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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2010-05-04 to 2010-07-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
other: Draft report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: USA EPA Health Effects Test Guideline OPPTS 870.3650, July 2000
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Not assignable, UVCB
EC Number:
641-088-6
Cas Number:
1229648-98-9
Molecular formula:
No molecular formula
IUPAC Name:
Not assignable, UVCB
Details on test material:
- Name of test material (as cited in study report): Polyram L200 (without solvent)
- Physical state: dark brown liquid
- Analytical purity: 88.6%
- Composition of test material, percentage of components:
Condensation product of N-C12-C18-alkylpropane-1,3-diamine, N-(3-aminopropyl)-N'-C12-C18-alkylpropane-1,3-diamine and formic acid: 88.6%N-(C12-C18)alkyl dipropylenetriamide and N-(C12-C18)alkylpropylenediamide: 10%
Other: 1.4%
- Lot/batch No.: 81222503
- Purity test date: 29 January 2008
- Expiration date of the lot/batch: 29 January 2011
- Storage conditions of test material: at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, l'Arbresle, France.
- Strain and Sanitary status: Sprague-Dawley Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®)
- Age at study initiation: males: approximately 11 weeks old; females: approximately 10 weeks old
- Weight at study initiation: Males: 380 to 468 g; Females: 201 to 262 g
- Housing: the males and females were individually housed, except during pairing, in wire-mesh cages. Towards the end of the gestation period and with their litter during lactation, the females were housed in polycarbonate cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 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): 12 h / 12 h

IN-LIFE DATES: From: 11 May 2010 To: 15 July 2010.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was mixed with the required quantity of vehicle in order to achieve the concentrations of 1, 3 and 9 mg/mL.
The dosage forms were prepared daily and were kept at room temperature in brown flasks. They were used within 4 hours of preparation.
The weighing print-outs were checked after every preparation and before delivery of the dosage forms to the animal room. The aspect of each dosage form was also recorded.

VEHICLE
- Justification for use and choice of vehicle :solubility
- Concentration in vehicle: 1, 3 and 9 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
After many months of work trying to validate the analytical method for analysis of the dosage forms, it was concluded that the method could not be validated. As a deviation to GLP, chemical analysis of the dosage forms was therefore not conducted, however the weighing prints-out from the daily preparation of the dosage forms were checked and the aspect of the dosage forms were also checked daily. This exception is considered not to impact on the overall status of the study. A discussion with all the analytical researches performed will be provided with the final report of the study.
Duration of treatment / exposure:
- Males: 15 days before mating, during the mating period (up to 2 weeks) and until sacrifice (i.e. at least 4 weeks in total)
- Females: 15 days before mating, during the mating period (up to 2 weeks), during pregnancy, during lactation until day 5 post-partum inclusive.
- Recovery animals: same treatment schedule as the principal male groups but without mating and kept for an additionnal 4-week treatment-free period.
(day 1 = first day of treatment period)

Frequency of treatment:
Once daily.
Doses / concentrations
Remarks:
Doses / Concentrations:
5, 15 and 45 mg/kg bw/day
Basis:
other: nominal
No. of animals per sex per dose:
- The low- and intermediate-dose groups were each composed of 10 males and 10 females.
- The high-dose group was composed of 15 males and 15 females.The first 10 animals/sex having been mated were sacrificed at the end of the treatment period ; the last 5 animals/sex not mated were dosed for 4 weeks and then retained for a 4-week treatment-free period.
- The control group was composed of 15 males and 15 females. As for the high-dose group, the first 10 animals/sex were mated and sacrificed at the end of the treatment period and the last 5 animals/sex were retained for a 4-week treatment-free period.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were selected on the basis of a 2-week toxicity study in the same species and strain in which dose-levels of 15, 45 or 150 mg/kg bw/day were administered. The dose-level of 150 mg/kg bw/day resulted in one premature sacrifice while at 45 mg/kg bw/day loud breathing, grey/white foci and thickening of forestomach wall were the only signs of toxicity (CIT 2010, study n°34507 TSR reported in chapter 7.5.1).

