Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
see Principles of method if other than guideline; The study integrity was not adversely affected by the deviations.
Principles of method if other than guideline:
List of protocol deviations:
1. Temporary deviations from the maximum and minimum level of relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations.
2. The sciatic nerve of a control female (no. 23) was not available for histopathology. Evaluation: Sufficient tissues were available for evaluation of the results.
3. On days 3, 7, 9 and 23 the maximum time between the earliest and latest dosing exceeded 4 hours with a maximum of approximately 35 minutes. Evaluation: Deviation was of an incidental nature, and was not considered to have adversely affected the study results.
4. No arena observation was performed at the start of week 3. Evaluation: Based on the available arena observations and clinical observations, an adequate assessment of the study results was considered possible.

The study integrity was not adversely affected by the deviations.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Clear colourless liquid, Batch WOP 04079D, 96.5 % purity, stored at room temperature in the dark, expiry date 01Feb2006

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Crl: (WI) BR (outbred, SPF-Quality)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: Charles River Deutschland, Sulzfeld, Germany.
Age at Start of treatment: Approximately 6 weeks.
Number of animals: 20 males, 20 females (females were nulliparous and non-pregnant)
Randomisation: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within +/- 20% of the sex mean.
Identification: Earmark and tattoo
Health inspection: A health inspection was performed prior to commencement of treatment to ensure that the animals are in a good state of health.

Conditions: Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 +/- 3.0 oC (actual range 19.7-22.8 oC), a relative humidity of 30-70% (actual range 26-93%) and 12 hours artificial fluorescent light and 12 hours darkness per day.
Accommodation: Group housing of 5 animals per sex in Macrolon plastic cages (MIV type, height 18 cm, during overnight activity monitoring individual housing in MIII type; height 15 cm) with sterilised sawdust as bedding material (Woody-Clean type 3/4, Tecnilab-BMI BV, Someren, The Netherlands) and paper as cage-enrichment (Enviro-dri, BMI, Helmond, The Netherlands). No cage-enrichment was provided during overnight activity monitoring. Certificates of analysis were examined and then retained in the NOTOX archives. Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany). Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.
Water: Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.

Results of analysis for ingredients and/or contaminants of bedding, diet and water were assessed and did not reveal any findings that were considered to have affected study integrity.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral gavage, using a stainless steel stomach tube. Formulations were placed on a magnetic stirrer during dosing.
Vehicle:
propylene glycol
Remarks:
Accuracy, homogeneity and stability over 5 hours of formulations of test substance in propylene glycol were demonstrated by analysis
Details on oral exposure:
Oral gavage, using a stainless steel stomach tube. Formulations were placed on a magnetic stirrer during dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations prepared for use on day 3 were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours was also determined (highest and lowest concentrations). The analytical method used was based on the results of a separate project for the development and validation of the analytical method (NOTOX project 433136).

Analysis of the accuracy of dose preparations revealed values within the range of 90-100 % of nominal (with one value of the group 2 formulation being slightly lower, i.e. 82% of nominal), which was considered to represent an acceptable level of accuracy for formulations of this type.
Duration of treatment / exposure:
See frequency of treatment
Frequency of treatment:
Once daily for at least 28 days, 7 days per week, approximately the same time each day with a maximum of approximately 4.5 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
male and female
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
male and female
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
male and female
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
male and female
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Details on study design:
Based on the results of a 5-day range finding study NOTOX Project 433226), the dose levels for the 28-day toxicity study were selected to be 0, 15, 150 and 450 mg/kg/day.

Dose volume: 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Examinations

Observations and examinations performed and frequency:
Mortality/Viability: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/2000/7). The time of death was recorded as precisely as possible.

Clinical signs: At least once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter (except inadvertently at start of week 3), this was also performed outside the home cage in a standard arena. The symptoms were gradred according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

Functional Observations: During week 4 of treatment, the following tests were performed on all animals (abbreviations mentioned in the respective tables indicated between brackets):
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).

