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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:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
the racemate is characterized by CAS-number 7696-12-0
Specific details on test material used for the study:
Lot/batch No.of test material: TT-SF-01-001

Test animals

Species:
rat
Strain:
other:
Details on species / strain selection:
HsdCpb: WU rats conventionally bred
(In-house random bred)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxicology Department , Rallis Research Centre , Rallis India Limited, Bangalore - 560 058, INDIA
- Age at study initiation: 8 weeks
- Housing: Two rats per sex per cage in sterilized suspended• polypropylene cages (size: approximately L 410 x W 282 x H 150 mm) with stainless steel mesh bottom and stainless steel top grill having facilities for holding pellet food and for drinking water in glass bottle with stainless steel sipper tube.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: After veterinary examination, for good health and the suitability for the study, the rats were acclimatized for five days before the start of the treatment.
- Females used in the study were nulliparous and non-pregnant.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): 30 - 70%.
- Photoperiod (hrs dark / hrs light): 12h fluorescent light, 12 hour dark

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The test item was administered orally as admixture with food. Doses are expressed as ppm in food . Oral route was chosen because it is the potential source of human exposure.
Vehicle:
acetone
Details on oral exposure:
- VEHICLE
Acetone is being used as vehicle because it quickly evaporates and availability of historical control data.
- PREPARATION OF DOSING SOLUTIONS: The experimental food has been prepared in quantities of 10 kg (G4/G4R) and 5 kg (G2 and 03) at each mixing. The required quantity of test item for each group has been weighed (G2: 2.5 g, G3: 5 g and 64/34R: 20 g) and dissolved in acetone (25 ml for G2 and G3 and 50 ml for G4/G4R) and mixed manually with a portion (approximately 0.5 kg for G2 and G3 and 1 kg for 34/04R) of powder food in stainless steel drum for 1 - 2 minutes to prepare the premix. This premix has been added in portions to the remaining bulk food (approximately 4.5 kg for G2 and G3 and 9 kg for G4/G4R) and mixed in a ribbon mixer for 20 minutes.
For control and control recovery group, 50 ml of acetone has been added and mixed manually with portion of powder food (approximately 1 kg) in stainless steel drum for 1 - 2 minutes to prepare the premix. This premix was added in portions to the remaining bulk food (approximately 9 kg) and mixed in a ribbon mixer for 20 minutes.

*500 ppm (G2)
1000 ppm (G3)
2000 ppm (G4 and G4R)
control groups (G1 and G1R)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A homogeneity test was carried out prior to initiation of the study and at the beginning of the treatment. Three samples i.e., from top, middle and bottom layers from the experimental food of each treatment group and for control group one composite sample were collected from the blender to determine the homogeneity of the test item in the experimental food which in turn reflect the test item concentration in the experimental food. Thereafter the test item fortified food was sampled at monthly intervals to determine the test item concentration in the experimental food.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
500 ppm
Remarks:
Low dose group
Dose / conc.:
1 000 ppm
Remarks:
Medium dose group
Dose / conc.:
2 000 ppm
Remarks:
High dose group
No. of animals per sex per dose:
10 males + 10 females for each group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment: The animals procured for the study were weighed and grouped into body weight ranges i.e., 221 - 280 g for males and 151 - 200 g for females. The males in the body weight ranges i.e., 231 - 270 g and females in the body weight ranges i.e., 161 - 190 were selected and distributed to all the study groups in equal numbers. The rats which were not selected for the study were discarded. Grouping was done on last day of acclimatization period.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Veterinary examination was done prior to the initiation of treatment and weekly thereafter (±1 day) during treatment and recovery periods.

DETAILED CLINICAL OBSERVATIONS: Yes
Clinical examination was done prior to initiation of treatment and once weekly (±1 day) during treatment and recovery period.

BODY WEIGHT: Yes
Individual body weights were recorded at the beginning of the treatment and weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE:
For determining weekly food consumption, the following method was adopted.
- Day 1*: Food input 1450 g (inclusive of mesh and hopper weight)
- Food output on day 8 (inclusive of mesh and hopper weight)
*: Day '1' denotes food input at the start of each week.

The cagewise food consumption was calculated by dividing the total food consumed in 7 days by the number of animals per cage to determine the food intake/rat/week. The visual estimation of the food spillage was added to the food output data. The weekly consumption/rat was divided by the number of days (7) to obtain food consumption (g)/rat/day. This was repeated throughout the treatment and recovery period.

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
Ophthalmological examination of all animals was done with an ophthalmoscope before the start of treatment and at the end of treatment for main groups and at the end of recovery period for recovery groups, prior to sacrifice. Mydriasis was induced before examination by using 1% Tropicamide.

