5,7-di-tert-butyl-3-[4-(2-hydroxyethoxy)phenyl]-1-heteropolycycl-2(3H)-one

Endpoint summary

Description of key information

ORAL

No NOAEL identified; Wistar rat; dose levels of 100, 300 and 1000/500 mg/kg; OECD 422

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 February 2013 - 14 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council (REACH).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar Han™:RccHan™:WIST
- Age at study initiation: Approximately twelve weeks old
- Weight at study initiation: males weighed 303 to 351 g; females weighed 185 to 217 g
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper. Following the pairing phase, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet: A pelleted diet was available ad libitum.
- Water: Mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 to 70 % (relative)
- Air changes: The rate of air exchange was at least fifteen air changes per hour
- Photoperiod: Low intensity fluorescent lighting was controlled to give twelve hours of continuous light and twelve hours of darkness.

IN-LIFE DATES: From: 03 April 2013 To: 27 May 2013

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The test material was prepared at the appropriate concentrations as a suspension in Corn oil. Formulations were prepared weekly and stored at approximately 4 °C in the dark.

VEHICLE
- Concentration in vehicle: 0, 25, 75 and 250/125 mg/mL
- Amount of vehicle (if gavage): Dose volume 4 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHODS
The test material concentration in the test samples was determined by high performance liquid chromatography with UV detection using an external standard technique. The test material gave a chromatographic profile consisting of a single peak; the test material itself was used as the analytical standard.
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test material in the vehicle. The method pf analysis was validated and proven to be suitable for use.

ANALYTICAL PROCEDURE
-Preparation of Standard Solutions
Stock solutions of the test material in methanol were prepared for external standard calibration. 100 mg was exactly weighed into a 100 mL volumetric flask and brought to volume with methanol to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in methanol with a concentration of 0.1 mg/mL.
On each occasion, standard solutions derived from two stock standard solutions were used for calculation.
- Analysis of Samples
The formulations were extracted with methanol. An aliquot of test material formulation was accurately weighed into a volumetric flask and brought to volume with methanol; this was then ultra-sonicated for 15 minutes and centrifuged at 500 rpm for 10 minutes. Where necessary, samples were further diluted with methanol to achieve the working concentration.
- Preparation of Accuracy Samples
Samples of corn oil were accurately fortified with known amounts of the test material equivalent to the highest and lowest concentrations. These samples were then prepared for analysis as the test samples (above).
- Preparation of Linearity Standards
A range of standard solutions were prepared in methanol from a stock solution of 1 mg/mL by serial dilution, covering the concentration range 0 to 0.1608 mg/mL.
- Instrumental Setup
HPLC: Agilent Technologies 1200, incorporating autosampler and workstation
Column: Sunfire C18 (150 x 4.6 mm i.d.)
Mobile phase: Methanol:water (80:20 v/v)
Flow rate: 1 mL/min
UV detector wavelength: 230 nm
Injection volume: 25 µL
Retention time: ~7.4 minutes

RESULTS - VALIDATION OF ANALYTICAL METHOD
- Specificity
The control dose samples and a solvent blank did not significantly affect the chromatogram at the retention time of the test material (whose area changed accordingly with known concentration).
- Linearity
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak area measured and working standard concentrations. The regression coefficients calculated were found to be 0.999.
- Accuracy
The fortified samples of corn oil were found to have a recovery value of ±2 %of the fortification.
- Test material Formulations
The test material was found to be stable in the formulations when kept for 37 days in the refrigerator (4 °C) due to results which met the variation limit of 10 % from the time-zero mean. The results indicate the accurate use of the test material and corn oil as the vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

Up to 8 weeks; 43 days for male animals, up to lactation day 5 for the females.

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 (reduced to 500) mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen by the study sponsor based on the results of previous studies on the test material. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one hour and five hours after dosing during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends and public holidays (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed individual clinical observations were performed for each animal using a purpose built arena to assess behaviour prior to the start of treatment and at weekly intervals thereafter. The following parameters were observed: gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post-coitum, and on Days 1 and 4 post-partum.
Additional body weight monitoring was performed on a daily basis for health monitoring purposes. Terminal body weights were also recorded for interim sacrifice animals and for full term animals.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post-coitum Days 0 to 7, 7 to 14 and 14 to 20. For females with live litters, food consumption was recorded on Days 1 and 4 post-partum.

FOOD EFFICIENCY: Yes
- Time schedule for examinations: The ratio of body weight change/dietary intake was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water intake was measured daily throughout the study (with the exception of the pairing phase).

