10/08/2008

Toxicity and Safety -Reproduction and post-natal toxicity

• Reproduction and Post-natal Toxicity in Rats

 

Animals

 

Wistar rats SPF (specific pathogens free) were used.

 

Experimental Protocol

 

The same protocol for 90 day (12 week multiple dose toxicity) was followed.

 

Groups of 10 maleand 10 female rats were injected sc in the nape with 1 (LD) or 10 (MD) or 100 (HD) mg/kg of a suspension of P-MAPA into 10 ml/kg for 90 consecutive days.

 

At the 91st and 96th days, mating was permitted within the same group.

 

At 97th day all the male were submitted to a sperm collection by electroshock method and then sacrificed.

 

Fecundation of all the females were denoted by the vaginal compress.

 

During the next 20 days (gestation period) and after the birth, and during 12 days of lactation, females continued with the P-M.A.P.A.. administration.

 

After the birth day the number of individuals of any matrix were determined and the descendants were marked for posterior evaluation.

 

At the end of the lactation period, after a 12 h starvation, females were anaesthetized, weighted and 5 ml of blood was collected for analysis.

 

Also urine was collected at the same time.

 

In the birth day and after 12 days of life, the following parameters were evaluated: It thoracic perimeter, interauricular distance, longitudinal length, body X-ray, weight, ocular diameter and transversal length.

 

At the 13th day, 1/3 of the offsprings of each group were sacrificed and the following organs were weighted and/or measured: spleen, kidney, thymus, heart and liver.

 

Results

 

MATRIXES

Weight evolution

 

The same results for 12 week multiple dose toxicity in rats were obtained.. The results after 128 days of P-MAPA treatment reinforced those of the sub-chronic 90 days study (12 week multiple dose toxicity).

 

Excretion and Water Consumption

 

No statistical differences in this study were found.

 

Hematological Parameters

 

In the red blood cells it was possible to observe that the hemoglobin and hematocrit levels were maintained stable.

 

From the morphological point of view, erythrocytes were macrocytic and hyperchromic, with clear increase of young erythrocytes, and this was dose-dependent in the P-MAPA administration.

 

No alterations in coagulation, trombine and protrombine were found.

 

P-MAPA administration induced a dose-dependent increase of total leukocytes, in function of increase of lymphocytes and neutrophils without any morphological alterations.

 

Monocytes and eosinophils did not undergo any variation in the five analyzed groups.

 

Biochemical Parameters

 

 

Bone Metabolism

 

The evident increase of phosphorous and the mild calcium decrease, proportional to the P-MAPA dose, reveal bone metabolism alterations probably caused by: paratiroids dysfunction, D-hypervitaminose, or magnesium salt, oxalates, phosphate or citrates administration.

 

The polymeric molecule of P-MAPA has magnesium and phosphate and this may explain the results obtained.

 

This hypothesis, however, was only confirmed after the negative results in the anatomopathological studies of bone and teeth of control and treated animals, and the special experiments with tooth germs and palate in embryonic mice; in both studies, no interference at high dose was observed on intrauterine development of these structures.

 

Energetic Metabolism

 

No alterations at this level were found, compared to the control group.

 

Biochemical Parameters

 

Same results as described in 12 week multiple dose toxicity in rats were found.

 

Reproductive Performance

 

A comparative evaluation between spermatozoids from the matrixes control and treated animals did not show any morphofunctional differences.

 

Anatomopathology

 

The histopathological examinations showed all the organs and structures were within the normal range.

 

Alterations related to the P-MAPA mode of action will be discussed in separate in the organs and/or structures which could be considered as targets.

 

The anatomopathological analysis of gross intestine of the P-MAPA treated animals revealed the absence of pathological alterations, however, Peyers patches were always prominent and with lymphoid tissue activity associated to intestine (GALT) augmented.

 

The hispathological examination of lymphonodes demonstrated to be within normality. However, the analysis showed a high activity in cortical and paracortical zones, since a dense cellular population was found, with preponderance of reticular cells and B?lymphocytes.

 

The cordons and medullar sinuses were always more dense in the P-MAPA treated animals than those lymphonodes from testimony and control animals.

 

Thymus in all the treated animals were normal as compared with the control animals.

 

Spleen analysis from the P-MAPA treated animals were normal, however, presented lymphoid cells (B and T) enhanced around the penicillata arthery and the germinative centres were evidently with the presence of numerous cells of the reticuloendothelial system (macrophages) and hematic pigmented cytoplasm, when compared with those of testimony and control animals.

 

Animals treated with a high dose of P-MAPA showed a hyperfunction of the phagocytic-macrophagic-splenic system, associated with necrosis of lymphoid elements and the presence of numerous megakariocytes.

 

These observations were proportional to the P-MAPA dose applied and must be due to an activation of the immunological system.

 

DESCENDANTS

 

Number of individuals

 

No statistically significant differences between the number of the descendants from matrix treated with P-MAPA and those of matrix control were found.

 

External Development Parameters

 

Thoraxic Perimeter

 

Male younglets from treated mothers with a high dose (100 mg/kg) and all the female younglets of all three P-MAPA doses exhibited an statistically significant decrease of thoracic perimeter after 12 days, but not at 24 h after birth.

 

Longitudinal length

 

The reduction of the dorsal longitudinal length (from nose to the tail) was significant only 24 h after birth in the mothers treated with high dose of P-MAPA. After 12 days, all the younglets exhibited less longitudinal length, proportional to the P-MAPA dose.

 

Transversal Length

 

The same results as the above parameters were obtained when the ventral transversal length (from the longest finger of left anterior foot to the right one) was determined.

 

Interauricular Distance

 

A decrease of interauricular distance was observed only in the first day after birth of the younglets from mothers treated with medium and high doses of P-MAPA; however, at 12 days, this decrease of female and male was not dependent of P-MAPA dose.

 

Ocular Diameter

 

No alterations were found at the first day and after 12 days in female younglets. However , males exhibited a decrease of ocular diameter in a dose/dependent manner.

 

Body Weight

 

All the younglets, males and females, presented a dose dependent reduction of body weight. These results are indicative that the decrease of all the parameters above were related with the fact that the younglets from treated mothers with P-MAPA presented smaller sizes than those of control group.

 

Internal Development Parameters

 

Organ Weight

 

In general, the female and male younglets from treated mothers with P-MAPA exhibited lower weights than the control. However, these values were only statistically significant for liver in all three doses and for heart, thymus, spleen and kidney in the high dose.

 

CONCLUSIONS :

 

• The analysis of the results of internal and external development, revealed that none of the alterations were related with P-MAPA administration before or during gestation or lactation period.

 

• The smaller size of the younglets probably was related with the long period of P-MAPA administration and the stress produced by the inoculation of the drug.