Registration Dossier

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:
from 2013-05-14 to 2013-10-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD guideline.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
2013-10-13
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: pale yellow liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, l’Arbresle, France
- Age at study initiation: 8 weeks old
- Weight at study initiation: The males had a mean body weight of 266 g (range: 218 to 309 g) and the females had a mean body weight of 168 g (range: 148 to 192 g).
- Fasting period before study: No
- Housing: The animals were housed in pairs, by sex and group, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm2) containing autoclaved sawdust (SICSA, Alfortville, France) (see § Study plan adherence). Each cage contained an object for the environmental enrichment (hut)
- Diet (e.g. ad libitum): All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch Nos. 4898480, 6318584, 3839086 and 1229567 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: The animals were acclimated to the study conditions for 28 days before the beginning of the treatment period.

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

IN-LIFE DATES: From:2013-03-04 To: 2013-10-10

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.003% (w/v) citric acid in drinking water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

The test item was prepared as a solution in the vehicle. Before use, the vehicle was degassed by sonication for 15 minutes, saturated by bubbling of argon for 5 minutes and then kept under an argon atmosphere until use.
The formulations were prepared under argon according to the following process:
+ weigh the required quantity of test item into a volumetric glass flask,
+ complete to final volume with vehicle.
After addition of the whole quantity of vehicle, the formulations were agitated for 15 minutes (under argon at room temperature and protected from light) by magnetic stirring.
The test item dose formulations were prepared daily and were used within 4 hours after preparation.
The test item dose formulations were delivered to the study room in sealed vials (under argon at room temperature and protected from light).

VEHICLE
- Justification for use and choice of vehicle (if other than water): this vehicle was used in previous studies
- Concentration in vehicle: 0, 2.5, 5 and 15 mg/kg
- Amount of vehicle (if gavage): 2 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test item in samples of each control and test item dose formulation prepared for use in weeks 1, 4, 8 and 13 was determined.
The determination of the differents concentration was made by titration
Duration of treatment / exposure:
The dose formulations were administered daily for 13 weeks (i.e. 91 to 92 days according to the necropsy schedule).
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2.5 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
5 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
15 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10 animal per sex and dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, based on the results of a previous study (CiToxLAB France/Study No. 39992 TSR) performed in the same species, in which the test item was administered daily by gavage at dose-levels of 5, 10, 20 or 50 mg/kg/day for 4 weeks. In this study, the clinical observations were limited to the dose-level of 50 mg/kg/day in males especially during Week 4, and consisted of slightly lower body weight gain/body weight loss, slightly lower food consumption and some clinical signs. Dose-related microscopic findings were observed in the liver (periportal vacuolar degeneration, single cell necrosis apoptosis and periportal hepatocytic hypertrophy) from 10 mg/kg/day onwards.

- Post-exposure recovery period in satellite groups: Yes; for 4 weeks after the end of treatment
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: every day


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study

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

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmological examinations were performed on all animals, before the beginning of the treatment period and on control and high-dose animals (except for the first ten animals per sex) on one occasion at the end of the treatment period.
As no treatment-related changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.
The pupils of the animals were dilated with tropicamide (Mydriaticum®, Laboratoires Théa, Clermont Ferrand, France). After assessment of the corneal reflex (at instillation of the tropicamide), the appendages, optic media and fundus were examined by indirect ophthalmoscopy (Oméga 180, Heine, Herrsching, Germany).

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
The following parameters were determined for all surviving animals sacrificed at the end of the treatment period and for prematurely sacrificed female B21685 (group 2).
As no treatment-related changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period

- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- Parameters checked in table N°1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Same time as haematology parameters
- Animals fasted: Yes
- How many animals: All anaimals
- Parameters checked in table N°2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
-All animals (except for the last ten group 1 and 4 animals per sex) were evaluated once in week 12.
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.
As no treatment-related changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.


OTHER:
Monitoring of estrous cycle
The estrous cycle stage was determined daily from a fresh vaginal lavage (stained with methylene blue) for all females (except for last ten group 1 and 4 females), daily for 14 consecutive days at the end of the treatment period.
As no treatment-related changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period

Epididymal sperm
Before sacrifice at the end of the treatment period, each male was anesthetized by an intraperitoneal injection of sodium pentobarbital.

The left epididymis was removed, weighed and sperm from the cauda was sampled for motility and morphology investigations
The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently kept at 20°C pending further investigation
As no treatment-related changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.