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to termination (Day 42 for males and Day 4 post-partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. A terminal blood sample was also taken for analysis from interim sacrificed animals. Blood was collected into tubes containing potassium EDTA anti-coagulant.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: Five males and five females selected from the control group and each surviving dose group
- Parameters examined: haemoglobin (Hb), erythrocyte count (RBC), haematocrit (Hct), erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC)), total leucocyte count (WBC), differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos) and basophils (Bas)), platelet count (PLT), reticulocyte count (Retic) (methylene blue stained slides were prepared but reticulocytes were not assessed).
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to termination (Day 42 for males and Day 4 post-partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. A terminal blood sample was also taken for analysis from interim sacrificed animals. Blood was collected into tubes containing lithium heparin anti-coagulant.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: Five males and five females selected from the control group and each surviving dose group
- Parameters examined: urea, calcium (Ca++), glucose, inorganic phosphorus (P), total protein (Tot.Prot.), aspartate aminotransferase (ASAT), albumin, alanine aminotransferase (ALAT), albumin/globulin (A/G) ratio (by calculation), alkaline phosphatase (AP), sodium (Na+), Creatinine (Creat), potassium (K+), total cholesterol (Chol), chloride (Cl-), total bilirubin (Bili) and bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all surviving animals were observed for signs of functional toxicity. Functional performance tests were also performed on five randomly selected males and females from group 1, 2 and 3 animals prior to termination, together with an assessment of sensory reactivity to various stimuli.
- Dose groups that were examined: All
- Battery of functions tested: In the functional performance tests, motor activity and forelimb/hindlimb grip strength were assessed. In the sensory reactivity tests, each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: grasp response, touch escape, vocalisation, pupil reflex, toe pinch, blink reflex, tail pinch, startle reflex and finger approach.

Sacrifice and pathology:

SACRIFICE
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post-partum. Surviving offspring were terminated via intra-cardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post-coitum.

GROSS PATHOLOGY
For all mated females, the uterus was examined. All adult animals and offspring, including those terminated early during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS
The following organs were dissected free from fat and weighed before fixation from all surviving males and females from each dose group: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary (post fixation), prostate, seminal vesicles, spleen, testes, thymus, thyroid (weighed post-fixation with parathyroid) and uterus (weighed with cervix).

HISTOPATHOLOGY
Samples of the following tissues were removed from five randomly selected males and females from the 300 mg/kg bw/day dose group and preserved in buffered 10 % formalin, except where stated: adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland, colon, duodenum, epididymides (preserved in Bouin’s fluid then transferred to 70 % industrial methylated spirits (IMS) approximately forty-eight hours later), eyes (fixed in Davidson’s fluid), gross lesions, heart, ileum (including Peyer’s patches), jejunum, kidneys, liver, lungs (with bronchi- inflated to approximately normal inspiratory volume with buffered 10 % formalin before immersion in fixative), lymph nodes (mandibular and mesenteric), mammary gland, muscle (skeletal), ovaries, pancreas, pituitary, prostate, oesophagus, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, thyroid/parathyroid, trachea, testes (preserved in Bouin’s fluid then transferred to 70 % industrial methylated spirits (IMS) approximately forty-eight hours later), thymus, urinary bladder, uterus/cervix and vagina.

The tissues listed below were preserved from all remaining animals with the following exceptions: at the request of the study sponsor the following modifications were applied to the routine histopathological investigations: histopathological examination of high dose group (500/1000 mg/kg bw/day) animals was limited to examination of the stomach of all twelve males and five females; routine histopathological investigations were extended to include examination of the liver and stomach from five randomly selected males and females from the 100 mg/kg bw/day treatment group together with the prostate and seminal vesicles from these males and ovaries from these females.
Coagulating gland, epididymides (preserved in Bouin’s fluid then transferred to 70 % industrial methylated spirits (IMS) approximately forty-eight hours later), gross lesions, mammary gland, testes (preserved in Bouin’s fluid then transferred to 70 % industrial methylated spirits (IMS) approximately forty-eight hours later) uterus/cervix and vagina.

The tissues from selected control and test group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination.
Since there were indications of treatment-related changes in the adrenal, lungs, mesenteric lymph node, pituitary and thyroid (both sexes) and testes, examination was subsequently extended to include similarly prepared sections of these tissues from five selected males and females from the 100 mg/kg bw/day dose group.

Other examinations:

Observations were also carried out to evaluate reproductive performance, pregnancy and parturition, and litter data.

Statistics:

Where appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at p<0.05. Data were analysed using the decision tree from the Provantis™ Tables and Statistics Module:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analysed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data not analysed by Provantis were assessed separately using the SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene’s test.
Where Levene’s test was shown to be non-significant (p≥0.05), parametric analysis of the data was applied, incorporating analysis of variance (ANOVA). If this data was shown to be significant this analysis was followed by pair-wise comparisons using Dunnett’s test. Where Levene’s test was significant, non-parametric analysis of the data was analysed incorporating the Kruskal-Wallis test, which if significant, was followed by the Mann-Whitney U test. Dose response relationship was also investigated by linear regression. Where the data was unsuitable for these analyses, then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below

Mortality:

mortality observed, treatment-related

Description (incidence):

see below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see below

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

see below

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY
Due to a marked weight loss and depreciation in general condition all high dose animals (1000/500 mg/kg bw/day) were terminated for reasons of animal welfare between Study Days 12 and 13.
There were no further treatment-related early terminations. However, one 100 mg/kg bw/day female (No. 40) was sacrificed on Day 13 due to a hind limb injury (the animal displayed signs of abnormal gait and distress when moving around its cage). Necropsy revealed a fracture of the tibia and fibula; while the cause of this trauma is uncertain given the nature of this finding it was considered unrelated to treatment.

