Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity - oral: van Otterdijk (2003) performed a subacute 28 -day oral toxicity test with the substance by daily gavage in the rat (50, 150 and 1000 mg/kg bw/d dose levels) according to OECD Guideline 407 and EU Method B.7. On the basis of the very slight and/or transient body weight effects at 150 mg/kg/day, and the sporadic appearance of clinical signs throughout the study that were in the absence of any histopathological findings, the NOAEL is considered to be 150 mg/kg/day (instead of 50 mg/kg bw/d as concluded in the study report).
In addition, Chase (2011) performed a subchronic 90 -day oral toxicity test with the test substance by daily gavage in Crj:CD(SD) rats according to OECD Guideline 408. The substance was administered for 90 consecutive days at dose levels of 15, 50 and 150 mg/kg/day. The NOAEL was considered to be 150 mg/kg/day.

Repeated dose toxicity - inhalation: Neither the properties of the substance nor exposure considerations trigger the need for a repeated dose toxicity study via inhalation or the dermal route.

Repeated dose toxicity - dermal: Neither the properties of the substance nor exposure considerations trigger the need for a repeated dose toxicity study via inhalation or the dermal route.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-04-28 - 2003-05-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): XTJ 568
- Physical state: Clear colourless liquid
- Analytical purity: 97.8%
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: 8191-34
- Expiration date of the lot/batch: 01-01-2004
- Stability under test conditions: no data
- Storage condition of test material: at room temperature in the dark, stable under storage conditions
- Other: specific gravity > 0.94 g/cm3
Species:
rat
Strain:
Wistar
Remarks:
Wistar Crl:(WI) BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males: 181-183 g (mean); females: 151-153 g (mean)
- Fasting period before study: not applicable
- Housing: Animals were housed in a controlled environment. Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height). During activity monitoring animals were individually housed overnight in Macrolon plastic cages (type III, height 15 cm) with sterilised sawdust (SAWI, Jelu Werk, Rosenberg, Germany) provided as bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 - 24.1°C
- Humidity (%): 34-69%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Method of administration:
Gavage.
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the test substance. Storage conditions: at ambient temperature.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy, homogeneity and stability were determined. Analytical method: quantitative analyses were based on the largest peak in the LC-MS chromatogram of XTJ 568.
From the results it was concluded that for formulations of Groups 1-4a relatively high spread was also noted during sample analysis. This spread was however lower than for the procedural recovery samples, particularly at the 200 mg/g level. These results indicate that the analysed concentrations in the formulations do not significantly deviate from 100ù and that the formulations were prepared accurately and homogeneously. Inhomogeneity of test substance formulations in water would be very unlikely since the test substance was found to be mixable with water during NOTOX project 375569. The results of the stability test revealed an apparent 12-14% increase in test substance concentration after 4 hours of storage. This apparent increase falls, however, well within the coefficient of variation of the method. Therefore it can be concluded that test substance formulations were stable during storage for 4 hours.
Duration of treatment / exposure:
28 days, 7 days per week
Frequency of treatment:
Once daily, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected on the basis of a 5-day dose range finding study (NOTOX project 375705).
- Rationale for animal assignment (if not random): at random (computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean)
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter (except at the end of week 14), this was also performed outside the home cage in a standard arena. The symptoms were graded 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

BODY WEIGHT: Yes
- Time schedule for examinations: On days 1, 8, 15, 22 and 28. Body weights of high dose animals were determined on day 19 prior to sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (weekly)

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION : No (subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Blood samples were collected under iso-flurane anesthesia immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Animals sacrificed on day 19 were not fasted. Blood samples were drawn from retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.25 mL), with citrate for clotting tests (1.0 mL) and Li-heparin treated tubes for clinical biochemistry parameters (1.0 mL). The following parameters were determined:
Hematology: erythrocytes count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, red cell distribution width, total leucocytes count, differential leucocyte count, clotting potential: prothrombin time, partial thromboplastin time

CLINICAL CHEMISTRY: Yes
Parameters checked: alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin, total, chloride, cholesterol, total, creatinine, glucose, phosphorus, protein, total, protein, albumin, urea, calcium, potassium, sodium.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
During week 4 of treatment, the following tests were performed on all animals:
- hearing ability, pupillary reflex, static righting reflex and grip strength (score 0 = normally/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).

