6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 February 2001 - 04 September 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

(adopted 1998)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoic acid

EC Number:

279-505-5

EC Name:

6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoic acid

Cas Number:

80584-91-4

Molecular formula:

C21H36N6O6

IUPAC Name:

6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoic acid

Details on test material:

- Physical state: solid
- Storage conditions: in the dark at ambient 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: Sprague Dawley (Cri: CD(SD) IGS BR) rats from Charles River (UK) Limited, Margate, Kent, UK
- Age at arrival: 6 weeks
- Weight at arrival: 155-217 g (males), 117-147 g (females)
- Housing: single housing in polypropylene cages
- Diet: rat and mouse No. 1 Expanded SQC Diet, supplied by Special Diets Services Limited, Stepfield, Witham, Essex, ad libitum
- Water: domestic mains quality water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 15
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared by simple mixing of appropriate weighed quantities of test item to measured volumes of 0.5% carboxymethyl cellulose (CMC; high viscosity) using a Silverson homogeniser, until the formulations were visibly homogeneous. No adjustment for test item purity was made. Vehicle formulation (without the test item under investigation) was prepared for the control animals. Dosing formulations were prepared weekly and dispensed daily prior to administration to the animals

DOSING VOLUME:
A constant volume of 5 mL/ kg bodyweight was used.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of dosing formulations was undertaken with regard to concentration and homogeneity. Triplicate samples of dosing formulations were taken from each formulation (including Control) immediately after preparation, taking care to magnetically stir the formulation whilst sampling, for dosing weeks 1, 6 and 13 of the study. The samples were assayed at Inveresk using methodology previously supplied by the Sponsor and validated in the Toxicology Support laboratories of Inveresk under a separate protocol (Inveresk Project No. 281146; Method No. 8114). The dosing formulation varied between -4.5% and +1.0% of the nominal concentrations.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 100, 1000 mg/kg bw
Basis:
other: nominal concentration

No. of animals per sex per dose:

Main study: 10 animals per sex and dose
Recovery study: 5 animals per sex and dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were agreed with the Sponsor after evaluation of a 4 week dose range finding study in rats with administration by gavage at Inveresk (Inveresk Project No. 455235). In that study, using dose levels of 0, 10, 100 and 1000 mg/kg bw\day there were no treatment related effects seen. Dose levels took into account the maximum tolerated dose in the test model and other factors such as anticipated human exposure.

- Post-exposure recovery period in satellite groups: 5 animals per sex (control and high dose group) were included in a 4 week post-exposure recovery period. One male animal originally assigned to the Recovery Study was moved to the Main Study Control group to account for one premature decedent male. Therefore, the recovery groups consisted only of 4 male animals.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
All animals were checked early morning and as late as possible each day for viability and reaction to treatment was checked pre-dose and immediate postdose throughout each day for any signs of ill health or reaction to treatment. The onset, intensity and duration of any signs were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
Once each week all animals received a detailed clinical examination, including appearance, movement and behavior patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
Body weights were recorded once during pretrial, daily up until the end of the dosing period and once weekly during the recovery period (only weekly data are reported). This included spare animals 101-106 during pretrial. Animals showing weight loss or deterioration in condition were weighed more frequently.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The quantity of food consumed by each animal was measured and recorded once at the end of the week prior to the start of treatment and then once weekly up until the end of the study. This included animals 101-106 during pretrial.

WATER CONSUMPTION AND COMPOUND INTAKE:
Water consumption was monitored by visual inspection of the water bottles on a weekly basis throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes were examined using an indirect ophthalmoscope after the application of 1% tropicamide. Anterior, lenticular and fundic areas were evaluated. An ophthalmoscopic examination was undertaken on all animals during pretrial (including animals 101-106) and on all control and high dose animals after approximately 12 weeks of dosing and during the last week of the recovery period. The timing of the recovery assessments, although covered in the final protocol, was omitted from amendment 1 (compilation error); this deviation does not affect the study integrity or outcome.

LABORATORY INVESTIGATIONS:
Samples were obtained during week 13 of treatment (Main Study) and during week 4 of the recovery period (Recovery Study). The animals were not deprived of food overnight prior to sampling. Samples for hematology, coagulation and clinical chemistry were collected via the orbital sinus and transferred into plastic tubes containing anticoagulant.