- Rationale for selecting satellite groups: Due to the strong irritant/corrosive properties of the test item, a satellite group of five animals/sex in the control and high-dose groups was included. Animals were not mated but were used for observation of reversibility, persistence or delayed occurrence of systemic/irritative toxic effects of the test item.

- Post-exposure recovery period in satellite groups: 4 weeks
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day during the acclimation and treatment-free periods and at least twice a day during the treatment period.

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:
- Males: on the first day of treatment (day 1), then once a week until sacrifice.
- Females: on the first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.
- Recovery animals: once a week throughout the study.

FOOD CONSUMPTION: Yes
- Males: once a week, over a 7 day period, from the first day of treatment until sacrifice.
- Females: once a week, over a 7 day period, from the first day of treatment through gestation (days 0-7, 7-14 and 14-20 post-coitum intervals) and lactation (days 1-5 post-partum intervals) until sacrifice.
During the pairing period, food consumption was not recorded for males or females.
-Recovery animals: once a week throughout the study.

FUNCTIONAL OBSERVATION BATTERY (FOB): Yes
The first five principal males and the first five principal females to deliver from each group were evaluated once at the end of the treatment period. For females, this was performed on day 5 post-partum after sacrifice of the pups.
This included a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity.

LABORATORY INVESTIGATIONS:
Hematology: Yes
The following parameters were determined on the first five males and the first five females to deliver of each group, on the day of sacrifice and were also carried out on the reverse animals at the end of the treatment-free period:
Erythrocytes (RBC), Hemoglobin (HB), Mean cell volume (MCV), Packed cell volume (PCV), Mean cell hemoglobin concentration (MCHC), Mean cell hemoglobin (MCH), Thrombocytes (PLT), Leucocytes (WBC), Differential white cell count with cell morphology, Prothrombin time (PT), Activated partial thromboplastin time (APTT), Fibrinogen (FIB).

Biochemistry: Yes
The following parameters were determined on the first five males and the first five females to deliver of each group, on the day of sacrifice:
Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (I.PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL.), Total proteins (PROT), Albumin (ALB), Albumin/globulin ratio (A/G), Total cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Bile acids (BIL.AC).

The following parameters were also carried out on the reverse animals at the end of the treatment-free period: Total proteins (PROT), Albumin (ALB), Albumin/globulin ratio (A/G).

Urinalysis:
The following parameters were determined for the first five males and the first five females to deliver of each group on the day of sacrifice:
volume, pH, Specific gravity, Proteins (PROT), Glucose (GLUC), Blood (hemoglobin) (BLOOD), appearance, color.
Sacrifice and pathology:
GROSS PATHOLOGY / HISTOPATHOLOGY

1. A complete macroscopic post-mortem examination was performed on all animals, also those found dead. Special attention was paid to the reproductive organs. A careful examination of the stomach lining was performed due to the strong irritant / corrosive properties of the test item.

2. Microscopic examination
* Preservation of tissues
The tissues listed in table 1 were preserved for all reverse animals, the first 5 sacrificed-as-scheduled males, the first 5 females to deliver and be sacrificed on day 6 post-partum of each group, and for animals found dead unless otherwise specified.

* Microscopic examination was performed on:
All macroscopic lesions
All animals found dead
All tissues listed in table 1 for the first 5 sacrificed-as-scheduled males and the first 5 females to deliver and be sacrificed on day 6 post-partum of the control- and high-dose groups
The forestomach, ileum and mesenteric lymph nodes from animals sacrificed on completion of the treatment-free period and from animals of the low- and intermediate-dose groups sacrificed at the end of the treatment period because of the results obtained at the end of the treatment period.