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy:
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:
aorta, caecum, (clitoral gland), duodenum, (eyes with optic nerve and Harderian gland), (femur including joint), ileum, kidneys, (lacrimal gland, exorbital), lung, infused with formalin, (nasopharynx), ovaries, peyer's patches (jejunum, ileium) if detectable, (preputial gland), rectum, sciatic nerve, (skeletal muscle), spinal cord - cervical, midthoracic, lumbar, sternum with bone marrow, testes, thyroid including parathyroid, trachea, uterus, all gross lesions, adrenal glands, brain (cereellum, mid-brain, cortex), (cervix), colon, epidimydes, (female mammary gland area), heart, jejunum, (larynx), liver, lymph nodes - mandibular, mesenteric, oesophagus, pancreas, pituitary gland, prostate gland, (salivary glands - mandibular, sublingual), (seminal vesicles), (skin), spleen, stomach, thymus, (tongue), urinary bladder, (vagina)
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
*: inadvertently not determined from animal no. 1

Histotechnology: All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- all tissues and organs collected at the scheduled sacrifice from all animals of the control group, group 3* and 4*: All surviving high dose animals were sacrificed on day 19 for humane reasons. Therefore, group 3 was also examined microscopically
- all tissues and organs from all animals of all dose groups which died spontaneously or were sacrificed in extremis;
- all gross lesions of all animals

Based on potentially treatment-related morphologic changes, the stomach was also examined from all rats of the intermediate dose groups. Tissues mentioned within brackets were not examined as there were no signs of toxicity or target organ involvement.
Statistics:
- 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 Fischer-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). 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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
High dose animals displayed multiple signs starting in the first treatment week, i.e. hunched posture, piloerection, salivation, chromodacryorrhoea, alopecia of the back region (only seen in females) and a lean appearance. Clinical signs observed subsequently in high dose animals included abnormal gait (most males and females), flat gait (one male and one female), transient hypothermia (all males) and swelling of the abdomen (most females and all males). Rales, quick breathing, gasping, ptosis, squeaking and dehydration were occasionally seen among high dose animals. Piloerection was also noted in most or all females dosed at 150 mg/kg/day between weeks 2 and 4. A hunched posture and general swelling of the skin was transiently observed in some mid dose females in weeks 2 and 3. A lean appearance was noted for one mid dose female (no. 34) in the last treatment week, which corresponds to the observed stagnant growth. There were no treatment-related clinical signs in mid dose males.