Sperm motility
The sperm was evaluated on a slide, after appropriate dilution. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope using a 40-fold magnification. Results were expressed as the proportion of motile and non-motile spermatozoa.


Sperm morphology
The morphology was determined from a sperm smear, after eosin staining and counting of 100 spermatozoa per slide. Results were expressed as the proportion of spermatozoa in each of the following categories:
+normal,
+normally shaped head separated from flagellum,
+abnormal head separated from flagellum,
+abnormal head with normal flagellum,
+abnormal head with abnormal flagellum,
+normally shaped head with abnormal flagellum.

Sperm count
After thawing, the left cauda epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron.
An aliquot of the suspension was sampled and the number of spermatozoa was counted in a microscope slide counting chamber.
Results were expressed as the number of spermatozoa per cauda and per gram of cauda.

Testicular sperm
After thawing, the left testis was weighed and ground. The resulting preparation was diluted and sperm heads resistant to homogeneization (i.e. elongated spermatids and mature spermatozoa) were counted in a Neubauer cell.
Results were expressed as a number of sperm heads per gram of testis and the daily sperm production rate was calculated (using a time divisor of 6.10).

Sacrifice and pathology:
On completion of the treatment or treatment-free period, after at least 14 hours fasting, all surviving animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.

GROSS PATHOLOGY: Yes (See table N°3)
HISTOPATHOLOGY: Yes (see table N°3)
Other examinations:
Bone marrow
Two bone marrow smears were prepared from the femoral bone (at necropsy) of each animal sacrificed 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 the smears were archived.
Statistics:
See Statistiques.docx

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
but unrelated to treatment
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
but unrelated to treatment
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY:
One female dead on day 76, but not related to treatment.

CLINICAL SIGNS:
All findings recorded during the study (i.e. unilateral mydriasis, nodosities at the urogenital region, soiled urogenital region, loud breathing, alopecia, thinning of hair and scabs) were considered to be unrelated to the test item treatment as they were present both in control and test item-treated animals, and/or were reported sporadically in only a few animals and/or without a dose-relationship

BODY WEIGHT AND WEIGHT GAIN:
The mean body weight changes were unaffected by the test item treatment during the study. Some statistically significant differences were recorded during the study between control and test item treated animals. As these differences were transient and sometimes showed opposite trends at a same dose-level, they were considered to be unrelated to the test item treatment

FOOD CONSUMPTION:
No relevant differences between control and test item-treated animals were noted during the treatment or treatment-free period.
The higher mean food consumption recorded in males given 15 mg/kg/day over the first 6 weeks of the treatment period was slight and considered to be of no toxicological importance

OPHTHALMOSCOPIC EXAMINATION
No test item-related ophthalmology findings were observed at the end of the treatment period.

HAEMATOLOGY
No effects on the hematology parameters were observed at the end of the treatment period.
The only statistically significant difference when compared to controls, i.e. higher mean leucocyte count associated with higher mean lymphocyte count in males given 5 mg/kg/day, was not attributed to the test item treatment as it was reported without any dose-relationship.

CLINICAL CHEMISTRY
At the end of the treatment period, mean alanine aminotransferase activity was higher (statistically significant) in males given 5 or 15 mg/kg/day compared to controls (1.4 to 2-fold, respectively). These changes, due to the contribution of a few individuals at 5 mg and almost all individuals at 15 mg/kg/day, were associated with some high individual aspartate aminotransferase activity. Females were not affected. The changes were attributed to the test item treatment and correlated with the microscopic findings observed in the liver from 5 mg/kg/day onwards (periportal vacuolar degeneration, hepatocellular hypertrophy and single cell necrosis/apoptosis).

Lower mean protein level was noted in females given 15 mg/kg/day (61 vs. 65 g/L in controls) associated with lower mean albumin level (37 vs. 41 g/L in controls). Although some individual values were low, the changes were isolated and not found in males; a relationship to the test item treatment was considered to be unlikely.

The other statistically significant differences observed when compared to controls (i.e. higher potassium level in high-dose males, lower calcium level in high-dose males and females, lower chloride level in low and intermediate dose males and higher glucose level in intermediate dose males) were of low magnitude and/or not dose related. Consequently, they were considered to be of no toxicological importance.