HAEMATOLOGY: Yes
About 0.5 mL blood was taken into EDTA and assayed for:
Haemoglobin, Red Blood Cell Count, Haematocrit, White Blood Cell Count, Mean Cell Volume, Mean Cell Haemoglobin, Mean Cell Haemoglobin Concentration, Platelets, Reticulocytes, Differential White Blood Cell Count, Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Large Unclassified Cells.
A blood film smear was made from all EDTA haematology samples and stained but not examined.
Coagulation:
Blood samples were taken into tubes containing 0.045 ml trisodium citrate (w/v). The final sample volume was as close as possible to 0.45 mL to give a final concentration of 0.38% (blood to citrate ratio of 9:1). Samples were assayed for Prothrombin Time and Activated Partial Thromboplastin Time.
Femoral bone marrow smears were taken at necropsy. These were not stained or evaluated as this was not required.

CLINICAL CHEMISTRY: Yes
At least 1.5 mL of blood (deviated from protocol as the body weight of some females did not permit 2.0 mL to be taken; does not affect study integrity or outcome) was taken into tubes containing lithium heparin, which was then centrifuged and the plasma was assayed for:
Urea, Glucose, Aspartate Aminotransferase, Alanine Aminotransferase, Alkaline Phosphatase, Sodium, Potassium, Chloride, Total Protein Albumin, AG Ratio, Creatinine, Calcium, Phosphate, Triglycerides, Total Bilirubin, Total Cholesterol.

URINALYSIS: Yes
Urine samples were collected from animals in metabolism cages. The samples were collected over a 4 hour period of food and water deprivation.
Samples were collected and assayed for:
Appearance (Turbidity and Colour), pH, Specific Gravity, Volume, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Blood Pigments, Microscopy of the Spun Deposit.

NEUROBEHAVIOURAL EXAMINATION: Yes
Once during the pre-treatment week (week -1) and once weekly thereafter, a more detailed examination was made of all animals. These examinations were conducted by a technician not involved in the dosing procedures or in the collection of the body weight or food consumption data, and were performed at an approximately standardized time of day with respect to the time of dosing. Before the independent technician entered the room on each occasion to perform the examinations, the cage card showing treatment group location was removed from each cage, leaving the second pre-prepared card as the cage identifier.

-Cageside observations:
Posture/condition on first approach (animal undisturbed), checking for:
Prostration, Lethargy, Writhing, Circling, Breathing abnormalities, Gait abnormalities, Tremor, Fasciculation, Convulsions, Biting (of cage components or self mutilation), Vocalisations, Piloerection, Ease of removal from cage.

-Body temperature:
A rectal temperature was taken for all animals. This deviation from the protocol did not affect the study integrity or outcome.

-Condition of the eyes, checking for:
Pupillary function, Miosis, Mydriasis, Exophthalmos, Encrustation, Lacrimation
Condition of the coat. Presence of salivation. Overall ease of handling.

-Observations in a standardized arena (3 min observation period):
Latency (time to first locomotory movement), level of mobility, rearing, grooming, urination/defecation, arousal (level of alertness), posture, tremor/convulsions, vocalisation, piloerection - recorded as for cageside observations, palpebral closure, gait abnormalities, stereotypy (excessive repetition of behaviours) and/or unusual behaviours.

Once during the pre-treatment week (week -1) and during week 12 of treatment, the following additional functional assessments were performed. Again, these assessments were performed at an approximately standardized time of day with respect to the time of dosing: Reaction to sudden sound (click above the head), reaction to touch on the rump with a blunt probe, grip strength, pain perception, landing foot splay, motor activity.

Other physical/functional abnormalities;
Any other abnormality not already recorded in the above screening battery.
A limited set of assessments were conducted on one day (day 1) during week 1, due to extreme weather conditions which resulted in a shortage of staff. The remainders of the assessments for week 1 were not conducted. The limited data are reported; the lack of a complete dataset for this time point does not affect the study integrity or outcome.

Sacrifice and pathology:

GROSS PATHOLOGY:
The scheduled terminations occurred after completion of 13 weeks of treatment (Main Study) and after completion of a further 4 weeks of the recovery period (Recovery Study). The animals including the premature decedent killed prematurely were humanely killed by exposure to carbon dioxide and had their terminal body weight recorded, followed by exsanguination. The premature decedent found dead also had its terminal body weight recorded.
Each animal was subjected to a detailed necropsy performed under the guidance of a veterinary pathologist. The necropsy consisted of an external and internal examination and recording of observations for all animals. All gross lesions were recorded in descriptive terms, including location(s), size (in mm), shape, color, consistency and number. Representative samples of the tissues detailed were taken from all animals and fixed in 10% neutral buffered formalin unless otherwise stated. Carcasses were discarded after checking the retained tissues against the protocol and a review of the necropsy report.

