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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Santoflex IP, concentration: 97.2% active ingredient

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: contained in the diet
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 180, 360, 720 ppm (males: 0, 13.5, 26.5, 54.0; females: 0, 15.6, 30.0, 59.0 mg/kg/d)
Basis:

No. of animals per sex per dose:
10 per sex and dose
Control animals:
yes, plain diet

Results and discussion

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
180 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: slight increase in liver weights , increase in albumin
Dose descriptor:
LOAEL
Effect level:
360 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: changes in hematology, clinical chemistry, significant increased liver weights
Dose descriptor:
LOAEL
Effect level:
180 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: significant increase in liver weight (+27%)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mortality

One high dose and one mid dose female were found dead on Test Day 93 following collection of terminal blood samples. These deaths were attributed to the stress of bleeding and not the administration of the test substance. All of the remaining and treated animals survived to the duration of the study.

Physical Observations

Soft stool was observed in 1 - 5 high dose males only during the study. This finding was not observed in any of the other control or treated animals. While this finding was seen in only a small number of high dose animals, its presence in the high dose only may suggest a relationship to test substance administration. Other clinical observations were of the type commonly seen in rodent studies of this duration.

Ophthalmology

Ophthalmoscopic examinations conducted at study termination were unremarkable. No treatment-related effects were evident.

Body Weights

The mean body weight and body weight gain of the treated males at 720 ppm were slightly lower than control throughout the study. The mean body weights of the treated females were considered comparable to or exceeded control throughout the study.

The differences from control noted in the mean body weight and mean body weight gain data suggest a treatment-related effect in the males at a dose level of 720 ppm.

Table: Mean body weight gain in treated male and female rats during the three month treatment

        Mean body weight gains (grams) months 0 - 3
 Group  Dose level (ppm)  Males  Females
 I  0  300  122
 II  180 303   139
 III  360  308  124
 IV  720  280  112

Table: Percent differences from control in the mean body weights of the treated males and females following 3 months treatment:

        Mean % difference from control (month 3)
 Group  Dose level (ppm)  Male  Female
 II  180  -0.1  +7.5
 III  360  +2.0  +1.8
IV   720  -4.7  -2.3

Food Consumption

Mean food consumption values in the treated males and females were unremarkable.

Test Substance Intake

Mean test substance intake was calculated based on individual body weight and food consumption data and nominal dose levels. Mean test substance intake values calculated over the 3 month duration of the study were as follows:

Table: Substance Intake:

        IPPD (mg/kg/d)
 Group  Dose level (ppm)  Male  Female
 II  180  13.5  15.6
 III  360  26.5  30.0
 IV  720  54.0  59.9

Dietary Analysis

Analysis of treated diets indicate that the diet mixing procedure resulted in homogeneously prepared diets and that the desired dietary

concentrations were achieved during the study.

Homogeneity analyses were performed on Group II and IV dietary batches. Mean nominal values of 107% for Group II and 100% for Group IV were obtained from the homogeneity analyses. During the course of the study, the following mean nominal values were obtained: Group II 112%, Group III 105% and Group IV 96.8%.

Hematology

Treatment-related findings were observed in several hematology parameters in the males and/or females at dose levels of 360 and 720 ppm. The affected parameters included significant reduced hemoglobin concentration and hematocrit values in males and females at 360 and 720 ppm at 6 weeks; significant reduced hemoglobin concentration in females at 720 ppm at 13 weeks; elevated platelet counts in males at 360 and 720 ppm at 6 weeks; and reduced mean erythrocyte counts in females at 360 and 720 ppm at 6 weeks and at 720 ppm at 13 weeks. These changes were statistically significant and were considered treatment-related. Based on these changes the authors suggested the no observed effect level for the hematology data to be 180 ppm.