At the end of the treatment-free period, higher mean alanine aminotransferase activity was still present in males, associated with higher mean aspartate aminotransferase activity (statistically significant), but the differences from controls were minimal, suggesting partial reversibility of these changes. This correlated with the minimal changes observed in the liver of these animals (minimal vacuolar periportal degeneration).

The other statistically significant differences observed when compared to controls were noted with an opposite trend in males and females (sodium and chloride levels) or were slight and the contribution of only a few individuals (cholesterol level in males ). Consequently, they were considered to be of no toxicological importance.

NEUROBEHAVIOUR
No behavioral or neurological abnormalities were observed during the tests in any treated animal.
Motor activity (60-minute recording period) was unaffected by the test item treatment.

ORGAN WEIGHTS
. At the end of the treatment period
Increased absolute and relative-to-body adrenal gland weights (not statistically significant) were observed in females treated at 15 mg/kg/day. In view of the microscopic changes (hypertrophy of cortical cells) observed in a few females treated at 15 mg/kg/day, this difference was considered to be related to possible stress and the test item treatment. At lower dose-levels, it was considered probable that the trend toward an increase in adrenal gland weights in females was unrelated to test item administration in view of the low magnitude of these changes and of the absence of microscopic correlates.
The other organ weight changes were considered not to be related to the test item as they were of low amplitude, were not dose-related and/or had no gross or microscopic correlates. These included the liver weights, statistically increased at 15 mg/kg/day in males (+7%; p<0.05). In view of the very low magnitude of this difference, a relationship was considered to be unlikely, although there were microscopic lesions in liver

GROSS PATHOLOGY
Unscheduled death
Female B21685 given 2.5 mg/kg/day and prematurely sacrificed on day 76 was emaciated and had a small spleen and enlarged adrenal glands correlating respectively with microscopic lymphoid atrophy and cortical cell hypertrophy (see microscopic examination). The cause of moribundity could not be established.
In view of the isolated occurrence of this moribundity, it was considered not to be related to test item administration.

Terminal sacrifice
At the end of the treatment period
There were no macroscopic post-mortem findings related to test item administration.
The few macroscopic findings had no histological correlates or correlated with common histological findings in control rats, and were thus considered to be incidental.

At the end of the treatment-free period
There were no macroscopic post-mortem findings related to test item administration.
The few macroscopic findings were considered to be incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
Unscheduled death
Female B21685, prematurely sacrificed on day 76, showed microscopic features suggesting stress. Specifically, diffuse lymphoid atrophy in the spleen (correlating with the small spleen observed at necropsy) and mandibular and mesenteric lymph nodes, marked hematopoietic hypocellularity in the sternal bone marrow, hypertrophy of adrenal glands (correlating with gross enlargement), atrophy of pancreas and salivary gland acinar cells, and atrophy of adipose tissue around the aorta were observed. These changes correlated with the hematology results (neutrophilia and lymphopenia) and body weight loss when compared to the other females in the groups on day 71, although these changes were of lower magnitude than those of histopathological findings. The cause of moribundity could not be established.
No test item-related vacuolar degeneration, hepatocellular hypertrophy or single cell necrosis/apoptosis were seen in the liver of this female.
In view of its isolated occurrence, the moribundity of this animal was considered not to be related to test item administration.

Terminal sacrifice
. At the end of the treatment period
Test item-related microscopic findings were seen in the liver of males treated at 5 mg/kg/day and males and females treated at 15 mg/kg/day, and in the adrenal glands of females treated at 15 mg/kg/day.

Minimal to moderate vacuolar degeneration was seen in the hepatocytes of periportal areas in males treated at 5 mg/kg/day and in males and females treated at 15 mg/kg/day. This was associated with minimal to slight hepatocellular hypertrophy and minimal single cell necrosis/apoptosis in these areas. In two males given test item at 15 mg/kg/day, focal necrosis associated with inflammation was also seen in the periportal areas. Although not related to increased liver weights, these lesions were considered to be related to the test item administration. Minimal to slight brown pigment in Kupffer cells was seen in 2/10 females treated at 15 mg/kg/day. In view of the associated degenerative/necrotic changes and the clinical pathology correlates (increased ALAT activity in males at 5 or 15 mg/kg/day), these findings were considered to be adverse at 5 and 15 mg/kg/day.

The single altered cell focus seen in one male treated at 5 mg/kg/day was considered as a fortuitous change in view of the poor dose-relationship, the low magnitude and the isolated occurrence of this change.