Lethargy, flat gait, general swelling of the skin and piloerection noted in one female dosed at 50 mg/kg/day (no. 27) were transient, and although a relationship to treatment can not be entirely excluded, these signs were not regarded to be in toxicological terms. Other infrequent findings among animals treated with the test substance included focal erythema or yellow staining of the genital region, scabs and bleeding on the snout (one high dose male no. 16), brown staining of the fur, a broken tail apex, alopecia and/or scales in the shoulder region, chromodacryorrhoea (in one male at 150 mg/kg/day and one female at 50 mg/kg/day) and watery discharge from the eyes. These findings are more frequently 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 among control animals.
Mortality:
mortality observed, treatment-related
Description (incidence):
Three males and one female at 1000 mg/kg/day were sacrificed for humane reasons on days 18 and 16, respectively. The remaining high dose animals were killed in extremis on day 19. In addition, one female (no. 40) was found dead on day 7. There was no mortality in other groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Markedly reduced weight gain was measured for males and females dosed at 1000 mg/kg/day up to death, attaining statistical significance for males only (p<0.01). The mean weight gain deficit by the end of week 2 was 48% and 40% for high dose males and females, respectively. One of the females (no. 36) showed weight loss (5%) in the first week of treatment. Lower weight gain was also noted for males dosed at 150 mg/kg/day by the end of week 4, resulting in a total weight gain deficit of 13% relative to male controls. In addition, stagnant growth was noted for two females at 150 mg/kg/day (nos. 34 and 35) in week 4, but the mean total weight gain value of this group remained similar to that seen in female controls. Body weights and body weight gain of low dose animals remained in control range over the 4-week study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Absolute food consumption of high dose animals was reduced up to death, and the effects were more marked in the first treatment week than in the second. Relative food intake (i.e. after correction for body weight) was reduced in week 1 only. Among other groups, food consumption before or after allowance for body weight was similar to control groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
High dose animals sacrificed on day 19 showed slightly lower red blood cell counts, haemoglobin values, haematocrit levels and/or mean corpuscular haemoglobin concentration levels. An increased mean corpuscular haemoglobin concentration (HB:HCT ratio) was noted for males dosed at 150 mg/kg/day. This deviation was considered to be incidental in nature and of no toxicological significance since both haemoglobin and haematocrit values were normal. No dose-response relationship was apparent for the statistically significant reduction of red cell distribution width in females dosed at 50 and 150 mg/kg/day. This deviation was also considered to be incidental in nature. Individual increases of neutrophil counts with concurrently reduced lymphocyte counts were noted in female no. 26 (50 mg/kg/day) and female no. 38 (1000 mg/kg/day). This shift in type of white blood cells was considered to be a secondary non-specific response to stress. Animals in a moribund condition often exhibit this pattern.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Pronounced changes noted in all high dose animals sacrificed on day 19 included:
- increased alanine aminotransferase (ALAT) and alkaline phosphatase activity values (ALP)
- increased glucose levels
Less pronounced changes among some or all intercurrently sacrificed animals included increased urea (animal no. 38), potassium (all males and female no. 38), inorganic phosphate (all animals, but within the normal range), chloride (slight in most animals), calcium (males) and cholesterol (all males but within the normal range), and reduced creatinine (animal nos 18, 36 and 39), sodium (all animals, but within the normal range), total protein and albumin (mainly in females). The statistically significant increase of alanine aminotransferase activity in females at 150 mg/kg/day was caused by a slightly high value for female no. 32. This animal also showed an increased alkaline phosphatase activity. Additionally, a high aspartate aminotransferase activity value was noted for female no. 33. There were no statistically significant changes in males at 50 or 150 mg/kg/day. The increased inorganic phosphate levels among females dosed at 50 and 150 mg/kg/day (p<0.05), were considered to be due to slightly lower control values and occurred with no clear dose-related trend. Accordingly, these deviations were considered to be incidental in nature. The higher potassium value of females dosed at 50 mg/kg/day was not accompanied by similar or higher deviations at 150 mg/kg/day and was therefore considered to be of no toxicological significance. No further differences were noted between treated and control animals that were considered to be an effect of treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observations: No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with XTJ 568, when compared to control animals. The variation in motor activity did not indicate a relation with treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Most pronounced organ weight changes noted in high dose animals consisted of:
- increased liver weights and liver to body weight ratios (males and females);
- reduced epididymides weight and epididymides to body weight ratios;
- reduced thymus weight and thymus to body weight ratios (females)

Other changes in organ weights of high dose females were absent when corrected for body weight. Due to low terminal body weights of high dose males, organ to body weight ratios were slightly higher than controls for most remaining organs. Lower absolute liver weights of males dosed at 150 mg/kg/day were absent when corrected for body weight. The changes in absolute testes weights and testes to body weight ratios of males dosed at 50 and 150 mg/kg/day occurred in the absence of a dose-response and considered to be due to lower terminal body weights at 150 mg/kg/day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings in high dose animals were noted in the stomach and consisted of:
- Thickening of the limiting ridge (2/5 males) or stomach wall (1/5 males and 1/5 females);
- Crateriform retractions of the glandular mucosa (1/5 males) or forestomach (1/5 males and all females)
- Red discoloration of the limiting ridge (1/5 males), glandular mucosa (1/5 females), or cardia (1/5 females)
- Irregular surface of the forestomach (2/5 males)
- Red foci on the glandular mucosa (1/5 females)
One high dose male (no. 18) did not have macroscopic abnormalities in the stomach. Other findings in these animals consisted of an emaciated appearance (1/5 males), red discoloration of the lungs (2/5 males) and red foci in the caecum (1/5 females). The female found dead on day 7 showed an advanced state of autolysis.