HAEMATOLOGY: Yes
In addition to determination of prothrombin time, the following haematological parameters were determined using Sysmex TM K-800 Automated Haematology Analyser (TOA Medical Electronics Co., Kobe, JAPAN).
1. Haemoglobin (Hb)
2. Red Blood Corpuscles (RBC) 4. Haematocrit (Hct)
3. White Blood Corpuscles (WBC)
5. Platelets (Plat)


CLINICAL CHEMISTRY: Yes
Plasma was separated in a refrigerated centrifuge at 5000 rpm for 15 minutes and analysed using Automatic clinical chemistry analyser (BM-HITACHI 704 Boehringer Mannheim, Mannheim, GERMANY) and Boehringer Mannheim (GERMANY) diagnostic kits were used for the assay of the following parameters (except urea, which was calculated manually by using BUN values):
1.Glucose (Glu) mmol/l
2. Creatinine (Creat) µmol/l
3. Inorganic Phosphorus (Pi) mmol/l
4. Total Plasma Protein (Tot.Pro.) g/I
5.Urea (mmol/l)
6. Albumin (Alb) g/l
7. Gamma Glutamyl Transpeptidase (GGT) U/l
8. Blood Urea Nitrogen (BUN) mmol/l
9. Alanine Amino Transferase (ALT) U/l
10. Aspartate Amino trasferase (AST) U/l
11. Total Cholesterol (Chol) mmol/l
12. Sodium (Na): mEq/l
Potassium (K): mEq/l
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Using specific computer programmes, body weights, net body weight gain, food consumption, laboratory investigations (haematology and clinical chemistry), organ weights and organ weight ratio data were compared by Bartlett's test for homogeneity of intra group variances.' When the variances proved to be heterogeneous, the data were transformed using appropriate transformation. The data with homogeneous intra group variances were subjected to one-way analysis of variance (ANOVA - Snedecor and Cochran, 1987). When 'F' was found to be significant, Dunnett's pairwise comparison (Scheffe 1953) of means of treated groups with the mean of the control group was done individually
Following a significant difference of a test group with the control group, the Dose Response correlation was estimated by including the control and all the treated groups and tested by 't' test.
In the case of recovery groups (reversal study), the data of treatment period and recovery period (no treatment period) were tested for homogeneity of intra group variances. When found heterogeneous, the data were transformed suitably to make the intra group variances homogeneous. The means of data with homogeneous intra group variances (two recovery groups) were compared by Students 't'-test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Veterinary examination did not reveal any test item related clinical signs.
However, incidences of local alopecia in one female of control group and hair thinning with hair regrowth in one male (at low dose) and 10 females (i.e., one in control, three in mid dose, four in high dose and two in control recovery groups) and blunt tail in one female at mid dose during treatment period were observed. During recovery period incidences of hair thinning with hair regrowth in 4 females (i.e., three in control recovery and one in high dose recovery group) was observed.
Mortality:
no mortality observed
Description (incidence):
There were no pre-terminal deaths
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The weekly mean body weights and net body weight gains were unaffected by the treatment at all the doses tested for both males and females
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food intake was unaffected by the treatment at all the doses tested for both males and females
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmological examination did not reveal any eye abnormalities.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematological parameters were unaffected by the treatment at all the doses tested for males.
For females, the value of WBC counts were significantly lower (dose-related) at all the doses tested. The value of MCHC at 1000 ppm and prothrombin time at 2000 ppm were significantly higher. The decreased value of WBC counts were considered incidental as there were no corresponding changes in platelet and differential leukocyte counts and the values were within the historical control data. The other changes were considered incidental as these changes were not in dose dependent manner. At 2000 ppm high dose recovery group an incidence of significantly lower value of neutrophils and significantly higher value of lymphocytes were observed and were considered incidental as these changes were not observed during treatment period.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
The value of sodium was significantly higher (dose-related) at all the doses tested. The increased level of sodium was considered incidental as the values were within the historical control data and of no toxicological significance. The value of creatinine was significantly higher at 1000 ppm. The value of total plasma protein and cholesterol were significantly higher at 2000 ppm.
These changes were considered incidental as these changes were not in dose dependent manner and of no toxicological significance. At 2000 ppm high dose recovery group significantly lower value of BUN, urea and potassium and significantly higher value of sodium were observed. The increased level of sodium was within the historical control data. The other changes were considered incidental as these changes were not observed during treatment period.

Females:
The values of cholesterol and sodium were significantly higher at all the doses tested and these were considered incidental as there was no dose correlation and no corresponding histopathological changes and of no toxicological significance. At 2000 ppm high dose recovery group the clinical chemistry parameters were unaffected.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Functional observation battery did not reveal any abnormalities in both sexes. However, in males a significantly higher value of landing foot splay at 2000 ppm high dose group was observed.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were no significant intergroup differences in the terminal fasting body weights in males and females
In males, there was a significant increase in the absolute and relative weight of livers in mid and high dose groups. This change was considered treatment related and microscopically this was associated with hepatocellular hypertrophy in liver. The significant increase in the relative weight of liver in high dose recovery group males was considered incidental as there were no corresponding microscopic changes.
In females, there was a significant increase in the relative weight of liver at high dose which was not considered treatment related as there were no corresponding microscopic changes. A significant decrease in the absolute weight of brain was observed in high dose recovery animals, this was considered incidental as there were no changes in the main groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment related gross pathological changes.
Petechiae in thymus were agonal hemorrhages, related to the type of death and not considered treatment related. Incidences of hair thinning and hair regrowth in skin were considered incidental as only a few incidences of epidermal hyperplasia were observed microscopically.
In females, dilatation of uterus was microscopically found to be minimal to severe dilatation. This was considered as a physiological change and not treatment related.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
More frequently observed histopathological changes were:
Liver: hepatocellular hypertrophy (minimal), necrobiotic foci (minimal) '
Lungs: mineralisation - blood vessels (minimal) lymphocytic infiltration (minimal and mild)
Spleen: increased hemosiderosis (minimal and mild)
Uterus: dilatation (minimal to severe)
Thymus: hemorrhage (minimal to moderate)