HISTOPATHOLOGY:
Tissues were processed from Control and all High dose animals and any animals found dead or killed prematurely in the Main Study. Sections were cut 4-6 µm thick, stained with haematoxylin and eosin (H&E) and evaluated by a pathologist.

Statistics:

Selected neurotoxicity (deviation from protocol; does not affect study integrity or outcome), and body weight, food consumption, hematology, clinical chemistry and urinalysis data were statistically analyzed for homogeneity of variance using the 'F-max' test, where appropriate. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, then a non-parametric test, such as Kruskal-Wallis ANOVA, was used. Organ weights were also analyzed as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. The following pairwise comparisons were performed:
Control group vs low dose
Control group vs intermediate dose
Control group vs high dose
All statistical tests were two-sided and performed at the 5% significance level. Histological incidence data were analyzed using Fisher's Exact Probability Test.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
There were two deaths, a control male which was found dead after blood sampling during week 13 and a low dose female, which was killed prematurely during week 13 due to a protruding right eye incurred after blood sampling during week 13.
There were notable intergroup differences in either sex. Typical clinical signs seen included agitation, a corrugated tail and staining, and were generally distributed evenly throughout the groups.

BODY WEIGHT AND WEIGHT GAIN
Week 1-13
There was a very slight increase in overall body weight gain in the main study females receiving 1000 mg/kg bw/day when compared to the control group (+5%). Due to the magnitude and the lack of statistical significance, this was not considered to be due to administration of test material.
Weeks 14-17
There was a slight decrease in body weight gain in the recovery study females previously dosed at 1000 mg/kg bw/day when compared to the control group (17.0 ± 4.1 g in the control group; 11.9 ± 5.3 g in the treatment group) . Due to the lack of a similar effect seen during Week 13, this was considered to be due to chance. There were no other intergroup differences.

FOOD CONSUMPTION AND COMPOUND INTAKE
There were no differences that were considered to be due to treatment with test substance.

WATER CONSUMPTION AND COMPOUND INTAKE
Visual assessment revealed no notable intergroup differences in either sex.

OPHTHALMOSCOPIC EXAMINATION
There were no findings considered to be due to treatment with test substance.

HAEMATOLOGY
Week 13
There was a very slight, statistically significant, increase in mean cell volume in the male 1000 mg/kg bw/day dose group (52 ± 1.9 to 54 ± 1.6). Very slight, non-statistically significant, decreases were also seen in red blood cell count (males receiving 100 or 1000 mg/kg bw/day) with an increase apparent in white blood cell count and lymphocytes (male 1000 mg/kg bw/day dose group). There were no intergroup differences in females.

Week 17
Very slight, statistically significant, increases in haemoglobin (14.4 ± 0.6 to 15.2 ± 0.3) and red blood cell count (7.41 ± 0.41 to 7.97 ± 0.21) were apparent in females previously dosed at 1000 mg/kg bw/day. Due to the lack of similar findings during week 13, they were considered to be due to chance. There were no other intergroup differences in either sex. The findings seen during week 13 were not evident at this time point.

CLINICAL CHEMISTRY
Weeks 13 and 17
There were no findings considered to be treatment related.

URINALYSIS
There were no findings considered to be treatment related.

NEUROBEHAVIOUR
Neurotoxicity clinical observations and motor activity: There were no intergroup differences in either sex.
Detailed functional observation battery:
There were several, statistically significant, changes in latency for males and for several parameters in female groups receiving the test substance during the course of the treatment period. However, they were generally of a slight magnitude, not dose related, and were therefore not considered to be attributable to administration of the test article.