CLINICAL OBSERVATIONS
Episodes of transient increased salivation (around the time of dosing) were observed in all test group animals during the study. This condition first became evident in high dose animals within the first few days of treatment and then developed in either sex of animals treated at 100 and 300 mg/kg bw/day from the second week of dosing; persisting through to the final day of treatment (Day 42) in males and was last identifiable during the final days of gestation. This condition was accompanied in one 100 and one 300 mg/kg bw/day male between Days 29 and 31 by noisy respiration, in two 300 mg/kg bw/day males by staining around the mouth (Day 12 only) and by staining around the eyes in one 100 mg/kg bw/day female (Days 36 and 40).
Increased salivation was also identified in one female control on Day 17.
High dose (1000/500 mg/kg bw/day) animals of either sex displayed occurrences of increased salivation, pilo-erection, staining around the mouth and ano-genital region. These findings were accompanied among the males by staining around the snout, hunched posture, abnormal respiration, ptosis, lethargy dehydration and emaciation and in females by staining around the eyes and generalised fur loss.

BODY WEIGHT
Low weight gains or weight loss developed in high dose (1000 mg/kg bw/day) males and females during the first week of treatment. Concerns over body weights were slightly alleviated by removing these animals from treatment for two days but once treatment was reinstated, at a reduced dose level (500 mg/kg bw/day), weight loss continued; this led to a decision by the Study Director for an early termination of these animals for reasons of animal welfare.
Males treated at 300 mg/kg bw/day also showed reduced weight gain in comparison with the controls (albeit to a much lesser degree than the high dose group) during the first week of treatment. This situation improved over the following weeks although group mean gains continued to remain lower (ca. 8 %) than that of the control throughout the treatment period.
Body weight development in 100 mg/kg bw/day males remained similar to that of the controls throughout the study.
Female test groups (100 and 300 mg/kg bw/day) did not vary greatly from that of the control females during maturation. However, slightly lower gains were evident in both surviving test groups (100 and 300 mg/kg bw/day) in comparison with controls during the gestation and lactation phases although there was no dose-dependent trend.

FOOD CONSUMPTION AND FOOD EFFICIENCY
Dietary intake in either sex of the high dose group was considerably lower than that of the concurrent controls. Group mean diet intake values for 300 mg/kg bw/day males were below that of the controls during the first two weeks of treatment with recovery thereafter. Dietary intake of males treated at 100 mg/kg bw/day remained unaffected by treatment. Food efficiency (the ratio of body weight gain to dietary intake) in both male test groups paralleled the trends seen for dietary intake. Food consumption in females treated at 100 mg/kg bw/day remained similar to that of the controls during the maturation phase. During the same period, 300 mg/kg bw/day test females consumed approximately 30 % less diet during the first week of dosing but showed recovery by Week 2. Diet intake in both test groups was noticeably lower than that of the control and significantly below that of the control during the lactation phase albeit during the gestation phase there was no dose-dependent trend. During the period of measurement of food efficiency in female treatment groups (i.e. maturation) group means remained similar to that of the controls.

WATER CONSUMPTION
Gravimetric measurement of water intake recorded throughout the study revealed males at 100 and 300 mg/kg bw/day to be consuming considerably more water than the controls with a similar trend evident during the brief period of treatment of high dose group animals of either sex. In females only the 300 mg/kg bw/day group showed increased water consumption during the maturation phase in comparison with the control females. However, both female test groups showed increased group means during gestation and lactation; attaining statistical significance during the gestation phase (100 mg/kg bw/day p<0.01 or p<0.05, 300 mg/kg bw/day p<0.001).

HAEMATOLOGY
No treatment-related changes were detected in haematological parameters.
Incidental changes were confined to a slight but statistically significant reduction in mean corpuscular haemoglobin concentration observed in males treated at 100 mg/kg bw/day (p<0.05) and in males at 300 mg/kg bw/day in comparison with the corresponding control. However, there was no supporting evidence of an anaemia and all individual values were within the normal ranges for rats of the strain and age used. As a consequence this isolated finding was considered to be unrelated to treatment with the test material.
Males treated with 300 mg/kg bw/day showed a statistically significant increase (p<0.01) in clotting (prothrombin) time compared to controls. However in the absence of any supporting evidence of an effect on the coagulation pathways and that all individual values were within the anticipated historical range, this finding was considered to be of no toxicological consequence.
Blood samples were also taken from high dose (1000/500 mg/kg bw/day) animals prior to termination. In consideration of the brief duration of treatment it is difficult to assess the impact of treatment on the haematological profile of these animals, however, a review of the data did not identify any adverse findings.