Incidental findings among control and/or treated animals included pelvic dilation of the kidneys, red discoloration of the mandibular lymph nodes, thymus or adrenal glands, red foci on the thymus, enlarged mandibular lymph nodes, fluid in the uterus, reduced size of the liver and spleen (one control female), and liver and pancreas grown together with diaphragm (one female at 150 mg/kg/day). These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-realted distribution they were considered changes of no toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic observations in high dose animals consisted of:
- moderate to marked squamous epithelial hyperplasia of the forestomach (4/5 males and 5/5 females);
- slight to moderate ulceration of the forestomach (4/5 males and 5/5 females), with associated slight to moderate acute or chronic inflammation;
- increased incidence and severity (slight to moderate) of limiting ridge epithelial hyperplasia (5/5 males and 5/5 females);
- slight to moderate ulceration (2/5 males) of the glandular mucosa of the stomach with associated acute inflammation;
- slight erosion of the glandular mucosa of the stomach (1/5 females);
- slight to moderate cortical lymphoid atrophy of the thymus (4/5 males)

There were no treatment-related microscopic observations in animals dosed at 50 or 150 mg/kg/day. In all groups a number of other histopathological observations were recorded but were considered to be within the normal range and severity of background alterations that may be seen in untreated animals of this age and strain.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
no

Analysis of dose preparations:

Analysis of the accuracy of dose preparations revealed values within the range of 68 -105% of nominal. This variation was considered to be related to the analytical method/test substance properties, and was smaller than the variation seen with the procedural recoveries. Also taking into account the high water solubility of the test substance, formulations in water (Milli-U) were considered to be stable for at least 4 hours and to be prepared homogeneously and accurately at the concentration tested.

Conclusions:
In the study report, a NOAEL of 50 mg/kg bw/day was derived. However, on the basis of the very slight and/or transient body weight effects at 150 mg/kg/day, and the sporadic appearance of clinical signs throughout the study that were in the absence of any histopathological findings, it is considered that 150 mg/kg/day should be the NOAEL for this study.
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-05-13 - 2011-11-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): XTJ 568
- Substance type: Clear colourless liquid
- Physical state: liquid
- Analytical purity: 97% (primary amine)
- Lot/batch No.: OG704
- Expiration date of the lot/batch: April 2012
- Storage condition of test material: At ambient temperature in the dark
- Other: Chemical name: Aliphatic polyetheramine C10H24N2O2
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: 44 to 50 days of age at start of treatment
- Weight at study initiation: 233 to 279 g (males) and 164 to 213 g (females) at start of treatment
- Fasting period before study: no
- Housing: Animals were housed inside a barriered rodent facility (Building 8, Room 1820). Sensory reactivity and grip strength assessment were performed in Room 1820 and motor activity was recorded in Room 1825. The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms. Before the study the room was cleaned and disinfected. The animals were housed five of one sex per cage. The cages were made of a polycarbonate body with a stainless steel mesh lid. Wood based material was used as bedding and was sterilised by autoclaving and changed at appropriate intervals each week. Cages, food hoppers and water bottles were changed at appropriate intervals. Each cage of animals was provided with an Aspen chew block for environmental enrichment. Chew blocks were provided throughout the study and were replaced when necessary. Each cage of animals was provided with a plastic shelter for environmental enrichment, which was replaced at the same time as the cages.
- Diet (e.g. ad libitum): The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance diet), except overnight before routine blood sampling. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light): Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: XTJ 568 was prepared for administration as a series of graded concentration in the vehicle. An appropriate volume of vehicle was added to the test substance and mixture by magnetic stirring until dissolution was achieved. The solution was diluted to volume with the vehicle and finally mixed using a high-shear homogeniser. A series of solutions at the required concentrations were then prepared by serial dilution with the vehicle. The test substance was used as supplied. All formulations were prepared weekly and stored refrigerated (approximately 4°C) before use.
Dose volume: 10 ml/kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A representative sample of test formulation (1 mL) was measured by accurate volume and dissolved in a suitable volume (100 mL) of diluent. The extract was diluted volumetrically using diluent to provide a solution containing XTJ568 at an expected concentration within the range 500 µg/mL to 2000 µg/mL, containing internal standard. The concentrations of XTJ568 in the final solution was quantified by high performance liquid chromatography using MS detection. The response ratio for XTJ568 and the internal standard in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response ratio versus calibration standard concentration. The area responses of the peaks observed at the characteristic retention times for XTJ568 and the internal standard in sample and procedural recovery chromatograms were measured and the response ratio calculated.
Results:
Linearity was confirmed over the nominal concentration range 50 ng/mL to 2500 ng/mL with a correlation coefficient > 0.990;
The precision of injection was < 2% for six replicate injections of standard solutions containing XTJ568 at nominal concentrations of 50 ng/mL and 2500 ng/mL;
Method accuracy and precision were confirmed: a mean procedural recovery value of 99.0% was obtained for 1.5 mg/mL and 98.0% was obtained for 15 mg/mL. The overal mean procedural recovery was 98.5%.