Statistics:
- Body weights and food consumption
Mean values were compared by one-way variance analysis and Dunnett test (mean values being considered as normally distributed, variances being
considered as homogeneous).
Percentage values were compared by Fisher exact probability test.

- Hematology and blood biochemistry
Citox software (version d.5) was used to perform the statistical analysis of hematology, blood biochemistry and urinalysis data

- Organ weights
PathData software (version 6.2b5) was used for 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, treatment-related
Description (incidence and severity):
clinical signs only
Mortality:
mortality observed, treatment-related
Description (incidence):
clinical signs only
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY

There were no unscheduled deaths during the study.

Hypersalivation was observed in males generally from weeks 2 to 4 of treatment at 45 mg/kg bw/day and in weeks 2 and 3 of treatment at 15 mg/kg bw/day, although in some males treated at 45 mg/kg bw/day it was observed as early as day 1 and sometimes lasted sporadically until week 6 of treatment. In females, hypersalivation was observed in the second week of the pre mating period and continued in most females throughout gestation and the beginning of the lactation period or until the end of the treatment period in females retained for the treatment-free period.

Loud breathing was observed in five males treated at 45 mg/kg/day between weeks 2 and 6 (from 2 days to 25 days) and in one male during the last 2 weeks of the treatment-free period. This last male also had round back and piloerection at the end of the treatment-free period. Six females treated at 45 mg/kg bw/dayalso had loud breathing; at the end of week 2 or week 4 for the two females which were mated, in weeks 2 to 5 in the females which were retained for the treatment-free period and towards the end of lactation period for two other females.

Half-closed eyes were observed in three males treated at 45 mg/kg bw/day for 2 to 4 days during weeks 2 or 4. Hypoactivity, half closed eyes, round back and piloerection were observed in up to five females of the high-dose group at the end of week 2 or the end of week 4 for females which were mated or around weeks 3, 4 and 5 for females which were retained for the treatment-free period. One of these females continued to have piloerection for the first week of gestation and another female, with no previous clinical signs other than hypersalivation, had hypoactivity and half-closed eyes for 2 days mid-gestation.
(see table 1 in remarks on results).

BODY WEIGHT AND WEIGHT GAIN

Males treated at 45 mg/kg bw/day gained statistically significantly less body weight during the first week of treatment when compared with the controls. Out of the 15 males, 7 males actually lost weight during this period (between -2 g and -19 g). Body weight gains were similar to those of the controls for the rest of the treatment period therefore the body weight deficit was not recouped and final mean body weight was 6% lower than that of the controls. Mean body weight remained lower throughout the treatment-free period because mean body weight gains were only similar to those of the controls and not higher, and therefore the mean body weight at the end of the treatment-free period was 10% lower than that of the controls.

Females treated at 45 mg/kg bw/day had mean body weight gains which were comparable with the controls during the treatment period but which were statistically significantly higher over the treatment-free period.

Mean body weights and mean body weight gains were comparable with the controls, or higher, in the male and female groups treated at 5 or 15 mg/kg bw/day.
(see table 2 in remarks on results).

FOOD CONSUMPTION

Correlating with the low body weight gain, during the first week of dosing males treated at 45 mg/kg bw/day had statistically significantly lower mean food consumption when compared with the controls (-22%, p<0.001). Food consumption was similar to that of the controls during the rest of the treatment period but was slightly lower during the treatment-free period. Females treated at the same dose level had slightly, but statistically significantly, lower mean food consumption during the first week of treatment (-11%, p<0.05). Mean food consumption remained slightly lower during the rest of the treatment period and during the gestation period but was comparable with that of the controls during the treatment-free and lactation periods.
The male and female groups treated at 5 or 15 mg/kg bw/day had comparable or slightly greater mean food consumption when compared with the
controls.

HAEMATOLOGY

Males, but not females, treated at 45 mg/kg bw/day had an approximately 3.5-fold and statistically significantly increased neutrophil count when compared with the controls. White blood cell count was increased as a consequence (+42%). Reversibility was observed at the end of the treatment free period for both parameters.