Mean hematology values (week 6 males)

 Group  HGB (g/dl)  HCT (%)  RBC (10E6/µl)PLAT (10E6/µl)  PLAT (10E5/µl)  WBC (10E3/µl)
 I (0 ppm)  16.3 ± 0.5  43 ± 1  7.44 ± 0.26  9.93 ± 1.73  13.8 ± 3.1
 II (180 ppm)  15.8 ± 0.8  42 ± 2  7.38 ± 0.38  9.64 ± 1.77  14.0 ± 3.7
 III (360 ppm)   15.0**± 0.7  41**± 2  7.08 ± 0.35  12.33*± 2.05  16.0 ± 2.2
 IV (720 ppm)  14.6**± 0.5  40**± 1  7.30 ± 0.39 13.09** ± 2.12  14.3 ± 2.1

Mean hematology values (study termination males)

 Group  HGB (g/dl)  HCT (%)  RBC (10E6/µl)PLAT (10E6/µl)  PLAT (10E5/µl)  WBC (10E3/µl)
 I (0 ppm) 16.3 ± 1.6  43 ± 4  7.50 ± 0.76  9.99 ± 0.76  10.9 ± 2.4
 II (180 ppm)  15.7 ± 1.1  42 ± 3  7.40 ± 0.63  10.11 ± 1.53  11.7 ± 4.1
 III (360 ppm)  15.8 ± 1.0  42 ± 3  7.55 ± 0.56  10.78 ± 1.54  14.8 ± 3.4
 IV (720 ppm)  15.2 ± 1.1  40 ± 3  7.58 ± 0.62 11.73  ± 2.16  12.8 ± 4.6

Mean hematology values (week 6 females)

 Group  HGB (g/dl)  HCT (%)  RBC (10E6/µl)PLAT (10E6/µl)  PLAT (10E5/µl)  WBC (10E3/µl)
 I (0 ppm)  15.7 ±  41 ± 1  6.82 ± 0.25 10.56 ± 1.30  8.7 ± 2.6
 II (180 ppm)  15.2 ± 0.4  41 ± 1  6.81 ± 0.22  10.32 ± 1.06  10.6 ± 2.5
 III (360 ppm)  14.5**± 0.5  40*± 1  6.53*± 0.23 11.86 ± 2.21  9.1 ± 2.4
 IV (720 ppm)  14.1**± 0.6  39**± 1  6.37**± 0.21  12.21 ± 1.72  10.3 ± 2.9

Mean hematology values (study termination females)

 Group  HGB (g/dl)  HCT (%)  RBC (10E6/µl)PLAT (10E6/µl)  PLAT (10E5/µl)  WBC (10E3/µl)
 I (0 ppm)  16.4 ± 1.2  43 ± 3  7.03 ± 0.55 10.32  ± 2.58  8.2 ± 2.1
 II (180 ppm)  15.7 ± 1.7  42 ± 5  6.99 ± 0.77  9.59 ± 2.55  8.1 ± 1.7
 III (360 ppm)  15.0 ± 1.6  40 ± 5  6.72 ± 0.69 9.83  ± 1.52  7.2 ± 1.6
 IV (720 ppm)  14.9**± 0.4  40 ± 1  6.68 ± 0.19 10.59  ± 0.86  8.7 ± 1.8

HGB: hemoglobin concentration, HCT: hematocrit, RBC: erythrocyte count, PLAT: platelet count, WBC: total leukocyte count

significant different: * p<0.05, ** p<0.01

Clinical Chemistry

Several clinical chemistry parameters were altered during the course of the study.

The effects included significant increases in total protein in males and females of the mid and high dose groups at week 6 and in males of the mid and high dose groups at study termination. A significant increase in albumin was noted in all treated males and in mid and high dose females at week 6; a significant increase in mid and high dose males at study termination and in mid and high dose females at week 6. Calcium levels were significant increased in all treated animals at week 6 and in low and high-dose males at termination. The chloride values were significantly decreased in mid and high dose males at study termination and in low and high dose females at week 6 and in all treated females at termination. Also noted but not considered biologically relevant were decreases in SGOT, SGPT and alkaline phosphatase. .

The authors suggested: while these differences from control did not exhibit a strong dose response in all cases and are generally not indicative of significant toxicity the consistency with which these differences from control occurred suggests that they were treatment-related.