BLOOD CHEMISTRY
An increase in the plasma concentrations of alanine aminotransferase (ALAT) was identified in males treated at 100 mg/kg bw/day (p<0.05) and 300 mg/kg bw/day (p<0.01). Further indication of liver function change was given by a reduction (p<0.01) in cholesterol levels in males treated at 100 and 300 mg/kg bw/day in comparison with the concurrent controls. A similar pattern (albeit of lesser severity) for cholesterol was also evident in 100 and 300 mg/kg bw/day females.
Both sexes exposed to 300 mg/kg bw/day were observed to have increased plasma levels of bile acid (males p<0.01, females p<0.05). Females treated at 300 mg/kg bw/day also showed an increase (p<0.01) in plasma urea levels in comparison with the corresponding controls.
Elevated albumin/globulin ratio mean values were seen in both sexes at 300 mg/kg bw/day, achieving statistical significance (p<0.01) in the males. Although this parameter may be an early indicator for impaired liver function, in the absence of any adverse effect on total plasma protein or albumin levels, this isolated change was considered of no toxicological consequence.
Incidental changes involved 300 mg/kg bw/day females that showed a slight but statistically significant increase (p<0.05) in inorganic phosphorus concentrations when compared to that of the female controls. Although a number of individual values were outside the normal ranges for animals of this age and strain there was no supporting evidence to indicate an effect on kidney function, therefore this isolated imbalance in plasma electrolyte levels was considered of no toxicological importance.
Blood samples were also taken from high dose (1000/500 mg/kg bw/day) animals prior to termination; while under the circumstances it is difficult to assess the impact treatment may have had on the blood chemistry a review of the data did not identify any noteworthy adverse findings.

FUNCTIONAL OBSERVATIONS
- Behavioural Assessments
The inter and intra group differences that were seen in behavioural scores were considered to be a result of normal variation for rats of the strain and age used and considered not to be related to treatment with the test material. Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.
All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.
- Functional Performance Tests
Males treated at 300 mg/kg bw/day showed a statistically significant increase (p<0.05) in one of three forelimb grip strength tests while females from this test group showed a statistically significant reduction (p<0.05) in one of three hind limb grip strength tests. In the absence of supporting evidence of neurotoxicity, these changes were considered attributable to biological variability.
Further findings in animals treated at 300 mg/kg bw/day involved a reduction (p<0.05) in the final 20 percentile activity measurement identified in the males and a reduction (p<0.05) for overall mobility and activity observed in the females from this test group in comparison with the controls. While the causes of these changes are uncertain as there was no supporting evidence of neurotoxicity in these animals, these intergroup variations were therefore considered not to be toxicologically significant.
- Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.

GROSS PATHOLOGY
Macroscopic examination of animals sacrificed at study termination revealed the following findings in a limited number of animals:
Small epididymides and testes were noted in one control male (No. 12) and in one 300 mg/kg bw/day male (No. 49). Small seminal vesicles were identified in one 100 mg/kg bw/day male (No. 35) and in three males (Nos. 49, 50 and 57) at 300 mg/kg bw/day. A small prostate was also identified in one male (No. 49).
Incidental findings involved one 100 mg/kg bw/day female (No. 47) observed to have an enlarged spleen and one male (No. 32) from this test group found to have flaccid kidneys and a fluid filled pelvic cavity confined to the left kidney. The isolated nature of both findings would suggest they are unrelated to treatment, furthermore, the lesions identified in the kidneys were consistent with hydronephrosis, a naturally low incidence occurrence found in rats.
Examination of early termination animals (1000/500 mg/kg bw/day) revealed local irritation in the stomach of 8 of 12 males and 5 of 12 females. Additionally for males, the prostate and/or seminal vesicle appeared small in one control, one 100 mg/kg bw/day and three 300 mg/kg bw/day males. Two 300 mg/kg bw/day males were also noted to have enlarged adrenals.

ORGAN WEIGHTS
Males and females treated at 300 mg/kg bw/day showed increased (p<0.01) liver weight (both absolute and relative to terminal body weight) in comparison with controls. Increased liver weight (p<0.01) was also identified in the males treated at 100 mg/kg bw/day.
Further findings in the surviving male test groups involved a reduction in prostate weights (100 mg/kg bw/day p<0.05; and 300 mg/kg bw/day p<0.01) accompanied in both test groups by low (p<0.01) seminal vesicle weights. Additional findings in females tested at 300 mg/kg bw/day included an increase (p<0.01) in relative brain weight (reduction in absolute weight) and both female test groups displayed a reduction for ovary and pituitary weights (100 mg/kg bw/day) p<0.05 and 300 mg/kg bw/day (p<0.01) in comparison with controls. However, individual values in the greater number of animals were within the normal historical ranges and as such considered not to be toxicologically significant.
Incidental findings in males that received 300 mg/kg bw/day involved a slight but statistically significant increase (p<0.05) in adrenal weights in comparison with controls. Individual values in the majority of animals remained within the historical ranges and as there were no degenerative histopathological changes identified it was concluded this finding was of no toxicological consequence. Females treated at 100 mg/kg bw/day were noted to have a slight reduction (p<0.05) in thyroid/parathyroid weight in comparison with the controls. However, in the absence of similar findings in females tested at 300 mg/kg bw/day this finding was considered to be a result of normal biological variation and not test material toxicity.
The organ weights of high dose (1000/500 mg/kg bw/day) animals were also recorded. However, in the absence of data to permit comparative assessment and given the short duration of treatment of this data can only be used in a speculative way.