Body weights: On days 1, 8, 15, 22 and 28.

Food consumption: Weekly.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Sacrifice and pathology:
All animals surviving to the end of the observation period and all moribund animals were deeply anaesthetised using iso-flurane and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4% formaldehyde solution:

Identification marks: not processed Ovaries
Adrenal glands Pancreas
Aorta Peyer's patches [jejunum, ileum] if detectable
Brain [cerebellum, mid-brain, cortex] Pituitary gland
Caecum (Preputial gland)
Cervix Prostate gland
(Clitoral gland) Rectum
Colon (Salivary glands - mandibular, sublingual)
Duodenum Sciatic nerve
Epididymides (Seminal vesicles)
(Eyes with optic nerve [if detectable] and (Skeletal muscle)
Harderian gland) (Skin)
(Femal mammary gland area) Spinal cord - cervical, midthoracic, lumbar
(Femur including joint) Spleen
Heart Sternum with bone marrow
Ileum Stomach
Jejunum Testes
Kidneys Thymus
(Larynx) Thyroid including parathyroid [if detectable]
(Lacrimal gland, exorbital) (Tongue)
Liver Trachea
Lung, infused with formalin Urinary bladder
Lymph nodes - mandibular, mesenteric Uterus
(Nasopharynx) Vagina
Oesophagus All gross lesions

Tissues mentioned within brackets were not examined microscopically as there were no signs of toxicity or target organ involvement

Organ weights: The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus

Histotechnology: All organs and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
Other examinations:
Clinical laboratory investigations: Blood samples were collected under iso-flurane anaesthesia immediately prior to scheduled post mortem examination, between 7:00 and 10:30 am. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.5 ml), with citrate for clotting tests (1.0 ml) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 ml).
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All surviving females and one male at 450 mg/kg/day showed an abnormal gait during the last week of treatment. One female sacrificed on day 3 showed clonic spasms prior to sacrifice. In addition, abdominal swelling was observed for one high dose male in week 4. Salivation was observed among all animals at 150 and 450 mg/kg/day, and among one male at 15 mg/kg/day.

Laboured respiration and rates were shown be the female at 15 mg/kg/day sacrificed on day 6, and were considered to have occurred due to a gavage accident. Other (incidental) findings that were noted included alopecia on the head, and swelling and dark colouration of the right eye. These findings are incidentally noted in rats of this age and strain, which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance. No clinical signs were noted in the control group.
Mortality:
mortality observed, treatment-related
Description (incidence):
Three females at 450 mg/kg/day (nos. 36, 38 and 40) were found dead or were sacrificed for humane reasons between days 3 and 19.

One female at 15 mg/kg/day (no. 30) was sacrificed for humane reasons on day 6. Histopathology revealed necrosis of the tracheal epithelia, which in combination with breathing difficulties seen prior to sacrifice (laboured respiration and rates) was considered indicative of a gavage accident. Also, no further mortality occurred in this dose group or the next higher dose group of 150 mg/kg/day.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in body weights and body weight gain were observed over the 4-week study period.

Higher body weights and body weight gain levels were recorded for males at 15 mg/kg/day throughout treatment (achieving a level of statistical-significance in most instances). Taking into account the nature of the effect (i.e. an increase) and since mean body weight (gain) of males at 150 and 450 mg/kg/day was similar to control levels, these changes were considered to be of no toxicological significance. Body weights and body weight gain of the other treated animals remained in the same range as control animals during the treatment phase.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Relative food intake was slightly reduced for high dose females in weeks 1 and 2.