A significant increase in the incidence of hepatocellular hypertrophy in liver was observed in high dose males. This change was considered treatment related and considered reversible as there was no incidence observed in the high dose recovery males.
Necrobiotic foci in liver were minimal consisting of 1 - 3 necrotic hepatocytes surrounded by inflammatory cells and could not be explained as there was no apparent cause.
The changes in kidneys in high dose males (basophilic tubules, urothelial hyperplasia) were considered incidental as the incidences were not significantly different from the control group.
In females, the higher incidente of increased hemosideroisis in spleen at high dose was not considered treatment related as the incidence was not significantly different from the control animals. The incidences of dilatation of uterus were considered as physiological changes and not treatment related.
All other changes were also considered incidental as the incidences were similar or higher in control animals.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
NOEL
Effect level:
38 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
NOEL
Effect level:
34.8 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
NOEL
Effect level:
41.2 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
Dose descriptor:
NOAEL
Effect level:
76 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test item racemic tetramethrin had no adverse effect on general health, growth, haematological and clinical chemistry parameters, organ weights, gross and histopathology at 500 ppm and 1000 ppm; The NOEL was set to 500 ppm, based on slight effects in males only, whereas the dose level of 1000 ppm is considered to be the No Observed Adverse Effect Level (NOEL) of racemic tetramethrin, which is equivalent to 76.0 mg/kg Bwt/day, for combined sex.
Executive summary:

Tetramethrin Tech. Grade was tested for 90 day oral toxicity in Wistar rats according to test method OECD 408.

The test item was mixed in the laboratory animal food at the concentrations of 500 (G2), 1000 (G3), 2000 (G4 and G4R) ppm and fed for 90 days to 4 groups i.e low, mid, high, and high dose recovery groups, respectively. Concurrent control groups (G1 and G1R) received laboratory animal food mixed with vehicle without the test item admixture.

All study groups consisted of 10 male and 10 female rats per group. The treatment was discontinued for the high dose recovery group (G4R) at the end of the 90th day of treatment and rats of both G1R and G4R groups were maintained on normal food for a further 28 day period without being exposed to the test item for the recovery observations.

Animals from all groups were observed for clinical signs, changes in body weights, food consumption and functional observation battery. Laboratory investigations on haematology and clinical chemistry were performed at sacrifice. The rats were subjected to a detailed necropsy at terminal sacrifice.

Histopathological evaluation was carried out on all the tissues collected from control and high dose group animals, lungs from low, mid, control recovery and high dose recovery groups, liver from males of the low, mid and recovery group dose animals and gross lesions from low, mid and recovery groups at the discretion of the pathologist. The data were statistically analysed.

Under the experimental conditions described in the Material and Method section, the following results were obtained:

A. SUMMARY OF RESULTS

1. Clinical Signs and Pre-terminal Deaths, Ophthalmological Examination and Neurological Examination: There were no test item related changes in clinical signs. There were no pre-terminal deaths. Ophthalmological examination did not reveal any eye abnormalities. Neurological examination did not reveal any test item related changes.

2. Body Weights and Net Body Weight Gains: Body weights and net body weight gains were unaffected by the treatment at all the doses tested in both sexes.

3. Food intake: Food intake was unaffected by the treatment at all the doses tested in both sexes.

4. Laboratory Investigations:

a. Haematology: The treatment did not show any treatment-related changes in haematological parameters at all the doses tested in both sexes.

b.Clinical Chemistry: The treatment did not show any treatment-related changes in clinical chemistry parameters at all the doses tested in both sexes.

5. Terminal fasting body weight, organ weight and organ weight ratios: There were no treatment related changes in the terminal fasting body weights in males and females. In males, a treatment related increase in the absolute and relative weight of liver in mid and high dose groups was observed. There were no treatment-related changes in the organ weights and organ weight ratios in females.

6. Gross and histopathology: There were no treatment related gross pathological changes. Microscopically, in males hepatocellular hypertrophy in liver at high dose was considered as a treatment related reversible change.

The test item racemic tetramethrin had no adverse effect on general health, growth, haematological and clinical chemistry parameters, organ weights, gross and histopathology at 500 ppm and 1000 ppm. The NOEL was set to 500 ppm, based on slight effects in males only, whereas the dose level of 1000 ppm is considered to be the No Observed Adverse Effect Level (NOAEL) of racemic tetramethrin, which is equivalent to 76.0 mg/kg bw/day, for combined sex.