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Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
according to guideline
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
Details on test animals or test system and environmental conditions:
Characterization of the test system: The range-finding and 28-day study were conducted with albino rats. The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies. Young adult, male and female rats, Wistar outbred (Crl:(WI)WU BR) were obtained from a colony maintained under SPF-conditions at Charles River Deutschland, Sulzfeld, Germany. The Wistar strain was used because it is routinely used at the testing facility for this type of studies.
At the commencement of the treatment period, the age of the rats was 5-7 weeks of age and the body weight variation did not exceed+/- 20% of the mean weight for each sex.
Animal allocation: Upon arrival on 5 March 2003 and 26 March 2003, the rats were taken to a quarantine room and checked for overt signs of ill health and anomalies. During the quarantine period serological investigation of the microbiological status was conducted in blood samples taken from random rats from each delivery. The results of serological examinations were passed on by telephone and indicated an acceptable microbiological status. All animals were accepted for use in either the range finding or 28-day study and the quarantine room was cleared for use as experimental room. The animals were acclimatised to the laboratory conditions for at least 5 days prior to the experimental start date. The animals assigned to the 28-day study were allocated (males and females separately) to the various groups by computer randomisation at arrival. Moreover, cages were also allocated to the experimental groups by computer randomisation. The additional animals for the range-finding (three males) and 28-day study (two animals of each sex) were kept in the same room as reserve animals during the study.
Identification of the test system: The studies were identified as study numbers 5042 (range-finding study) and 5042/01 (28-day study).
Each group of rats was coded by a letter and a colour. At arrival, all rats were identified by a transient mark on their tail. The 28-day study animals were identified by a unique animal identification number tattooed in the ears shortly after start of treatment. Each cage was provided with a card showing the colour code, the animal identification numbers, the cage number, the group letter and the study number.
Animal maintenance: The rats were housed under conventional conditions in one room, in macrolon cages with sterilised wood shavings (Woody Clean, Type 3/4) as bedding material and environmental enrichment (shreds of paper), three (range-finding study) or five (28-day study) rats per cage, separated by sex. The optimal range for temperature of 22±3 °C was not exceeded during the study period, whereas the upper limit of the optimal range of 30-70% for humidity was exceeded on several occasions. Maximum values for humidity of 70.6-99.9% were recorded for periods less then two hours, mainly during periods of wet cleaning of the room. The room was ventilated with about 10 air changes per hour. Lighting was artificial with a sequence of 12 hours light and 12 hours dark (lights on from 7.00 a.m. to 7.00 p.m.).
Feed and drinking water: Feed and drinking water were provided ad libitum from the arrival of the rats until the end of the study. In the 28-day study, the animals had no access to feed and water during the neurobehavioural observations in week 4 and feed was withdrawn (water was available) overnight prior to necropsy.
The rats were fed a commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3, batches 2413 and 2414 (range-fmding study) and batches 2413,2656 and 2789 (28-day study). Each batch of this diet was analysed by the supplier (SDS Special Diets Services, Witham, England) for nutrients and contaminants. The results of the analyses were made available to TNO, examined and retained in the archives. All batches were accepted for use. The feed was provided as a powder, in stainless steel cans, covered by a perforated stainless steel plate that serves to prevent spillage. The feed in the feeders was refreshed once per week and filled up when necessary.
The drinking water (tap water) was given in polypropylene bottles, which were cleaned weekly and filled as needed. Tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC) was supplied by N.V. Hydron Midden-Nederland. The supplier routinely examines the physical, chemical and microbiological variables of the drinking water. In addition, the supplier periodically (twice per year) analyses water samples taken at the premises of TNO Nutrition and Food Research in Zeist for a limited number of physical, chemical and microbiological variables, The results of the routine examinations and of the samples taken were made available to and retained in the archives of TNO Nutrition and Food Research.
Certificates of analyses of food and water are available on request.
Route of administration:
oral: gavage
Details on oral exposure:
Water was selected as a suitable vehicle based on trial formulations prepared at TNO.
The test substance was suspended in MilliQ-water. All formulations were prepared on a weight/weight basis at concentrations of 0 (MilliQ water only), 20, 60 and 200 mg/ml. A density of 1.0 g/ml for the test substance1 was assumed. In the absence of stability data in water at the time of performance of the dose range-finding study, formulations were freshly prepared daily within 4 hours prior to each dosing on 10-14 March 2003. Study, formulations were freshly prepared daily within 4 hours prior to each dosing on 10-14 March 2003.
On 26 March 2003, representative formulations were prepared according to the same method. These latter formulations were not used for dosing, but for sampling for test substance analysis only.
As formulation analysis indicated a stability of the test substance in water for at least 7 days, formulations were prepared once per week for 7 days in advance during the 28-day study. On 4, 11, 17 and 24 April2003, stock formulations at each concentration were prepared, thoroughly homogenised by magnetic stirring for at least 10 minutes and subsequently divided in 7 equal portions. The portions not used for dosing on the day of preparation were stored in a refrigerator (2-10 °C) for a maximum of 7 days. For practical reasons, during week 3 of the study only 6 out of 7 portions were used from each concentration prepared on 17 April 2003, and fresh formulations were prepared for the 28th day of treatment, i.e. 1 May 2003. On each day of dosing, the formulations stored in the refrigerator were allowed to accommodate to ambient temperature for at least 30 minutes prior to start of dosing.
All formulations were thoroughly homogenised immediately before use and homogeneity was maintained during dosing by constant stirring.
Samples for test substance analysis were taken from the first batch of formulations prepared on 4 April 2003 (nominal day 0).
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Test substance: A sample of T-1063FM, a white powder (lot number 10123U, TNO dispense number 030027), was received by the Residue Analysis Department on 12 February 2003 and stored at ambient temperature in the dark.
Test samples: Test samples were taken in duplicate from formulations of T-1063FM in MilliQ-water prior to start and from the first batch of formulations prepared for the 28-day study. All samples weighed approximately 0.5 g (accurately weighed) and were stored in glass containers until analysis. The samples taken on 26 March 2003 were analysed for contents and homogeneity. Two additional duplicate samples from the low-dose and the high dose concentrations were stored in the refrigerator (2-10 °C) for analysis of stability over 7 days. The latter samples were analyzed on 2 April 2003. On 4 April2003, test samples were taken from the first batch of formulations that were used in the 28-day study. These samples were stored in the refrigerator (2-10 °C) until analysis for contents and homogeneity on 7 April2003.

