end of the treatment period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [3] were examined.
Sacrifice and pathology:
ORGAN WEIGHTS: see table 4.
The body weight of each animal was recorded before euthanasia at the end of the treatment 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 euthanasia) 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 the potential determination of the bone marrow differential cell count were prepared from the femur of each animal euthanized 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 (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately 4 microns and stained with hematoxylin-eosin.

HISTOPATHOLOGY:
A microscopic examination was performed on all tissues listed in the Tissue Procedure Table (table 4):
- for the control- and high-dose animals (groups 1 and 4) euthanized at the end of the treatment period,
- for all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period.
Other examinations:
BONE MARROW:
Two bone marrow smears were prepared from the femoral bone (at necropsy) of each animal euthanized on completion of the treatment period and stained with May Grünwald Giemsa.
As no relevant abnormalities were observed during the hematological investigations, the bone marrow differential cell count was not determined and smears were archived.

THYROID HORMONES:
An additional blood sample was taken from each animal at the end of the treatment period into tubes containing K3-EDTA as anticoagulant. The levels of the thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) was not determined as there was no indication for an effect on the pituitary-thyroid axis.
Statistics:
Citox software will be used to perform the statistical analysis of body weight, food consumption, hematology, blood biochemistry and urinalysis data.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only test item-related clinical sign, ptyalism, was observed in one female given 75 mg/mg/day and in all animals given 300 mg/kg/day. This sign, commonly observed when a test item is administered by gavage, was considered not to represent an adverse effect.
The other clinical signs recorded during the study, i.e. thinning of hair, alopecia, wound, chromodacryorrhea, chromorhynorrhea, abnormal growth of teeth, soiled neck or head, increased hindlimb, scabs and reflux at dosing, were isolated and/or observed both in control and test item-treated animals, with no dose-relationship. These signs were considered to be unrelated to the test item treatment.
Mortality:
no mortality observed
Description (incidence):
No deaths occurred during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights and mean body weight gains were unaffected by the test item treatment.
When compared to controls, statistically significant higher mean body weight was noted during the second week of treatment in females given 300 mg/kg/day (+6% when compared to the controls). This difference was considered to be unrelated to the test item treatment as they was of low magnitude.
Statistically significant lower mean body weight gain was observed between Weeks 7 and 8 in females given 25 mg/kg/day. This difference was considered to be unrelated to the test item treatment as they was of low magnitude and
observed without any dose relationship.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Mean food consumption was unaffected by the test item treatment in both sexes.
When compared to controls, statistically significant higher mean food consumption was noted during the last week of treatment in males given 75 mg/kg/day and from Week 9 to Week 12 included (i.e. 4 weeks) in males given 300 mg/kg/day. These differences were considered to be unrelated to the test item treatment as they were of low magnitude (less than 15% when compared to the controls), without any impact on body weight, and observed in one sex only.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes were observed in high-dose animals at the end of the treatment period, when compared to pre-test.
Chorioretinopathy was observed unilaterally in two control males.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
See table 5.
At 300 mg/kg/day, changes in hematology parameters recorded at the end of the treatment period consisted of higher mean neutrophil counts in males and higher mean monocyte counts in both sexes. These changes were considered to be of no toxicological importance as there were of low magnitude. In addition, mean leucocytes count was not affected in males and the slight increase in mean leucocytes count observed in females was observed without any dose relationship and was of low magnitude.

At 25 or 75 mg/kg/day, no changes were observed at the end of the treatment period in both sexes.

Statistically significant differences between control and test item-treated animals, namely in mean packed cell volume (higher in males given 75 or 300 mg/kg/day) and mean cell hemoglobin concentration (lower in males given 75 mg/kg/day) were considered to be unrelated to the test item treatment as they were of low magnitude, inconsistent between sexes and/or had no dose relationship.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
See table 6.
At 300 mg/kg/day, changes in blood bichemistry parameters recorded at the end of the treatment period consisted of higher mean inorganic phosphorus in both sexes and potassium, urea and total bilirubin levels in males.
At 75 mg/kg/day, changes consisted of higher mean potassium and total bilirubin levels in males and inorganic phosphorus level in females.
These changes were considered to be of no toxicological importance as there were of low magnitude and without any dose relationship, and/or obsersed in one sex only.
At 25 mg/kg/day, no relevant changes were observed at the end of the treatment period in both sexes.