Food consumption before or after allowance for body weight was similar between other treated and control animals.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes distinguished treated from control animals:
- Increased red blood cell distribution width (RDW) in ales and females at 450 mg/kg/day;
- Reduced red blood cell counts (RBC) in females at 150 and 450 mg/kg/day;
- Reduced haemoglobin level (HGB) in females at 150 and 450 mg/kg/day;
- Reduced haematocrit level (HCT) in females at 150 and 450 mg/kg/day;
- Reduced mean corpuscular haemoglobin concentration (MCHC) in females at 450 mg/kg/day;
- Increased reticulocyte counts in males and females* at 450 mg/kg/day;
- Reduced prothrombin time (PT) in females at 450 mg/kg/day
*no statistical significance.

Minor statistically significant differences included increased platelet counts and lower white blood cell count in males and femals at 150 mg/kg/day respectively. These findings were considered to have arisen by chance and in the absence of a treatment-related distribution considered to be of no toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) differences were noted between control animals and treated animals:
- Increased urea level in males and females* at 450 mg/kg/day;
- Increased creatinine levels in males at 450 mg/kg/day;
- Increased potassium level in males at 150 and 450 mg/kg/day, and females at 150 and 450* mg/kg/day;
- Increased inorganic phosphate level in males and females* at 450 mg/kg/day;
- Increased cholesterol levl in females at 450 mg/kg/day*
*no statistical significance.

The lower alkaline phosphatase (ALP) level in males at 150 mg/kg/day was considered to be of no toxicological significance, since no dose-related response was apparent and taking into account the nature of the effect (i.e. a decrease).
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals, surviving to the end of the study. The variation in motor activity did not indicate a relation with treatment.

The high value for the total high sensor readings of one high dose male no. 17 was considered to have occurred by chance and occurred in the absence of any supportive clinical signs. As this did not form part of a group response, no toxicological relevance was ascribed to this change.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weight of males and females at 450 mg/kg/day were increased, while liver to body weight ratios were increased in males and females at 150 and 450 mg/kg/day. In addition, a slightly increased kidney to body weight ratio was measured for males at 450 mg/kg/day.

The statistically significant increase of absolute heart and kidney weights of males at 15 mg/kg/day was considered not to be a sign of toxicity since no dose response relation was apparent.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Crateriform retractions in the stomach and emaciation were recorded for one high dose female kileed in extremis on day 3.

Two high dose females that died spontaneously on days 11 and 19 showed advanced autolysis and several organs were missing due to cannibalism. Incidental findings among control and/or treated animals included pelvic dilation of the kidneys, enlarged mandibular lymph node, diaphragmatic hernia of the liver and fluid in the uterus. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The following treatment related microscopic findings were observed:
- Bile duct proliferation of minimal or slight degree (2/5 males and 2/5 females at 150 mg/kg/day, 4/5 males and 4/5 females at 450 mg/kg/day);
- Diffuse midzonal/centrilobular hypertrophy of the liver at minimal or slight degree (4/5 males at 450 mg/kg/day);
- Increased severity of cortical hyaline droplets in the kidneys to slight or moderate degree (4/5 males at 150 mg/kg/day and 5/5/ males at 450 mg/kg/day);
- Very slight increase in the severity of splenic hemopoiesis (primarily erythropoiesis) to a moderate degree (1/5 males at 450 mg/kg/day).

All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, the effects observed at 150 mg/kg/day were not indicative of clear organ dysfunction and were not regarded to be adverse in toxicological terms. Deaths at 450 mg/kg/day were considered treatment-related, and as such considered to be clear evidence for an adverse effect of the test substance at this dose level. Therefore, from the results presented in this report, a definitive No Observed Adverse Effect Level (NOAEL) for Pentamethyl-trioxepane of 150 mg/kg/day was established.
Executive summary:

In conclusion, the effects observed at 150 mg/kg/day were not indicative of clear organ dysfunction and were not regarded to be adverse in toxicological terms.  Deaths at 450 mg/kg/day were considered treatment-related, and as such considered to be clear evidence for an adverse effect of the test substance at this dose level.  Therefore, from the results presented in this report, a definitive No Observed Adverse Effect Level (NOAEL) for Pentamethyl-trioxepane of 150 mg/kg/day was established.