Analysis of T-1063FM
Principle: Samples containing T-1 063FM were determined using liquid chromatography (LC) with Mass spectrometric Detection (LC/MS). The mass spectrometer was used with an atmospheric pressure Turbo Ion Spray ionization (TIS) interface, operating in the positive ion and full scan mode.
Quantification was obtained by comparing the peak area of mass fragment 87.0 m/z in the sample dilutions with those of calibration solutions containing known amounts of T-1 063FM (external standard).
Chromatography: The validation samples, the study samples and the calibration solutions were analysed using LC/MS. After each sample and or standard solution a blank sample (mobile phase) was injected. The following conditions were used:
HPLC system:Knauer Wellchrom HPLC-pump(s) K-1001
Auto sampler: Spark Midas, combined with a Spark Prospect apparatus
Column: Hypersil BDS 3 C18, 50 x 4.6 mm and a pre-column of the same stationary phase
Column temperature:n.a.
Flow: 0.7ml/min
Mobile phase: methanoV MilliQ water/ acetic acid= 49.5: 49.5: 1 (v/v)
Detection: mass spectrometry with atmospheric pressure TIS (MSITIS, Perkin Elmer SCIEX API 2000 LC/MS system)

MSITIS API-interface settings:
Ionization: Turbo Ion Spray
Split->MS: 0.3 ml/min
Source parameters
-Nebulizer gas (gas 1):20 psi
-Turbo gas (gas 2): 60 psi
-Ion spray voltage: 5500V
-Temperature: 400°C
API conditions
-Curtain gas: 20 psi
-Interface heater: on
Scan parameters
-Polarity: positive
-Scan type: Multiple ion scan
-Q1 mass: 87.0 m/z AMU
-DP: 20V
-FP: 400V
-EP: -10V
-CEP: 14.56 v
-Scan time: 200msec
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Administration of the test substance: The oral route was used because this is an anticipated route of human exposure. The test substance was administered by oral gavage using a stainless steel stomach tube at a dose volume 5 ml/kg body weight/day. Administration was once daily for 5 days in the range-finding study or for 28 consecutive days in the 28-day study. The animals were dosed for the last time on the day prior to necropsy. The rats of the various groups were dosed with different concentrations of the test substance in the vehicle, to ensure a constant dose-volume at all dose levels. Controls were treated with the vehicle only. In the 28-day study, the dose volumes were adjusted to the latest weekly recorded body weight for each individual rat, to maintain a constant dose level in terms of the animal's body weight.
Experimental groups and dose levels: The range-finding study comprised three groups of three males each, a vehicle control group and two groups receiving the test substance at a dose level of 300 or 1000 mg/kg bw.
The 28-day study comprised four groups of five males and five females each, viz. one carrier control group and three groups receiving different levels of the test substance for 28 consecutive days. These groups were intended to provide information on the subacute oral toxicity of the test substance and to establish a no-observed-adverse-effect level (NOAEL). The dose levels of the 28-day study were 0, 100,300 and 1000 mg/kg bw, and were also based on the results of the range-finding study.
Positive control:
Positive control not used in this study.
Observations and examinations performed and frequency:
Clinical signs: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimise loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.
Neurobehavioural observations (28-day study only): Detailed clinical observations outside the home-cage were conducted on all animals once weekly, starting in the week prior to exposure. Neurobehavioural observations and motor activity assessment were conducted on all animals per sex per group in the fourth week of treatment.