Statistically significant differences between control and test item-treated animals, namely in mean sodium (lower in all test item-treated males), mean chloride (lower in males and females given 300 mg/kg/day), mean inorganic phosphorus (lower in males given 25 mg/kg/day) and mean urea levels (higher in females given 75 mg/kg/day), and mean aspartate aminotransferase (lower in males given 25 mg/kg/day and in females given 75 mg/kg/day) and mean alanine aminotransferase activities (lower in females given 75 mg/kg/day) were considered to be unrelated to the test item treatment as they were of low magnitude, inconsistent between sexes and/or had no dose-relationship.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No differences between control and test item-treated animals were observed at the end of the treatment period.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
See table 10.
Motor activity and Functional Observation Battery
When compared to control mean values, there were no relevant differences in the motor activity of test item-treated animals at the end of the dosing period. The apparent higher mean number of horizontal and rearing movements in males given 300 mg/kg/day were not considered to be test item treatment-related since these numbers were slight in amplitude and without any correlating clinical signs.
No neurologic, autonomic or behavioral changes that could be related to the treatment were observed during the FOB assessment phase (e.g. touch escape, reactivity to handling, grooming).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See table 7.
Liver weights were minimally increased in high dose males and females, as well as in low dose males. Although minor, these differences correlated histologically with periportal vacuolation and were considered treatment-related.
All other differences were considered unrelated to treatment. This included a statistically significant increase in uterus weight at the dose of 75 mg/kg, reflecting differences in cycle compared to controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See table 8.
Accentuated lobular pattern was observed at all dose levels, mainly in males. It correlated histologically with periportal vacuolation.
In addition, white discoloration or thickening of the forestomach were observed in a few high dose rat, correlating with acanthosis.

All other observations belonged to the spectrum of spontaneous changes in rats of this age and strain, including various discolorations noted in the liver, which appeared in treated as in control animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See table 9.
Treatment related changes were observed in the liver and stomach.
A dose-related higher incidence and severity of periportal vacuolation was present in the liver at all dose levels in males and from the dose of 75 mg/kg/day in females. In the absence of relevant clinical pathology differences between control and treated groups, and in absence of evidence of liver degeneration or necrosis, this change, which is frequently observed with a low severity in control rats, is considered non-adverse.

Dose-related minimal to moderate acanthosis of the squamous portion of the stomach was observed in both sexes from the dose of 75 mg/kg/day. In two high-dose males, it was accompanied by minimal erosion. These changes most probably reflect a mild irritant effect of the test item. Given the relatively low scores and incidences, they are not regarded as adverse.

All other changes belonged to the spectrum of spontaneous pathology in rats of this age and strain.

In conclusion, once daily oral administration of the test item at doses of 25, 75 or 300 mg/kg/day to rats for 13 weeks produced dose-related minimal to moderate periportal vacuolation in the liver at all doses, and minimal to moderate acanthosis in the forestomach (associated with erosion at the high-dose) from the dose of 75 mg/kg/day. These changes were not considered as adverse.
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 5: Hematology

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

25

75

300

0

25

75

300

Neutrophils (G/L)

1.54

1.57

1.48

2.18**

0.78

0.65

0.62

0.81

% from controls

NA

+2

-4

+42

NA

-17

-21

+4

Monocytes (G/L)

0.27

0.28

0.44

0.57**

0.15

0.14

0.16

0.23*

% or fold from controls

NA

+4

+63

x2.1

NA

-7

+7

+53

Statistically significant from controls (in bold): *: p < 0.05; **: p < 0.01; NA: not applicable.

 

Table 6: Blood biochemistry

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

25

75

300

0

25

75

300

Potassium (mmol/L)

3.88

4.01

4.33**

4.26*

3.56

3.73

3.87

3.83

% from controls

NA

+3

+12

+10

NA

+5

+9

+8

I. phos. (mmol/L)

2.04

1.87*

2.12

2.21*

1.40

1.52

1.70*

1.71**

% from controls

NA

-8

+4

+8

NA

+9

+21

+22

Urea (mmol/L)

3.5

3.8

3.8

4.1*

4.2

4.8

5.0*

4.7

% from controls

NA

+9

+9

+17

NA

+14

+19

+12

Total bilirubin (µmol/L)

0.35

0.37

0.83

0.85

1.18

1.17

1.35

1.37

% from controls

NA

+6

x2.4

x2.4

NA

-1

+14

+16

I. phos.  : inorganic phosphorus

Statistically significant from controls (in bold): *: p < 0.05; **: p < 0.01; NA: not applicable.

 

Table 7: Organ weights

 

Selected differences in organ weights (% from controls)

Gender

Males

Females

Group

2

3

4

2

3

4

Dose level (mg/kg/day)

25

75

300

25

75

300

Number of animals

10

10

10

10

10

10

Liver

 

Absolute (%)

+11

+6

+15

-2

+1

+12*

Relative to body weight (%)

+8*

+2

+10**

-4

0

+7#

          *: p = 0.05; **: p = 0.01 (Dunnett’s test) (based on actual values and not on the percentages presented in the table)

 #: p = 0.05 (Dunn’s test) (based on actual values and not on the percentages presented in the table)


 

Table 8: Macroscopic observations

 

Incidence of selected macroscopic observations

Gender

Males

Females

Group

1

2

3

4

1

2

3

4

Dose level (mg/kg/day)