Males treated at 45 mg/kg bw/day also had a minimal reduction in red blood cell count, hemoglobin concentration and hematocrit at the end of the treatment period. At the end of the treatment-free period, these parameters were statistically significantly increased when compared with the controls, indicating recovery of any eventual effects.

There were no effects in males treated at 5 or 15 mg/kg/day or in females at any dose-level.
(see table 3 in remarks on results).

CLINICAL CHEMISTRY

Males treated at 45 mg/kg bw/day had a statistically significantly lower protein concentration at the end of the treatment period when compared with the controls (-6%). Albumin concentration was also decreased when compared with the controls (-8%) and since the albumin: globulin ratio was unaffected, globulin concentration must also have decreased. At the end of the treatment-free period, protein and albumin concentrations were comparable with those of the controls.

There were no effects in males treated at 5 or 15 mg/kg/day or in females at any dose-level.
(see table 4 in remarks on results).

URINALYSIS

There were no effects of treatment on urine parameters.

NEUROBEHAVIOUR

There were no treatment-related observations noted during the Functional Observation Battery and there were no effects on spontaneous locomotor activity at any dose-level.

ORGAN WEIGHTS

In the animals sacrificed at the end of the treatment period, there were no changes in the mean organ weights which were considered to be associated with administration of the test item. The mean absolute and relative thymus weights were lower in both males and females given 45 mg/kg bw/day and in females given 5 or 15 mg/kg bw/day. As these values were not statistically significant, were poorly dose-related (females), were found in isolated animals, and in the absence of histopathological changes, these variations were considered to be fortuitous.

In the animals sacrificed at the end of the treatment-free period, there were no changes in the mean organ weights which were considered to be associated with the test item. The mean absolute and relative weights of the thymus were lower in both sexes previously given 45 mg/kg bw/day, reaching astatistically significant level for the absolute value in males (p<0.05).
Any relationship with the treatment was considered to be unlikely since differences were due to isolated animals from control and high-dose group.

GROSS PATHOLOGY

There were no treatment-related macroscopic findings.

HISTOPATHOLOGY: NON-NEOPLASTIC

At 45 mg/kg bw/day, changes were observed in the gastro-intestinal tract (forestomach, ileum and jejunum) and in the mesenteric lymph node of male and female rats.
In the forestomach, pronounced signs of irritation characterized by acanthosis with hyperkeratosis, ulceration and inflammation were observed in males given 45 mg/kg/day. These changes were considered to be adverse. In females, only minimal acanthosis was observed in one animal. None of these changes were observed at 5 or 15 mg/kg bw/day.
At 45 mg/kg bw/day, enlarged foamy macrophages were observed in the lamina propria of the ileum of all but one animals and in the jejunum of only one animal. None of these changes were observed at 5 or 15 mg/kg bw/day.
Enlarged foamy macrophages (occasionally forming aggregates) associated with dilation of sinuses were observed in the mesenteric lymph nodes with a dose-related trend at all dose-levels in females and from 15 mg/kg bw/day in males . In males given 45 mg/kg bw/day, this was associated with granulocytes. In the absence of degenerative changes, this change was considered to be non adverse.

After the treatment-free period, signs of recovery were observed in animals previously given 45 mg/kg bw/day, particularly in the forestomach where only minimal acanthosis was observed in a single male. Enlarged macrophages (occasionally forming aggregates) were observed in the ileum and the mesenteric lymph nodes, but with a slightly lower incidence and/or severity than at the end of the treatment period suggesting a partial recovery.