Table: Mean clinical chemistry (males week 6)

 Group  SGOT (IU/l)  SGPT (IU/l)  ALKPHOS (IU/l)  T PROT (g/dl)  ALB (g/dl)  GLOB (g/dl)  CL (mEq/l)  Ca++ (mg/dl)
 I (0 ppm)  115 ± 19  35 ± 5  120 ±18  6.3 ± 0.9  3.9 ± 0.2  2.4 ± 0.1  102±2  10.4±0.2
 II (180 ppm)  128 ± 29  38 ± 8  98 ± 13 6.3  ± 0.3  4.1* ±0.2 2.2 ± 0.4  100±2 10.7* ±0.3
 III (360 ppm)  98 ± 10  32 ± 4  94*±26  6.7*±0.2 4.1* ±0.1  2.6±0.2  100±2  10.9**±0.3
 IV (720 ppm)  98± 20  29 ± 6  94*±23  6.8**±0.3  4.2**±0.1  2.6±0.3  100±2  10.9**±0.2

Table: Mean clinical chemistry (males study termination)

 Group  SGOT (IU/l)  SGPT (IU/l)  ALKPHOS (IU/l)  T PROT (g/dl)  ALB (g/dl)  GLOB (g/dl)  CL (mEq/l)  Ca++ (mg/dl)
 I (0 ppm)  95 ± 22  35 ± 8  82 ± 16  6.3 ±0.2  3.5 ±0.2  2.8±0.2  102±1 9.7 ±0.3
 II (180 ppm)  93 ± 24  38 ± 8  74 ± 18  6.5±0.3  3.6±0.2  2.9±0.3  101±1  10.1*±0.4
 III (360 ppm)  81± 18  32± 4  64 ± 20  6.9**±0.3  3.7±0.1  3.2*±0.3 101* ±2  10.0±0.3
 IV (720 ppm)  68*±14  25**±2  62*±11  6.8**±0.3  3.7±0.1  3.1*±0.3  100*±1  10.1*±0.3

Table: Mean clinical chemistry (females week 6)

 Group  SGOT (IU/l)  SGPT (IU/l)  ALKPHOS (IU/l)  T PROT (g/dl)  ALB (g/dl)  GLOB (g/dl)  CL (mEq/l)  Ca++ (mg/dl)
 I (0 ppm) 90 ±17  34±8  58±18  6.2±1.0  3.7±0.3  2.5±0.8  103±1  10.3±0.5
 II (180 ppm)  71±14  29±8  61±22  6.7±0.5  4.0±0.4  2.6±0.5  101**±2  10.8*±0.4
 III (360 ppm)  74±22  25*±5 755± 18  7.0*±0.6  4.1*±0.4 2.9**± 0.3  102±1  10.8**±0.3
 IV (720 ppm)  60**±14 24**±3   52±14 7.1* ±0.3 4.3** ±0.2 2.8*± 0.2  101**±2  11.3**±0.2

Table: Mean clinical chemistry (females study termination)

 Group  SGOT (IU/l)  SGPT (IU/l)  ALKPHOS (IU/l)  T PROT (g/dl)  ALB(g/dl)  GLOB (g/dl)  CL (mEq/l)  Ca++ (mg/dl)
 I (0 ppm)  89 ±9  32±9  41±17  6.6±0.4  3.8±0.3  2.8±0.4  104±1  10.1±0.4
 II (180 ppm)  84±20  31±15  35±10  6.9±0.8  4.1±0.5 2.8 ±0.5  102*±1  10.2±0.7
 III (360 ppm)  81±16  26±6  35±9  7.0±0.6  4.0±0.4  2.9±0.3 102*±1  10.2±0.6
 IV (720 ppm)  65**±13 21**± 3  35±7  7.3±0.5  4.2±0.4  3.0±0.4  102*±2  10.5±0.5

SGOT: Serum Glutamic Oxaloacetic Transaminase, SGPT: Serum Glutamic Pyruvic Transaminase, ALK PHOS: Alkaline Phosphatase, T PROT: Total proteimn, ALB: Albumin, GLOB: Globulin (calculated), CL: Chloride, Ca++: Calcium

statistical significant: * p<0.05, ** p<0.01

(parmeter not included in tables, which showed no significant differences from control: blood urea nitrogen, crearinine, fasting glucose, total bilirubin, sodium, potassium, inorganic phosphorusGamma Glutamyl Transpeptidase)

Terminal Organ and Body Weights, Organ to Body Weight and Organ to Brain Weight Ratios

The mean liver weights, liver to body weight and liver to brain weight ratios were significantly increased in the treated males at dose levels of 360 and 720 ppm and in the treated females at dose levels of 180, 360 and 720 ppm.