HISTOPATHOLOGY
The following treatment-related histopathological findings were present:
- Centrilobular hepatocyte hypertrophy of the liver in males receiving 100 mg/kg bw/day or above and in females receiving 300 mg/kg bw/day.
- Follicular cells: hypertrophy/hyperplasia in the thyroid of males receiving 300 mg/kg bw/day and females receiving 100 mg/kg bw/day or above.
- Pituitary: hypertrophic/vacuolated cells (males receiving 100 mg/kg bw/day or above).
- Testicular Sertoli cell vacuolation in males receiving 100 mg/kg bw/day or above and Leydig cell atrophy in males receiving 300 mg/kg bw/day.
- Prostate: atrophy in males receiving 300 mg/kg bw/day.
- Atrophy in the coagulating glands and seminal vesicles of males receiving 100 mg/kg bw/day or above.
- Epithelial hyperplasia in the stomach of both sexes receiving 300 mg/kg bw/day or above accompanied by erosion, inflammation and congestion in the males treated at 1000/500 mg/kg bw/day).
- Haemorrhaging of the mesenteric lymph nodes in both sexes receiving 300 mg/kg bw/day.
- Adrenal cortex vacuolation in males receiving 100 mg/kg bw/day or above.
- Alveolar macrophages in the lungs of males receiving 300 mg/kg bw/day and females receiving 100 mg/kg bw/day or above.

DISCUSSION
There were no clinical signs to indicate toxicity; however, oral administration of the formulated test material to all test animals induced persistent episodes of excessive salivation around the time of dosing, indicating the test material to have irritative properties. Confirmation of this hypothesis was evident at necropsy in early termination high dose animals that showed inflammatory gastric changes. Subsequent histopathological examination revealed changes characterised by epithelial hyperplasia in both sexes at 300 mg/kg bw/day and the high dose group, with erosion, inflammation and congestion present in high dose males. There was also microscopic evidence of mesenteric lymph node haemorrhage in animals receiving 300 mg/kg/day. Based on these findings it is reasonable to assume the increased water consumption observed in test animals in this study, together with any adverse body weight fluctuations and reduced food intake/utilisation also to have been associated with the irritant characteristics of the test material and may also have led to common and stress-related responses to the test material (the increase and incidence/severity of vacuolation in the adrenal cortex of males receiving 100 mg/kg bw/day or above was considered to be secondary to stress). This reasoning may also help explain a number of the organ weight changes such as those seen for the reduced brain, pituitary and ovary weights (females treated at 300 mg/kg bw/day). It is also a reasonable assertion that the cumulative effects of treatment were sufficient to have prompted stress-related responses in the females treated at 100 and 300 mg/kg bw/day which may have been a factor in the number of animals treated at 100 mg/kg bw/day showing a deficit of attention to their offspring (the litters of three of these females did not survive to Day 5 post-partum). However, the reproductive contrast between the 100 and 300 mg/kg bw/day females and controls showed no clearly discernible dose-related differences in reproduction; there is therefore justification to conclude that there were no direct reprotoxicological adverse changes identified in females in this study. Other noteworthy findings involved the blood chemistry results that gave some evidence to suggest changes in liver function with an increase in plasma alanine aminotransferase levels together with fluctuations in group mean cholesterol and bile acid values among males and/or females treated at 100 and 300 mg/kg bw/day. In relation to the histopathological findings for the liver, a treatment-related increase in incidence/severity of thyroid hypertrophy/hyperplasia in males receiving 100 mg/kg bw/day or above and females receiving 300 mg/kg bw/day and pituitary: hypertrophic/vacuolated cells in males receiving 100 mg/kg/day or above were considered likely to be secondary to alterations to hormonal feedback mechanisms due to liver enzyme induction.
A similar justification may be made for thyroid follicular cells (male 100 mg/kg bw/day or either sex treated at 300 mg/kg bw/day). However, females receiving 100 mg/kg bw/day were also identified to have hypertrophy/hyperplasia of thyroid follicular cells this was considered less likely to be due to a secondary effect of liver enzyme induction, as there were no other findings at this dose level to support this reasoning and on this basis a primary adverse response to treatment cannot be discounted in females and on this basis possibly also males from this treatment group.
To conclude, the evidence of adverse treatment-related responses is greater in animals treated at 300 mg/kg bw/day than for those seen in animals exposed to 100 mg/kg bw/day and while a significant contributing factor was undoubtedly the irritative properties of the test material there is sufficient evidence remaining, including those identified at histological examination, to preclude classification of a No Observed Adverse Effect Level in either sex tested at 100 and 300 mg/kg bw/day in this study.

Dose descriptor:

NOAEL

Basis for effect level:

other: Treatment related effects were detected in animals of either sex from all treatment-groups, precluding classification of a NOAEL for systemic toxicity.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Table 1: Group Mean Body Weight Values (g) - Males

Day Relative to Start	Dose Group (mg/kg)
	0	100	300	1000/500
1	327.4	326.4	325.2	326.8
8	341.3	341.1	320.6	295.8
9	343.7	343.0	319.1	291.7
10	344.5	345.2	320.9	286.8
11	346.8	346.6	320.5	281.0
12	349.8	349.3	320.1	273.9
13	350.7	351.4	321.1	274.7*
14	352.5	353.6	322.8	-
15	355.5	355.8	324.5	-
16	351.2	352.5	319.0	-
17	353.7	353.2	324.5	-
18	353.8	353.5	325.0	-
19	356.4	355.8	326.3	-
20	358.7	357.9	329.3	-
21	360.6	359.8	332.2	-
22	363.7	362.6	335.5	-
23	366.0	364.0	336.0	-
24	367.1	365.1	339.3	-
25	369.3	369.9	340.3	-
26	369.9	371.3	339.6	-
27	373.0	374.4	344.1	-
28	375.6	378.2	346.0	-
29	375.3	373.4	345.8	-
36	386.4	389.6	358.0	-
43	394.8	399.6	365.0	-