Test item-related minimal to slight diffuse hypertrophy of cortical cells was observed in the adrenal glands of 3/10 females treated with test item at 15 mg/kg/day. The change correlated with the minimal increase in adrenal gland weights in females treated at 15 mg/kg/day. In view of the low magnitude and incidence of this change, and the absence of associated degenerative changes, this was considered not to be adverse.

The other microscopic findings were considered not to be related to the test item since they were seen with a similar incidence in controls or were considered to be consistent with spontaneous background lesions in rats of these strain and age.

. At the end of the treatment-free period
Minimal vacuolar periportal degeneration was seen in the livers of 10/10 males and 1/10 females previously treated at 15 mg/kg/day. No hepatocellular hypertrophy or single necrosis/apoptosis was recorded in these animals, suggesting partial reversibility of the findings noted at the end of the treatment period, and correlating with the minimal increased ALAT and ASAT activity in the males previously treated at 15 mg/kg/day when compared to controls sacrificed at the end of the treatment-free period.
In females previously treated at 15 mg/kg/day, no hypertrophy of adrenal gland cortical cells was recorded, suggesting complete reversibility of this change

OTHER FINDINGS
SEMINOLOGY:
No test item-related effects were noted at seminology investigations.
The lower mean sperm motility and lower mean number of spermatozoa in epididymides and testes of high dose males were due to the contribution of a few low individual values when compared to controls. As the differences were of low magnitude, are known physiological variations and were not supported by microscopic changes, they were considered to be unrelated to the test item treatment.

ESTROUS CYCLE:
No effects were observed on the estrous cycle.

Effect levels

Dose descriptor:
NOAEL
Effect level:
2.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on Liver effects observed at 5 and 15 mg/kg bw/day AI.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mean body weight change/Mean body weight (expressed in g)

Sex

Male

Female

Dose-level (mg/kg/day)

0

2.5

5

15

0

2.5

5

15

Treatment period

Weeks 1 to 5

+77

+84

+84

+88*

+44

+38

+42

+42

Weeks 5 to 9

+44

+44

+45

+40

+15

+21**

+18

+20**

Weeks 9 to 13

+30

+26

+31

+26

+20

+22

+23

+17

Weeks 1 to 13

+151

+154

+160

+153

+79

+82

+82

+78

% from controls

-

+2

+6

+1

-

+4

+4

-1

Mean body weight in Week 13

413

424

427

422

248

249

251

246

Treatment-free period

Weeks 13 to 17

+20

-

-

+17

+8

-

-

+5

Mean body weight in Week 17

436

-

-

448

257

-

-

249

Statistically significant from controls: *: (p<0.05) or **: (p<0.01),

-: not applicable.

 

Mean food consumption (g/animal/day)

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

2.5

5

15

0

2.5

5

15

Treatment period

Weeks 1 to 4

24.7

26.0

25.2

27.3

17.6

17.4

18.0

17.8

Weeks 5 to 8

23.4

25.0

24.9

25.1

17.8

17.8

17.8

17.8

Weeks 9 to 13

23.1

24.0

24.1

23.7

17.7

17.7

17.1

17.4

Weeks 1 to 13

23.7

24.9

24.7

25.2

17.7

17.6

17.6

17.7

% from controls

-

+5

+4

+6

-

-1

-1

0

Treatment-free period

Weeks 14 to 17

22.4

-

-

23.0

17.5

-

-

17.0

% from controls

-

-

-

+3

-

-

-

-3

a: no statistical analysis was performed.

-: not applicable.

 

Changes in blood biochemistry parameters in weeks 13 and 17

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

2.5

5

15

0

2.5

5

15

End of treatment period

ASAT (IU/L)

70

60

79

102

69

72

67

68

ALAT (IU/L)

42

45

58*

85**

40

39

36

40

End of treatment-free period

ASAT (IU/L)

73

-

-

81*

74

-

-

66

ALAT (IU/L)

49

-

-

56*

41

-

-

44

ASAT:aspartate aminotransferase; ALAT:alanine aminotransferase,

Statistically significantfrom controls*: p<0.05; **: p<0.01.