In addition mean spleen and kidney weights, organ to body weight, and organ/brain weight ratios were also elevated in the females only at a dose level of 720 ppm. There were no microscopic findings associated with these organ weight changes and while these increased organ weights were attributed to treatment they were not considered indicative of significant toxicity.

Table: Terminal organ and weight, organ/body weight and organ/brain weight (males)

 Group (ppm)  Terminal body weight (g)  Liver Wt (g)  Liver/body weight (x/100)  Liver/Brain (x/1)
 I (0 ppm)  505 ± 28  13.497 ± 1.116  2.67 ± 0.13  6.34 ± 0.52
 II (180 ppm)  508 ± 67  15.742 ± 3.287 (+17%)  3.08 ± 0.26  7.42 ± 1.30
 III (360 ppm)  514 ± 42  16.929* ± 1.762 (+25%)  3.29** ± 0.12  7.99** ± 0.79
 IV (720 ppm)  478 ± 50  18.158** ± 3.073 (35%) 3.78** ± 0.31   8.63** ± 1.57

Table: Terminal organ and weight, organ/body weight and organ/brain weight (females)

roup (ppm)  Terminal body weight (g)  Liver Wt (g)  Liver/body weight (x/100)  Liver/Brain (x/1)
 I (0 ppm)  266 ± 23  7.378 ± 0.694 2.78 ± 0.17
 II (180 ppm) 289 ± 35   9.382** ±1.403 (27%)  3.25** ± 0.24  4.82** ± 0.66
 III (360 ppm)  270 ± 19 9.004* ± 0.467 (22%)  3.35** ± 0.25  4.54** ± 0.22
 IV (720 ppm)  258 ± 20 10.950** ± 1.430 (48%) 4.24** v 0.24  5.51** ± 0.58

Females terminal spleen weights (g): control: 0.389 v 0.046, 720 ppm: 0.480* ±0.103; organ/body weight ratio: control: 1.47 v0.19, 720 ppm: 1.85** ± 0.32; organ/brain ratio: control: 1.98± 0.24, 720 ppm: 2.41* ± 0.46

Females terminal kidney weights (g) : control 1.910 ± 0.177, 720 ppm:2.221* ± 0.249; organ/body weight ratio: control: 7.21

±0.71, 720 ppm: 8.63** ± 0.91; organ/brain ratio control:0.97 ± 0.09, 720 ppm:1.12* ± 0.10

Pathology

All animals were examined postmortem for the presence of grossly visible morphologic abnormalities. Those observed occurred sporadically and were not considered to be related to the test substance.

Histology

Tissues from all animals in the control and high dose groups were examined microscopically. In addition the lungs, liver, spleen and kidneys from the low and mid dose animals were evaluated. There were no microscopic findings evident which were attributed to the administration of the test substance.

Males:

Testes: germinal epithelium: atrophy: control 1/10, high dose: 0/10

Epidymides: lymphoid cell infiltrate: control: 1/10, high dose: 3/10

prostate: lymphoid cell infiltrate: control: 5/10, high dose: 2/10

seminal vesicles: no findings control 0/10, high dose: 0/10

Females:

ovaries:congestion: control: 0/10, low: 1/10, mid: 1/10, high dose: 0/10

ovaries: minieral deposition: control: 0/10, high dose: 1/10

uterus: eosinophil infiltrate: control 9/10, low: 3/3, mid: 2/2, high: 10/10

uterus: hydrometra: control: 3/10, low 2/3, mid: 1/2, high: 5/10

uterus: lymphoid cell infiltrate: control: 0/10, high: 2/10

Applicant's summary and conclusion