ORGAN WEIGHTS
Week 14
There was a very slight, non-statistically significant, increase in mean adrenal weight in the male 1000 mg/kg bw/day dose group. There was a very slight, non-dose related, decrease in mean thyroid weight in all female groups receiving the test material, achieving statistical significance at all dose levels. The slight, non-statistically significant, increase in mean testes weight in the male 1000 mg/kg bw/day was due to an unusually high testes weight for one animal and therefore not considered attributable to administration of test substance. There were very slight, statistically significant, decreases in mean kidney and pituitary weights in the female 10 and 100 mg/kg bw/day dose groups. Due to the lack of similar effect in the female 1000 mg/kg bw/day dose group, these findings were not considered to be due to administration of test material.
Week 18
The findings seen at week 14 were not evident at this time point. The very slight, statistically significant, decreases in mean thyroid (0.0294 ± 0.0019 g to 0.0230 ± 0.0017 g) and liver weights (20.41 ± 0.63 g to 17.71 ±0.56 g; male 1000 mg/kg bw/day) and very slight, statistically significant increase in mean pituitary weight (female 1000 mg/kg bw/day) were not evident at week 14 and so were considered to be due to chance.

GROSS PATHOLOGY
Weeks 14 and 18
All necropsy findings were typical of spontaneously arising background findings in rats of this strain and age, on this kind of study. There were no necropsy findings that could be attributed to dosing with test material.

HISTOPATHOLOGY:
Weeks 14 and 18
Premature Decedents
One control animal was found dead post orbital sinus bleeding during week 13. There were no significant histology findings and the cause of death was undetermined. A 10 mg/kg bw/day dose female was euthanized due to a protruding right eye incurred during the week 13 blood sampling. There were no significant histology findings for the eye.

Survivors: Main and Recovery Study Animals
A deciduoma was found in one control female. All other histology findings were typical of spontaneously arising background findings in rats of this strain and age, on this kind of study. There were no histology findings that could be attributed to dosing with test material.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Dosing male and female Sprague-Dawley rats with the test article via gavage for 13 weeks at dose levels of 0, 10, 100 or 1000 mg/kg body weight did not result in any effects related to administration of the test item at any dose level. The No Observed Effect Level (NOEL) was therefore considered to be 1000 mg kg\day.

Executive summary:

The toxicity of the test article was assessed in the rat after oral administration by gavage for 13 weeks followed by a 4 week recovery period. Two groups of 10 male and 10 female (Low and Intermediate dose groups) and one group of 15 male and 15 female (High dose group) Sprague-Dawley rats were dosed orally by gavage once daily at dose levels of 10, 100 and 1000 mg/kg/ respectively. A further group of 15 male and 15 female Sprague-Dawley rats received vehicle alone and acted as a Control group. After 13 weeks of treatment (Main Study) the first 10 numbered males and females from the Control and High dose groups and all animals from Low and Intermediate dose groups were killed and necropsied. The remaining animals from the Control and High dose groups were retained for a further 4 weeks without treatment (Recovery Study) to assess the reversibility of any effects seen, whereupon they were killed and necropsied. Analysis of dosing formulations was undertaken from formulations for dosing during Weeks 1, 6 and 13 of dosing. The animals were regularly monitored for any signs of ill health or reaction to treatment. Detailed functional observations were performed weekly, with additional functional observations performed during Pretrial and Week 12. Body weights were recorded during Pretrial, daily during dosing and once weekly during the Recovery period (reported once weekly), with food consumption recorded once weekly. Ophthalmoscopic assessments were undertaken during Pretrial, Weeks 13 and 17. Urine and blood samples were both collected for laboratory investigations during Weeks 13 (Main Study) and 17 (Recovery Study). Designated animals were killed and subjected to a detailed necropsy examination after completion of 13 weeks of treatment (Main Study) and after a further 4 week recovery period (Recovery Study). Representative samples of tissues were taken from all animals and fixed in 10% neutral buffered formalin unless otherwise stated, with a selection of tissues being weighed and/or preserved. A full list of tissues from Control and High dose animals were subjected to a comprehensive histological examination. There were no effects in body weight, food consumption, treatment related clinical signs, ophthalmoscopy, or any indication that the test article was neurotoxic. There were some changes in some hematology parameters in males dosed at 100 or 1000 mg/kg body weight. These changes were of a very slight magnitude, not dose related and not associated with any histopathological changes, and so were not considered treatment-related. The increase in mean adrenal weight in the male 1000 mg/kg body weight dose group and decrease in mean thyroid weight in all female groups receiving the test substance were of a very slight magnitude, not dose related and not associated with any histopathological findings. These effects were therefore not considered to be due to administration of test material. In conclusion, dosing male and female Sprague-Dawley rats with the test article via gavage for 13 weeks at dose levels of 0, 10, 100 or 1000 mg/kg body weight did not result in any effects related to administration of the test item at any dose level. The No Observed Effect Level (NOEL) was therefore considered to be 1000 mg kg\day.