0

25

75

300

0

25

75

300

Number of animals

10

10

10

10

10

10

10

10

Liver

 

Accentuated lobular pattern

0

5

5

9

0

1

0

4

 Forestomach                
 Thickening  0  0  0  2  0  0  0  0
 White discoloration  0  0  0  1  0  0  0  1

 

Table 9: Microscopic observations

 

Incidence and severity of selected histological changes

Gender

Males

Females

Group

1

2

3

4

1

2

3

4

Dose level (mg/kg/day)

0

25

75

300

0

25

75

300

Number of animals

10

10

10

10

10

10

10

10

Liver

 

Vacuolation, periportal

 

Minimal

4

4

2

4

1

3

4

6

Slight

0

5

5

2

1

0

1

2

Moderate

0

0

0

1

0

0

0

0

Stomach

 

Erosion

 

Minimal

0

0

0

2

0

0

0

0

Acanthosis

 

Minimal

0

0

2

0

0

0

2

3

Slight

0

0

0

2

0

0

0

1

Moderate

0

0

0

2

0

0

0

2

 

Table 10: Motor activity

 

Sex

Male

Female

Dose level (mg/kg/day)

0

25

75

300

0

25

75

300

Horizontal movements

402

406

426

484

823

721

838

710

% from controls

-

+1

+6

+20

-

-12

+2

-14

Rearing

98

87

97

129

162

180

179

148

% from controls

-

-11

-1

+32

-

+11

+10

-9

-: not applicable.

Applicant's summary and conclusion

Conclusions:
The toxicity of the test item was evaluated after daily administration (gavage) to rats at dose levels of 25, 75 or 300 mg/kg/day for 13 weeks.
The test item was clinically well tolerated at all dose levels and induced only non adverse changes in pathology.
Consequently, under the experimental conditions of the study, based on the absence of adverse effects, the No Observed Adverse Effect Level (NOAEL) after the 13-week treatment period was established at 300 mg/kg/day in both sexes.
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.

 

Methods

Three groups of 10 males and 10 females Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item at dose levels of 25, 75 or 300 mg/kg/day for 13 weeks. The test item was administered as a solution in the vehicle (corn oil) under a constant dosage volume of 5 mL/kg/day.

Another group of 10 males and 10 females Sprague-Dawley rats received the vehicle alone under the same experimental conditions and acted as a control group.

The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 4, 9 and 13 were determined using a Gas Chromatography with FID detection analytical method.

The animals were observed daily for mortality and clinical signs.Detailed clinical examinations were performed weekly and a Functional Observation Battery (FOB) was conducted in Week 12.

Body weight was recorded once pre-test, on the first day of treatment and then at least once a week. Food consumption was recorded once a week.

Ophthalmology examinations were performed pre-test on all animals and at the end of the treatment period on all control- and high-dose animals.

Hematology, blood biochemistry and urinalysis investigations were performed at the end of the treatment period.

On completion of the treatment period, the animals were sacrificed and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues for the control- and high-dose animals and on liver and stomach with forestomach from the low- and intermediate-dose animals sacrificed at the end of the treatment period.

 

Results

Actual concentrations of the test item in the administered dose formulations analyzed in Weeks 1, 4, 9 and 13 remained within the acceptable range of ± 10% when compared to the nominal values.

No unscheduled deaths occurred during the study.

Ptyalism was the only test item-related clinical sign observed during the study. It was considered not to represent an adverse effect.

No neurologic, autonomic or behavioral changes were observed in Week 12 during the FOB assessment phase.

No test item-related effects were observed on body weight, body weight change or food consumption.

No test item-related ophthalmological changes were observed.

In clinical pathology, no changes that could be related to treatment were noted in treated animals.


Liver weights were minimally increased in high dose males and females, as well as in low dose males and accentuated lobular pattern was observed at all dose levels, mainly in males. These differences correlated histologically with periportal vacuolation. In addition, white discoloration or thickening of the forestomach were observed in a few high dose rat, correlating with acanthosis.

 

At microscopic examination, a dose-related higher incidence and severity of periportal vacuolation was present in the liver at all dose levels in males and from the dose of 75 mg/kg/day in females.

Dose-related minimal to moderate acanthosis of the squamous portion of the stomach was observed in both sexes from the dose of 75 mg/kg/day. In two high dose males, it was accompanied by minimal erosion. These changes most probably reflect a mild irritant effect of the test item and were considered to be non adverse.

 

Conclusion

The toxicity of the test item was evaluated after daily administration (gavage) to rats at dose levels of 25, 75 or 300 mg/kg/day for 13 weeks.

The test item was clinically well tolerated at all dose levels and induced only non adverse changes in pathology.

Consequently, under the experimental conditions of the study, based on the absence of adverse effects, the No Observed Adverse Effect Level (NOAEL) after the 13-week treatment period was established at 300 mg/kg/day in both sexes.