Effect levels

Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the reduction of body weight and body weight gain, the significant changes in hematological and blood biochemical parameters observed in males treated at 45 mg/kg/day.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Summary of clinical signs

Dose-level (mg/kg/day)

0

5

15

45

Ptyalism

 

1♂

10♂, 10♀

15♂, 15♀

Loud breathing

 

 

 

5♂, 6♀

Round back

 

 

 

2♂, 3♀

Piloerection

 

 

 

2♂, 4♀

Cold to the touch

 

 

 

1♂

Hypoactivity

 

 

 

1♂, 6♀

Half-closed eyes

 

 

 

3♂, 6♀

Table 2: Summary of body weights and body weight gains (in g)

Sex

Male

Female

Dose-level (mg/kg/day)

0

5

15

45

0

5

15

45

Treatment period

 

 

 

 

 

 

 

Day 1

412

411

418

408

233

230

228

229

Day 15

452

462

473

425

253

253

256

246

Day 36

499

528

541*

470

268

 

 

271

Days 1-8

+22

+26

+26

+1#

+8

+13

+17**

+9

Days 8-15

+19

+25

+30*

+16

+11

+11

+11

+9

Days 1-36

+87

+117*

+123*

+61

+40

 

 

+37

Treatment-free period

 

 

 

 

 

 

 

Day 43

554

 

 

500*

279

 

 

286

Day 64

609

 

 

550

293

 

 

311

Days 36-64

+74

 

 

+73

+25

 

 

+40*

Table 3: Summary of hematological parameters

Sex

Male

Female

Dose-level (mg/kg/day)

0

5

15

45

0

5

15

45

White blood cell (G/L)

 

 

 

 

 

 

 

end of treatment period

10.97

11.29

10.41

15.61

11.26

12.17

11.12

11.04

end of treatment-free period

12.68

 

 

9.56*

6.89

 

 

5.90

Neutrophil (G/L)

 

 

 

 

 

 

 

 

end of treatment period

1.54

1.45

1.77

5.28*

3.33

3.31

3.24

3.51

end of treatment-free period

1.60

 

 

1.53

0.77

 

 

1.01

Red blood cell (T/L)

 

 

 

 

 

 

 

 

end of treatment period

8.72

8.43

8.51

8.22

7.19

7.24

7.24

7.42

end of treatment-free period

8.79

 

 

9.56*

8.41

 

 

8.04

Hemoglobin (g/dL)

 

 

 

 

 

 

 

 

end of treatment period

15.7

14.8

15.3

14.9

13.6

13.6

13.5

13.7

end of treatment-free period

15.2

 

 

16.4*

15.0

 

 

14.7

Hematocrit (L/L)

 

 

 

 

 

 

 

 

end of treatment period

0.46

0.44

0.45

0.44

0.40

0.40

0.41

0.41

end of treatment-free period

0.45

 

 

0.48*

0.43

 

 

0.42

Statistically significant *: p<0.05.

Table 4: summary of blood biochemical parameters

Sex

Male

Female

Dose-level (mg/kg/day)

0

5

15

45

0

5

15

45

Protein (g/L)

 

 

 

 

 

 

 

 

end of treatment period

63

62

64

59*

63

63

64

63

end of treatment-free period

65

 

 

66

70

 

 

68

Albumin (g/L)

 

 

 

 

 

 

 

 

end of treatment period

36

36

36

33

36

37

38

36

end of treatment-free period

36

 

 

36

39

 

 

39

A/G ratio

 

 

 

 

 

 

 

 

end of treatment period

1.30

1.41

1.26

1.30

1.32

1.31

1.41

1.37

end of treatment-free period

1.28

 

 

1.21

1.24

 

 

1.32

Statistically significant *: p<0.05.