The limit of detection was estimated as 1.15 ng/mL.

The specificity of the HPLC assay was demonstrated by the absence of a peak at the characteristic retention time for XTJ 568 in the control sample.

The mean concentrations of XTJ568 in test formulations analysed for weeks 4 and 13 of the study were within ± 10% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily, seven days per week.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study were selected in conjunction with the Sponsor based on the results of a 4-week toxicity study performed on behalf of the Sponsor in which doses of 50, 150 and 1000 mg/kg/day were investigated.

The high doses in this study of 150 mg/kg/day was expected to result in some toxicity but was not anticipated to cause undue toxicity or mortality. The mid-dose of 50 mg/kg/day was expected to be well tolerated although it may have resulted in some non-adverse toxicity. No effects of treatment were anticipated at the low dose of 15 mg/kg/day.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-heath amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

Daily during the first week of treatment, detailed observations were recorded at the following times in relation to dose administration:
Immediately before dosing
Between one and two hours after completion of dosing of all groups
as late as possible in the working day

Once each week for weeks 2 to 13, detailed observations were recorded at the following times in relation to the dose administration:
Immediately before dosing
Between one and two hours after completion of dosing of all groups

During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

BODY WEIGHT: Yes
- Time schedule for examinations: the weight of each rat was recorded one week before treatment commenced (Week -1), on the day that treatment commenced (Week 0), weekly throughout the treatment period and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The weight of food supplied to each cage, that remaining and an estimated of any spilled was recorded for each week throughout the treatment period. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Fluid intake was assessed by daily visual observation. No effect was observed and, consequently, quantitative measurements were not peformed.

OPHTHALMOSCOPIC EXAMINATION: Yes
Before treatment commenced, the eyes of all animals allocated to the study (including spare animals) were examined by means of a binocular indirect ophthalmoscope. During Week 12 of treatment the eyes of all animals of Groups 1 (Control) and 4 (150 mg/kg/day) were similarly examined.

Prior to each examination, the pupils of each animal were dilated using tropicamide opthalmic solution (Mydriacyl). The Adnexae, conjunctiva, cornea, sclera, anterior chamber, iris, (pupil dilated), lens, vitreous and fundus were examined.

As no treatment-related changes were observed, the examination was not extended to animals of Groups 2 or 3 (15 or 50 mg/kg/day).

HAEMATOLOGY: Yes
During week 13 treatment (before dosing), blood samples were obtained from all animals after overnight withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the sublinqual vein.