 

Mean sperm analysis data

 

Sex

Male

Dose-level (mg/kg/day)

0

2.5

5

15

% of motile sperm

95.4

93.8

92.2

90.8

% of morphologically normal sperm

96.1

95.8

96.9

95.6

Mean number of spermatozoa
(106/cauda of epididymis)

130.6

117.2

120.6

106.1

Mean number of sperm heads
(106/g of testis)

131.7

125.6

115.3

117.8

 

Main organ weight differences (in percentages) at the end of the treatment period

 

Sex

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

2.5

5

15

2.5

5

15

Exam. animals / Num. of animals

10/10

10/10

10/10

9/10

10/10

10/10

Body weight

+3

+4

0

+1

+2

+1

- Adrenal glands

 

 

 

 

 

 

  . absolute

-7

-4

-1

+9

+9

+12

  . relative

-10

-8

0

+7

+7

+10

 

Incidence and severity of test item-related findings at the end of the treatment period

 

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

2.5

5

15

0

2.5

5

15

liver/n

10

10

10

10

10

9

10

10

Degeneration; vacuolar; periportal

0

0

7

10

0

0

0

8

. grade 1 (minimal)

0

0

3

1

0

0

0

8

. grade 2 (slight)

0

0

4

7

0

0

0

0

. grade 3 (moderate)

0

0

0

2

0

0

0

0

Single cell necrosis/apoptosis

0

0

4

7

0

0

0

1

. grade 1 (minimal)

0

0

4

7

0

0

0

1

Hypertrophy; hepatocellular

0

0

3

10

0

0

0

8

. grade 1 (minimal)

0

0

3

3

0

0

0

7

. grade 2 (slight)

0

0

0

7

0

0

0

1

Adrenal glands/n

10

10

10

10

10

9

10

10

Hypertrophy; cortex; diffuse

0

0

0

0

0

0

0

3

. grade 1 (minimal)

0

0

0

0

0

0

0

2

. grade 2 (slight)

0

0

0

0

0

0

0

1

n: number of organs submitted for microscopic examination.

 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) was establihsed at 2.5 mg/kg bw/day (expressed in terms of active content).
Executive summary:

In a 13 week toxicity study performed according to OECD 408 guideline and in compliance with GLP (CIT, 2014), Tetrakis [hydroxymethyl]phosphonium chloride oligomeric reaction products with urea and tetradecylamine was administered to male and female wistar rats, at the dose levels of 0, 2.5, 5.0 and 15 mg/kg bw/day (expressed in terms of active content). This treatment period was followed by a 4 week recovery period for an half of animal of group 1 and 4.

Clinical signs, functional observations, body weight change and dietary intake were monitored during the study. The estrous cycle was determined daily for 14 consecutive days at the end of the treatment period. Heamatology and blood chemistry were evaluated for all animals at the end of the treatment, and blood biochemistry investigations were performed at the end of the treatment –free period. Ophthalmological examination was also performed on all animals before the beginning of the study and on control and high-dose animals at the end of the treatment period. Sperm analysis was also performed at the end of the treatment. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

No test-item-related mortality occurred and no test item-related clinical signs were observed during the study. The functional observation battery was unaffected by the test item treatment. No relevant differences between control and test item-treated animals were noted in body weight evolution or food consumption during the treatment or treatment-free period. No ophthalmology findings were observed at the end of the treatment period. Estrous cycle was not altered by the test item treatment. The epididymal sperm motility and morphology and the spermatozoa count in epididymides or testes were unaffected by the test item treatment.

At hematology investigations, no relevant changes were noted.

At blood biochemistry investigations, alanine aminotransferase activity was higher (statistically significant) in males given 5 or 15 mg/kg/day compared to controls (1.4 to 2-fold, respectively) at the end of the treatment period, associated with some high individual aspartate aminotransferase activity. These changes, which correlated with the microscopic findings observed in the liver from 5 mg/kg/day onwards, had partially reversed after a 4-week treatment-free period.

At pathology investigations at the end of the treatment period, adverse microscopic findings were noted in the liver (periportal vacuolar degeneration, hepatocellular hypertrophy and single cell necrosis/apoptosis) of males treated at 5 mg/kg/day and both sexes treated at 15 mg/kg/day. These changes were seen to a lesser extent at the end of the treatment-free period, thus suggesting partial reversibility.

Non adverse hypertrophy of cortical cells was observed in the adrenal glands of a few females given 15 mg/kg/day, correlating with minimally increased adrenal weights observed in these females when compared to controls. These changes were not seen at the end of the treatment-free period, suggesting total reversibility.

Under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) was establihsed at 2.5 mg/kg bw/day (expressed in terms of active content).

This subchronic toxicity study in rats is acceptable and was conducted comparably to the guideline requirements for a subchronic oral study (OECD 408) in rats.