Applicant's summary and conclusion

Conclusions:
Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxic effects was considered to be 15 mg/kg bw/day because of the reduction of body weight and body weight gain, the significant changes in hematological and blood biochemical parameters observed in males treated at 45 mg/kg bw/day.
The No Observed Adverse Effect Level (NOAEL) for local effects was considered to be 15 mg/kg/day because irritation was observed in the forestomach of males treated at 45 mg/kg/day. However, in the absence of a similar structure in the stomach from human beings, the effect was not considered relevant for Human health.
Executive summary:

The potential toxicity of the test substance was evaluated following repeated oral administration to rats according to OECD Guideline 422 and EPA guideline OPPTS 870.3650. A satellite group was included in the control and high-dose groups for observation of reversibility, persistence or delayed occurrence of systemic/irritative effects for 4‑week post-treatment. The study was conducted in compliance with the principles of Good Laboratory Practice regulations.

Three groups of Sprague-Dawley rats received the test substance daily, by gavage, before mating and through mating and, for the females, through gestation until day 5 post-partum. The dose-levels were 5, 15 or 45 mg/kg bw/day. The low- and intermediate-dose groups were each composed of 10 males and 10 females and the high-dose group was composed of 15 males and 15 females. In the high-dose group, the first 10 animals/sex having been mated were sacrificed at the end of the treatment period ; the last 5 animals/sex not mated were dosed for 4 weeks and then retained for a 4-week treatment-free period.

Another group of 15 males and 15 females received the vehicle, sesame oil, alone, under the same experimental conditions and acted as a control group. As for the high-dose group, the first 10 animals/sex were mated and sacrificed at the end of the treatment period and the last 5 animals/sex were retained for a 4-week treatment-free period. The dosing volume was 5 mL/kg bw/day.

 In all animals from the principal and satellite groups, clinical signs and mortality were checked at least once daily during the treatment period and detailed clinical examinations were performed once before the beginning of the treatment period and then once a week until the end of the study. Body weight and food consumption were recorded weekly and at designated intervals throughout gestation and lactation. A Functional Observation Battery, including assessment of responses, reflexes, forelimb grip strength, landing foot splay and rectal temperature, was performed on the first five males and the first five females to deliver from each principal group at the end of the study. At the end of the Functional Observation Battery, motor activity was measured over a 1-hour period.

Blood and urine samples were taken from the first five males and the first five females to deliver from each principal group at the end of the treatment period for analysis of hematology, blood biochemistry and urine parameters. In addition, hematological and selected blood biochemical parameters were measured at the end of the treatment-free period in the animals of the satellite group.

In the principal groups, males were sacrificed after completion of the mating period and females on day 6 post-partum. Animals from the satellite group were sacrificed at the end of the treatment-free period. All animals were subjected to a complete macroscopic post-mortem examination with a careful examination of the stomach lining due to the strong irritant / corrosive properties of the test item. A microscopic examination was performed on selected organs of the first five males and the first five females to deliver from the control and high-dose groups sacrificed at the end of the treatment period, on the one non-pregnant female and on all macroscopic lesions. In addition, the forestomach, ileum and mesenteric lymph nodes from the low- and intermediate-dose groups sacrificed on completion of the treatment period and from the control and high-dose groups sacrificed at the end of the treatment-free period were examined.

 

There were no unscheduled mortalities. Hypersalivation was observed in all males and females treated at 15 or 45 mg/kg bw/day. In addition, some males and females treated at 45 mg/kg bw/day had loud breathing, round back, piloerection, hypoactivity and half-closed eyes on occassions during the study. No relevant clinical signs were observed at 5 mg/kg bw/day.

Males treated at 45 mg/kg bw/day had statistically significantly lower mean body weight gains and food consumption in the first week of treatment (seven males lost weight). The deficit was not recouped during the treatment period or during the treatment-free period. Females treated at 45 mg/kg bw/day also had statistically significantly lower mean food consumption in the first week of treatment.There were no effects at 5 or 15 mg/kg bw/day.

There were no treatment-related observations noted during the Functional Observation Battery and there were no effects on spontaneous locomotor activity at any dose-level.