Blood samples (nominally 0.5 mL) were collected into tubes containing EDTA as anticoagulant and examined for the following characteristics:
hematocrit, haemoglobin concentration, erythrocyte count, mean cell haemoglobin, mean cell haemoglobin concentration, mean cell volume, total white cell count, differential WBC count: neutrophils, lymphocytes, eosinophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells, platelet count.
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyser. Confirmation or a written description from the blood film was made where appropriate.

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined in respect of:
Prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
At the same time and using the same animals as for peripheral haematology, further blood samples (nominally 0.7 mL) were collected into tubes containing lithium heparin as anticoagulant. all tubes were mechanically agitated for at least two minutes and the sample subsequently centrifuged at 2000 g for 10 minutes in order to separate the plasma. After separation, the plasma was examined in respect of:
Alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, urea, creatinine, glucose, total cholesterol, triglycerides, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein, albumin; albumin/globulin ratio (A/G ratio) was calculated from total protein concentration and analysed albumin concentration

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of the observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the number of animals and the times of testing were balanced across the groups on each day of testing. The following measurements, reflexes and responses were recorded:
approach response, touch response, auditory startle reflex, tail pinch response, grip strength and motor activity
Sacrifice and pathology:
- Necropsy: Animals surviving until the end of the scheduled treatment period were killed by carbon dioxide asphyxiation and subsequent exsanguination. The sequence in which the animals were killed after completion of treatment was selected to allow satisfactory inter-group comparison.
All animals were subject to detailed necropsy.

After review of the history of each animal, full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interacion was recorded.

The requisite organs were weighed and external and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

The retained tissues were checked before disposal of the carcass.

Organ weights:
The following organs, taken from each animal killed after 13 weeks of treatment, were dissected free of adjacent fat and other contiguous tissue an weights recorded:
adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus with cervix
left and right epididymides and testes were weighed separately.

Bilateral organs were weighed together with the exception of the testes and epididymides. Organ weights were also adjusted for terminal body weight, using the weight recorded on the day of necropsy.

Histology: Tissue samples were dehydrated, embedded in paraffin wax, sectioned at approximately four to five microns thickness and stained with haematoxylin and eosin.
Those tissues subject to histological processing included the following regions:
adrenals, brain, femur with joint, heart, kidneys, liver, lungs, spinal cord, sternum, stomach, thyroid, uterus

Pathology: light microscopy: all tissues preserved for examination were examined for all animals of group 1 (control) and 4 (150 mg/kg/day) sacrificed on completion of the scheduled treatment period.
Tissues reported at macroscopic examination as being grossly abnormal were examined for all animals in line with current practice.
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used: minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings
Other examinations:
Sperm analysis: Immediately after scheduled sacrifice of each male killed after 13 weeks of treatment, the left vas deferens, epididymis and testis was removed and the epididymis and testis were weighed.
The following parameters were tested: sperm motility, sperm morphology, sperm count, homogenisation-resistant spermatids count
Statistics:
All statistical analyses were carried out separately for all males and females. All analyses were carried out using the individual animal as the basic experimental unit. The following sequence of statistical tests was used for grip strength, motor activity, bodyweight, clinical pathology, organ weight and sperm analysis data:
- a parametric analysis if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.

a non-parametric analysis was performed if Barlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.

For grip strength, motor activity, clinical pathology and sperm analysis data, if 75% of the data (across all groups) were the same value, for example c, Fisher's exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against both control for i) values < c versus values > or = c, as applicable.

For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate. The treatment comparisons were made on adjusted group means in order to allow for differences in bodyweight which might influence the organ weights.