*N = 10

- = All animals dead

 

Table 2: Group Mean Body Weight Values (g) - Females

Day Number	Dose Group (mg/kg)
	0	100	300	1000/500
Relative To Start
1	206.3	197.7	200.1	198.4
8	208.2	201.8	201.9	187.3
9	208.7	200.6	202.9	185.4
10	207.5	200.0	203.7	183.3
11	207.8	202.8	204.3	182.3
12	211.9	204.9	206.7	179.8
13	212.8	204.3	206.7	178.3
14	210.3	203.7	207.1	-
15	211.0	206.1	205.9	-
Gestation*
0	219.4	209.5	212.0	-
7	242.1	229.7	232.4	-
14	268.4	253.9	255.4	-
20	328.1	305.1	302.4	-
Lactation*
1	247.8	227.9	230.3	-
4	259.3	234.6	236.4	-

For the control and 300 mg/kg dose group, N = 10; for the 100 mg/kg dose group N = 11

- = All animals dead

 

Table 3: Group Mean Clinical Chemistry Values

Dose Group (mg/kg)	Sex	Urea (mg/dL)	Glucose (mg/dL)	Tot.Prot (g/dL)	Albumin (g/dL)	A/G Ratio	Na+ (mmol/L)	K+ (mmol/L)	Cl- (mmol/L)	Ca++ (mmol/L)	P (mmol/L)	ASAT (IU/L)	ALAT (IU/L)	AP (IU/L)	Creat (mg/dL)	Chol (mg/dL)	Bili (mg/dL)	BA (µmol/L)
0	M	46.6	150.4	7.166	3.96	1.238	146.8	4.456	100.8	2.722	2.02	78.8	56.4	179.0	0.692	83.8	0.130	7.02
	F	51.2	124.8	6.512	3.74	1.372	145.8	4.210	103.0	2.654	1.06	122.8	97.2	149.6	0.868	74.0	0.054	7.56
100	M	44.0	159.8	7.372	4.16	1.310	146.8	4.502	101.6	2.658	2.10	92.8	70.6*	194.8	0.742	53.4**	0.120	6.76
	F	53.4	131.2	6.692	3.78	1.352	146.6	4.012	102.6	2.600	0.94	94.2	74.6	98.0	0.748	67.6	0.084	17.44
300	M	48.8	153.2	6.748	4.10	1.554**	144.8	4.474	101.8	2.672	1.68	87.2	82.2**	176.4	0.688	32.2**	0.108	19.86**
	F	81.6**	144.4	6.522	3.80	1.410	142.8	4.464	100.0	2.702	1.68*	153.4	120.0	141.4	0.710	58.0	0.088	20.46*
1000/500	M	45.2	159.6	5.198	3.35	1.834	145.2	4.908	102.9	2.485	1.72	97.6	82.3	46.4	0.607	27.7	0.073	4.98
	F	53.3	165.5	6.193	3.98	1.812	145.8	4.889	100.6	2.729	1.63	121.0	68.1	39.8	0.688	29.1	0.057	13.40

**p<0.01

*p<0.05

 

Table 4: Group Mean Organ Weights with Corresponding Relative Organ weights

Parameter		Dose Group (mg/kg)
		Males				Females
		0	100	300	1000/500	0	100	300	1000/500
Terminal body weight	Mean (g)	394.8	399.6	365.0	274.7	257.1	238.7	234.5	178.3
Adrenals	Mean (g) Mean (%)	0.07423 0.019	0.07260 0.018	0.07963* 0.022*	0.11019 0.040	0.08979 0.035	0.07765 0.033	0.09516 0.040	0.10243 0.058
Brain	Mean (g) Mean (%)	2.08628 0.530	2.04157 0.513	2.00358 0.551	1.96117 0.719	1.85020 0.721	1.80494 0.759	1.72560** 0.729**	1.77099 1.001
Epididymides	Mean (g) Mean (%)	1.52090 0.385	1.44850 0.364	1.34723 0.370	1.04219 0.380	-	-	-	-
Heart	Mean (g) Mean (%)	1.06949 0.272	1.04629 0.262	0.9628 0.263	0.72641 0.265	0.84446 0.327	0.78995 0.332	0.88490 0.374	0.56182 0.316
Kidneys	Mean (g) Mean (%)	2.26862 0.575	2.25666 0.566	2.17250 0.594	1.69883 0.619	1.52727 0.593	1.46866 0.618	1.48501 0.627	1.11771 0.627
Liver	Mean (g) Mean (%)	12.7228 3.220	13.9166** 3.482**	14.3625** 3.929**	9.96443 3.634	10.6813 4.130	10.5402 4.421	12.0003** 5.053**	7.60416 4.249
Ovaries	Mean (g) Mean (%)	-	-	-	-	0.12403 0.048	0.11025* 0.047*	0.09662** 0.041**	0.08500 0.048
Pituitary	Mean (g) Mean (%)	0.01134 0.003	0.01182 0.003	0.01091 0.003	0.00903 0.003	0.01651 0.006	0.01330* 0.006*	0.01451* 0.006*	0.01128 0.006
Prostate	Mean (g) Mean (%)	0.62648 0.159	0.51280* 0.129*	0.46103** 0.126**	0.29197 0.105	-	-	-	-
Seminal vesicles	Mean (g) Mean (%)	2.45358 0.621	1.92570** 0.483**	1.55276** 0.424**	0.64716 0.234	-	-	-	-
Spleen	Mean (g) Mean (%)	0.73403 0.186	0.68886 0.172	0.67545 0.185	0.51214 0.186	0.66617 0.260	0.64281 0.269	0.62130 0.263	0.40305 0.225
Testes	Mean (g) Mean (%)	3.69015 0.933	3.55399 0.893	3.38957 0.932	3.19231	-	-	-	-
Thymus	Mean (g) Mean (%)	0.43589 0.110	0.46139 0.115	0.33921 0.093	0.27380 0.099	0.23458 0.090	0.21850 0.091	0.18896 0.080	0.21787 0.121
Thyroid/Parathyroid	Mean (g) Mean (%)	0.023533 0.006	0.026483 0.007	0.027150 0.007	0.01450 0.008	0.021942 0.009	0.017503* 0.007*	0.021067 0.009	0.015250 0.009
Uterus and cervix	Mean (g) Mean (%)	-	-	-	-	0.74188 0.287	0.71135 0.300	0.64748 0.272	0.45658 0.251