Laboratory investigations showed that males treated at 45 mg/kg bw/day had a statistically significantly increased neutrophil count and a non-statistically significantly increased white blood cell count at the end of the treatment period. In addition, red blood cell count, hemoglobin and hematocrit were all slightly decreased. Protein and albumin concentrations were also decreased but at the end of the treatment-free period, all parameters were comparable with controls. There were no treatment-related effects on hematological and biochemical parameters at 5 or 15 mg/kg/day in males or in females at any dose-level.

No treatment-related effects on organ weights and no particular macroscopic findings were observed.

Microscopic examination revealed enlarged foamy macrophages graded as minimal or slight in the ileum of males and females treated at 45 mg/kg bw/day. No particular findings were found in the ileum of animals treated at 5 or 15 mg/kg bw/day. Enlarged foamy macrophages, occasionally forming aggregates at high dose and associated with dilation of sinuses, were also observed in the mesenteric lymph nodes with a dose-related trend at all dose-levels in females and from 15 mg/kg bw/day in males. At the lower doses, the finding was marginal: 1 male treated at 15 mg/kg bw/day and 1 female treated at 5 mg/kg bw/day had a minimal infiltration in the mesenteric lymph nodes. As no degenerative changes were associated, this effect was therefore considered to be non-adverse. Partial recovery was observed by the end of the treatment-free period.

Symptoms related to the strong irritant / corrosive nature of the test substance were observed in the forestomach of males treated at 45 mg/kg bw/day at the end of the treatment period. Males rats showed acanthosis and edema of the forestomach mucosa and ulcerations and hyperplasia in the forestomach.This was considered to be treatment-related and adverse. At the end of the treatment-free period, only 1/5 males had minimal acanthosis indicating recovery.

In conclusion, clinical signs of poor condition were observed in some males and females at 45 mg/kg bw/day. Week 1 body weight gain was markedly lower in males (some males lost weight) and food consumption was reduced in males and females. Males did not

recoup the low body weight gain and mean body weight remained lower at the end of the treatment-free period. A significant leucocytosis and a slight anemia together with a decrease in protein and albumin concentrations were observed in males but all showed recovery by the end of the treatment-free period. There were no treatment-related macroscopic findings or treatment-related effects on organ weights. At microscopic examination, the strong irritating/corrosive properties of the test item were confirmed through the findings in the forestomach of all males. These findings were regarded as an irritant/corrosive effect and not as symptoms of a cumulative-systemic toxicity but were considered to be adverse. Both males and females had enlarged foamy macrophages in the ileum and mesenteric lymph nodes. They probably represent histiocytes containing test-item lipid complexes which are poorly degraded by lysosomal enzymes. As no degenerative changes were associated, this effect was therefore considered to be non adverse. Recovery was observed at the end of the treatment-free period in all organs, particularly in the forestomach, where only minimal acanthosis was observed in one male.

 At 5 and 15 mg/kg/day, there were no adverse clinical signs and no effects on body weight or food consumption. None of the hematological or blood biochemical parameters were affected by treatment and no abnormal responses, reflexes or behavior were observed during the Functional Observation Battery. There were no treatment-related macroscopic findings and no treatment-related effects on organ weights.There were no particular findings in the forestomach and ileum at microscopic examination. Mesenteric lymph nodes revealed only minimal or slight enlarged foamy macrophages in three females and one male treated at 15 mg/kg/day and in one female treated at 5 mg/kg/day.

 Based on the experimental conditions of this study,  the No Observed Adverse Effect Level (NOAEL) for systemic toxic effects was considered to be 15 mg/kg/day because of the reduction of body weight and body weight gain, the significant changes in hematological and blood biochemical parameters observed in males treated at 45 mg/kg/day.

The No Observed Adverse Effect Level (NOAEL) for local effects was considered to be 15 mg/kg/day because irritation was observed in the forestomach of males treated at 45 mg/kg/day. However, in the absence of a similar structure in the stomach from human beings, the effect was not considered relevant for Human health.