Body weight: In the range-finding study, the body weight of each animal was recorded at the start of the study (day 0) and on day 4.
In the 28-day study, the body weight of each animal was recorded at the start of the study (day 0) and once per week thereafter, including the day of scheduled sacrifice in order to calculate the correct organ to body weight ratios.
Food consumption and food conversion efficiency: Food consumption was measured per cage by weighing the feeders. The results are expressed in g per animal per day. The consumption was measured over a successive 5-day period during the range-finding study, and over successive periods of 7 days or 6 days (week prior to autopsy) during the 28-day study. The efficiency of food utilisation was calculated and expressed in g weight gain per g food consumed.
Haematology and Clinical biochemistry (28-day study only): Haematology and clinical biochemistry were conducted at the end of the treatment period on all surviving rats at scheduled necropsy on day 28. Blood samples were taken from the abdominal aorta after overnight fasting of the animals. Animals were deprived of food for approximately 16 hours prior to sampling.
The haematological determinations were carried out in blood samples with K2-EDT A as anticoagulant. The clinical biochemistry determinations were carried out in plasma prepared by centrifugation of blood collected in heparinised plastic tubes.
The following measurements were made in blood of all animals:
Haemoglobin; packed cell volume; red blood cell count; reticulocytes; thrombocyte count; total white blood cell count; differential white blood cell count; prothrombin time
The following parameters will be calculated:
mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin concentration (MCHC)
Clinical chemistry:
alkaline phosphatase activity (ALP); bilirubin (total); aspartate aminotransferase activity (ASAT); cholesterol (total); alanine aminotransferase activity (ALAT); triglycerides; gamma glutamyl transferase activity (GGT); phospholipids; total protein; calcium (Ca): albumin; sodium(Na); ratio albumin to globulin; potassium (K); urea; chloride (Cl); creatinine; inorganic phosphate; glucose.
Sacrifice and pathology:
Gross necropsy: The animals were sacrificed on nominal day 4 (range-finding study) or day 28 (28-day study) in such a sequence that, on the average, time of killing was approximately the same for each group. The animals were anaesthetised with a mixture of CO2/O2, followed by exsanguination from the abdominal aorta and then examined grossly for pathological changes.
In the range-finding study, the liver and kidneys of all animals were weighed and subsequently discarded together with the carcasses.
In the 28-day study, samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde.
The underlined organs were weighed (paired organs together) as soon as possible after dissection to avoid drying.
Adrenals; Axillary lymph nodes; Bone marrow (femur): Brain (brain stem, cerebrum, and cerebellum): Caecum: Colon: Epididvmides: Eyes*; Heart; Kidneys; Liver; Lungs; Mammary gland (females)*; Mesenteric lymph node; Nerve peripheral (sciatic nerve); Ovaries; Parathyroids; Peyer's patches; Pituitary; Prostate; Rectum; Seminal vesicles* + coagulation glands*; Small intestines (duodenum, ileum, jejunum); Spinal cord (at three levels); Spleen; Stomach (glandular and non-glandular); Testes; Thymus; Thyroid; Trachea/bronchi; Urinary bladder; Uterus; Vagina*; All gross lesions.
(*The tissues marked with an asterisk were preserved but not processed for histopathological examination. There was no indicated based on gross observation that histopathological examination was considered necessary in the first instance.)