**p<0.01

*p<0.05

Conclusions:

The oral administration of the test material to rats by gavage, at dose levels of 100, 300 and 1000/500 mg/kg bw/day, resulted in treatment-related effects detected in animals of either sex from all treatment-groups, precluding determination of a ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity.

Executive summary:

A study was conducted to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) in accordance with the standardised guideline OECD 422 under GLP conditions. The study was also designed to be compatible with Commission Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on REACH.

The test material was administered by gavage to three groups of twelve male and twelve female Wistar rats for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 (reduced to 500) mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Corn oil).

Due to a marked adverse response that developed in high dose group (1000 mg/kg bw/day) animals during the first week of treatment, these animals were at first removed from treatment for two days, then recommenced at 500 mg/kg bw/day. However, the condition of the animals deteriorated further soon after resumption of treatment and a decision was then taken on welfare grounds to sacrifice these animals.

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study. Extensive functional observations were performed on five randomly selected males from the control, 100 and 300 mg/kg bw/day dose groups after the completion of the pairing phase, and for five randomly selected parental females from each mated dose group on Day 4 post-partum.

Haematology and blood chemistry were evaluated prior to termination on five randomly selected males and females from each dose group. A terminal blood sample was also taken from the 1000/500 mg/kg bw/day test group.

Surviving males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post-partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post-coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Treatment-related findings involved episodes of transient increased salivation (around the time of dosing) in either sex treated at 100 and 300 mg/kg bw/day between the second week of treatment and final week of gestation (females) or study termination (males). Animals of either sex treated at 1000/500 mg/kg bw/day displayed instances of increased salivation, pilo-erection, staining around the mouth and ano-genital region. These findings were accompanied, in the males only, by staining around the snout, hunched posture, abnormal respiration, ptosis, lethargy, dehydration and emaciation. Additional findings in the females included staining around the eyes and generalised fur loss.

Treatment with the test material led to low weight gains or weight loss in all male animals. Weight gains of the female test groups (100 and 300 mg/kg bw/day) did not vary greatly from that of the control females during maturation.

Food consumption was reduced in the high and 300 mg/kg dose groups; Food efficiency in both male and female test groups paralleled the trends seen for dietary intake in the controls. All treatment groups showed an increase in water consumption, though for the 100 mg/kg females this was only during gestation and lactation.

No treatment-related changes were detected in haematological parameters. Statistically significant findings were observed in alanine aminotransferase (increase) and cholesterol (decrease) in either sex at 100 and 300 mg/kg bw/day. These changes were accompanied in males from both test groups by elevated values for albumin/globulin ratio and in 300 mg/kg bw/day females by an increase in plasma urea levels. In addition, both sexes that received 300 mg/kg bw/day showed heightened levels of bile acids.

There were no treatment-related changes in the behavioural parameters measured or functional performance assessments or sensory reactivity assessments.

The predominant finding at necropsy in early termination high dose (1000/500 mg/kg bw/day) animals of either sex was local irritation in the stomach.

Males and females treated at 300 mg/kg bw/day showed increased liver weight (both absolute and relative to terminal body weight) in comparison with controls. This was accompanied in the males by a slight but statistically significant increase in adrenal weights. Females from this test group showed a slight reduction in pituitary weight and reduced ovary weights in comparison with controls together with minor changes in brain weight. In addition, males treated at 100 and 300 mg/kg bw/day showed reduced absolute and relative prostate and seminal vesicle values in comparison with control males.