Histopathological examination: The tissues to be examined microscopically were embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin.
Histopathological examination was performed on all tissues and organs listed above - except those marked with an asterisk - of all animals of the control group and the high-dose group. Based on treatment related changes observed in the kidneys of high dose females and in the stomach and duodenum of high dose male and female rats, the histopathology of these organs and tissues was extended to the respective males and/or females of the mid- and low-dose groups. In addition, gross lesions were examined microscopically in all rats of all dose groups.
The statistical procedures used in the evaluation of data were generally as follows:
- Body weights: one-way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests;
- Analytical results of homogeneity and stability: one-way analysis of variance (ANOVA).
- Red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values and organ weights: one-way ANOV A followed by Dunnett's multiple comparison tests;
- Reticulocytes and relative differential white blood cell counts: Kruskal-Wallis nonparametric ANOVA followed by Mann-Whitney U-tests;
- Functional observational battery and motor activity results:
- continuous measures and motor activity data: one-way analysis of variance followed by Dunnett's post-hoc group comparisons in case of a significant result;
- rank order data: Kruskal-Wallis analysis of variance followed by planned multiple comparisons between dose groups in case of a significant result followed by multiple comparison tests;
- categorical data: Pearson chi-square test;
- Histopathological changes: Fisher's exact probability test.
All tests were two-sided. Probability values of p<0.05 were considered significant, except for the analytical results where probability values op p<0.01 were considered significant.
Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See 'Figures_bodyweight_28d_T-1063FM.pdf' attached as background material.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
See 'Table_food_consumption_28d_T-1063FM.pdf' attached as background material.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
See 'Tables_haematology_clin_biochemistry_28d_T-1063FM.pdf' attached as background material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See 'Tables_haematology_clin_biochemistry_28d_T-1063FM.pdf' attached as background material.
Urinalysis findings:
not examined
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
Description (incidence and severity):
See 'Summary of microscopic observations-28 day toxicity.pdf' attached as background material.
Histopathological findings: neoplastic:
not specified
Details on results:
Clinical chemistry: Clinical chemistry values showed the following statistically significant differences between groups treated with T -1063FM and the vehicle control group:
- Decreased total protein in high dose males
- Decreased albumin in high dose males and high dose females
- Decreased urea in high dose males
- Decreased triglycerides in low dose females. This finding was not confirmed at the higher dose levels and, therefore, considered a fortuitous finding unrelated to treatment.
Individual changes in clinical chemistry values were observed in one high dose male (no.34), i.e. a decreased alkaline phosphatase activity, and in one high dose female (no.3), comprising decreased activities of alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase and an increased activity of gamma glutamyl transferase.

Microscopic findings: Upon microscopic examination, rats treated with 1000 mg/kg T-1063FM showed statistically significant changes in the incidences of histopathological lesions in the stomach, the duodenum and the kidneys, compared to controls. All rats showed very slight to moderate (high dose female no.3) gastritis in the stomach, characterized by an increased (sub)mucosal polymorphonuclear cell infiltrate of the fundic and pyloric mucosa, incidentally accompanied by focal intra-epithelial (limiting ridge) or submucosal oedema. In the duodenum of all males and 4/5 females deposition of small amounts of amorphous grayish (H&E-staining) material, denoted in the table as 'duodenal pigment deposits', was seen in the proximal villi of the mucosa. The pigment was present as 'small lakes' in the lamina propria and/or deposited at the basal membranes of the epithelium, blood-, or lymphatic vessels and was not accompanied by any other morphological change.
Treatment with 300 mg/kg T-1063FM revealed (very) slight gastritis in three male and one female rat and very slight duodenal pigment deposition in three male and two female rats. In two of these rats (male no.8 and female no.13) pigment deposition was reduced to a trace (denoted as 'very slight').
Treatment-related corticomedullary mineralization was seen in the kidneys of all female rats of the high-dose and three female rats of the mid-dose group, achieving a statistical significance in the high dose group when compared to control females. This renal change was characterized by very slight to moderate tubular mineralization at the corticomedullary junction. In addition, a statistically significant increased incidence of basophilic tubules was seen in the kidneys of females of the high-dose group.
All other histopathological observations mentioned were common findings in rats of this strain and age and occurred only incidentally or at similar incidences amongst the groups, including controls. Therefore, they were not considered to be related to treatment.
Key result
Dose descriptor:
Effect level:
> 100 - < 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
histopathology: non-neoplastic
serum/plasma biochemistry
Key result
Critical effects observed:

Summary of microscopic findings are attached under background material.

Under the conditions used in this study, oral treatment of Wistar rats with T-1063FM resulted in changes in clinical chemistry parameters and histopathological changes in stomach, duodenum and kidneys observed at dose levels of 1000 and 300 mg/kg bw. No treatment related changes were noted at the dose level of 100 mg/kg bw, which was therefore considered the No Observed Adverse Effect Level (NOAEL) for T-1063FM in this study.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
100 mg/kg bw/day
Study duration:

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated oral treatment of rats at a dose level of 1000 mg/kg bw resulted in changes in albumin contents in blood and histopathological changes in stomach and duodenum in both sexes and in the kidneys of the females. In rats treated at 300 mg/kg bw, histopathological changes in the stomach, duodenum and kidneys were still apparent, but the incidence had decreased in comparison with high dose animals. All other observations made in mid dose animals were considered to be similar to that for controls. No treatment related findings were observed in animals treated at 100 mg/kg bw.

The histopathological changes consisted of gastritis (stomach) and duodenal pigment deposition. The gastro-intestinal tract (GI-tract) is the primary target for the test substance after administration via oral gavage. No evidence from histopathological examination was found for liver damage that might explain the changes in albumin, and corroborative total protein, and urea observed in the high dose animals. One of the effects in the kidneys of females, i.e. corticomedullary mineralization, was probably caused by the large amount of phosphorus, in the form of pyrophosphate, in the test substance, which may have disturbed the Ca:P ratio in the animals. Renal basophilic tubules, another change observed in females, are part of common background pathology with rather large variations in incidence. Though, therefore, the higher incidence in high dose females may be a fortuitous finding, a relationship with treatment cannot be excluded.

Justification for classification or non-classification

The substance T-1063 FM is a physical mixture of piperazine and pyrophosphate. Piperazine is a strongly basic amine known to be corrosive to skin (EU Risk Assessment Report, Piperazine, CAS No: 110-85-0, 2005) after application of high concentrations. T-1063 FM has cytotoxic properties as well, as described in the OECD 473 and 476 studies (high concentrations).

Therefore, the gastrointestinal findings observed after application of T-1063 FM by oral gavage (OECD 407) are considered local effects at the site of first contact. This is further supported by the observation that oral application of T-1063 FM in carboxy methylcellulose (CMC, NOAEL = 1000 mg/kg bw/day, OECD 421 study) apparently induced irritant effects instomach and duodenum at much lower incidence, compared to administration in water (OECD 407 study, NOAEL = 100 mg/kg bw/day). The toxic effects observed are considered as general and not specific organ toxicity.

As basis for classification and labelling regarding repeat dose toxicity according to REGULATION (EC) 1272/2008, therefore a 90-day feeding study in rats with T-1063 FM according to guideline OECD 408 is proposed.