Treatment-related histopathological findings were present: centrilobular hepatocyte hypertrophy of the liver in males (100 mg/kg or above) and females (300 mg/kg); follicular cells: hypertrophy/hyperplasia in the thyroid of males (300 mg/kg) and females (100 mg/kg or above); pituitary: hypertrophic/vacuolated cells in males (100 mg/kg or above); testicular Sertoli cell vacuolation in males (100 mg/kg or above) and Leydig cell atrophy (300 mg/kg); prostate: atrophy in males (300 mg/kg); atrophy in the coagulating glands and seminal vesicles of males (100 mg/kg or above); epithelial hyperplasia in the stomach of both sexes (300 mg/kg or above) accompanied by erosion, inflammation and congestion in the males treated at 1000/500 mg/kg; haemorrhaging of the mesenteric lymph nodes in both sexes (300 mg/kg); adrenal cortex vacuolation in males (100 mg/kg or above); and alveolar macrophages in the lungs of males (300 mg/kg) and females (100 mg/kg or above).

The oral administration of the test material to rats by gavage, at dose levels of 100, 300 and 1000/500 mg/kg bw/day, resulted in treatment-related effects detected in animals of either sex from all treatment-groups, precluding determination of a ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted in accordance with standardised guidelines under GLP conditions. The quality of the database is therefore considered to be high.

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

The substance was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 (reduced to 500) mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Corn oil). Due to a marked toxic response that developed in the high dose group (1000 mg/kg bw/day) animals during the first week of treatment; these animals were at first removed from treatment for two days dosing, then dosing recommenced at 500 mg/kg bw/day. However, the condition of the animals deteriorated further soon after resumption of treatment and a decision was then taken on welfare grounds to terminate these animals. The histopathology results showed that these effects were caused by severe local irritation to the stomach resulting in erosion, inflammation and congestion in the high dose group and epithelial hyperplasia at the mid-dose group. In addition, all test animals induced persistent episodes of excessive salivation around the time of dosing indicating the test item to have irritative properties.

 

Systemic Effects and Classification

 

The oral administration of the substance to rats by gavage, at dose levels of 100, 300 and 1000/500 mg/kg bw/day, resulted in treatment related effects detected in animals of either sex from all treatment-groups precluding the definition of a No Observed Adverse Effect Level (NOAEL) for systemic toxicity by the Study Director. The effects that precluded the definition of a systemic NOAEL were the observation of follicular cell hypertrophy/hyperplasia in the thyroid gland of both males and females at 100 and 300 mg/kg bw/day. Thus, the Study Director identified treatment-related effects that may potentially result in classification only in the thyroid; not in the cardiovascular organs, the liver, adrenal glands or pituitary. However, all of these organs will be addressed below.

 

Cardiovascular organs. No effects of any kind were observed in the cardiovascular organs.

 

Liver. Males and females treated at 300 mg/kg bw/day showed increased (p<0.01) liver weight (both absolute and relative to terminal body weight) in comparison with controls. Increased liver weight (p<0.01) was also identified in the males treated at 100 mg/kg bw/day.Centrilobular hepatocyte hypertrophy of the liver in males receiving 100 mg/kg bw/day or above and in females receiving 300 mg/kg bw/day. Toxicity studies have demonstrated that exposure of laboratory animals to liver enzyme inducers during safety assessment results in a signature of toxicological changes characterized by an increase in liver weight, hepatocellular hypertrophy and cell proliferation. Recent advances over the last decade have revealed that for many xenobiotics, these changes may be induced through a common mechanism of action involving activation of the nuclear hormone receptors CAR, PXR, or PPARa. The generation of genetically engineered mice that express altered versions of these nuclear hormone receptors, together with other avenues of investigation, have now demonstrated that sensitivity to many of these effects isrodent-specific. These data are consistent with the available epidemiological and empirical human evidence and lend support to the scientific opinion that these changes have little relevance to man and are considered as an adaptive and a non-adverse reaction (Hall et al, 2012).

 

Adrenal Glands. Increased adrenal gland weights were observed in males receiving 300 mg/kg bw/day, which correlated with increased vacuolation of the adrenal cortex. These observations were considered by both the Study Director and the Study Pathologist to be stress-related and associated to the strong local irritation properties of the substance (Everds, 2013).

 

Pituitary Gland. In females a reduction for pituitary weights (100 mg/kg bw/day) p<0.05 and 300 mg/kg bw/day (p<0.01) in comparison with controls was reported but without any histopathologic correlates. Also, individual values in the greater number of animals were within the normal historical ranges and as such considered not to be toxicologically significant. In fact, the values at 0.006% for the relative pituitary weights given in Table 22 were identical for the control, 100 and 300 mg/kg bw/day groups, so the statistical significance shown for the two treatment groups (p<0.05) must be either a reporting error or a statistical chance based on very small within group standard deviations. Furthermore, the study report (page 49) indicates that hypertrophy and vacuolation of pituitary cells were observed in males at 100 and 300 mg/kg bw/day, but in fact this was not the case according to the pathology report and so must be considered to be a reporting error.

 

In conclusion, the data of this screening study do not justify any classification for STOT RE 1.

 

References

 

Everds, N E, et al.(2013) Interpreting Stress Responses during Routine Toxicity Studies: A Review of the Biology, Impact, and Assessment. Toxicologic Pathology. Mar;41(4):560-614

 

Hall A P, et al. (2012) Liver Hypertrophy: a review of adaptive (adverse and non-adverse) changes. Toxicologic Pathology. Oct;40(7):971-994

 

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance is not considered to require classification with respect to repeat dose toxicity.