Significant differences between control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs observed that were related to the time of dosing.
Mortality:
no mortality observed
Description (incidence):
There were no deaths.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight gain was not affected by treatment. The overall gain of males receiving 15 or 150 mg/kg/day was slightly higher than the controls. These inter-group differences were small and there was no dose-relationship, consequently they were considered to have arisen by chance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Fluid intake was assessed by daily visual observation. No effect was observed and, consequently, quantitative measurements were not peformed.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ophthalmic lesions in Week 12 that were attributable to treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematology investigations during week 13 of treatment did not reveal any findings that were considered to be of any toxicological significance. Slightly low neutrophil and lymphocyte counts (with associated low total white cell counts) were apparent, when compared to the controls, for females receiving 150 mg/kg/day and platelet counts were also slightly high for males receiving 150 mg/kg/day. These changes were minor in nature, restricted to one sex and were not considered to be of any toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Biochemical investigations during Week 13 of treatment did not reveal any findings that were considered to be of any toxicological significance. All inter-group differences that attained statistical significance were considered minor in nature, lacked dose-relationship or were restricted to one sex and therefore considered of no toxicological significance. These included the slightly high potassium concentrations for treated females, slightly low creatinine concentration for males receiving 50 mg/kg/day and slightly high calcium concentrations for females receiving 150 mg/kg/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity and grip strenght values were considered to be unaffected by treatment.
Compared with controls, hindlimb grip strength values for males receiving 15 mg/kg/day were low (p<0.01) during Week 12 of treatment but this was attributed to natural variation as hindlimb values for males receiving the higher dose levels of 50 or 150 mg/kg/day were unaffected. Forelimb grip strength values for males and values for forelimbs and hindlimbs in females were also unfaffected.
Motor activity: Motor activity scores for males and females were considered to be unaffected by treatment. High beam and low beam motor activity scores (rearing and cage floor activity respectively) for males receiving 15 or 150 mg/kg/day were low (approximately 0.77 X and 0.69 W controls respectively) throughout most of the 1-hour recording period. Although some of the differences attained statistical significance (p<0.05) and some of the group mean values in these two groups were below the historical control data range, an association with treatment was considered unlikely because scores for males receiving 50 mg/kg/da were unaffected. in addition, the differences in group mean values were due largely to the scores of just two control males, which were considerably higher than those for all other controls. These inter-group differences in motor activity scores for males were therefore considered to be due to natural variation. Scores for females were unaffected by treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Organ weights were unaffected by treatment. All inter-group differences that attained statistical significance were considered minor in nature, lacked dose-relationship or were restricted to one sex and therefore considered of no toxicological significance. These included the slightly low heart and thymus weights for treated males and the low brain weights for males which received 150 mg/kg/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination revealed no changed associated with treatment. All macroscopic findings reported were of the type encountered normally in CD rats at these laboratories.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The microscopic examination revealed no changes associated with treatment.
The incidence and distribution of all findings were consistent with the commonly seen background microscopic changes in rats of this age and strain.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
An adenocarcinoma of the mammary gland was observed in a control female and a female receiving 15 mg/kg/day. The finding was considered to be part of spontaneously occurring background pathology findings in female rats of this age and strain and therefore unrelated to treatment.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
- Sperm analysis: there were no effects of treatment on sperm number or morphology. There were slight reductions in % motile and progressively motile sperm at 50 and 150 mg/kg/day however these were largely due to one animal in each group (male Nos. 21 and 6) with low values. The reasons for these low values could not be established however they were not considered to be related to treatment with the substance.
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no target organs identified at histopathological examination. Minor changes seen in blood parameters were considered to be of no toxicological importance and not adverse in nature.
Key result
Critical effects observed:
no
Conclusions:
The oral administration of the substance to Crl:CD(SD) rats for 13 weeks at doses up to 150 mg/kg/day was well tolerated. There were no target organs identified at histopathological examination. Minor changes seen in blood parameters were considered to be of no toxicological importance and not adverse in nature. Consequently the No Observed Adverse Effect Level (NOAEL) was considered to be 150 m/kg/day. The substance is considered not classified as STOT RE.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: oral

van Otterdijk (2003) performed a subacute 28 -day oral toxicity study with the substance by daily gavage in the rat. Based on the results of a 5-day range finding study, the dose levels for the 28 -day toxicity study were selected to be 0, 50, 150 and 1000 mg/kg/day.

The study was based on the following guidelines:

- EC Directive 96/54/EEC, B.7 Repeated Dose (28 days) Toxicity (oral), 1996

- OECD 407, Repeated dose 28 -day Oral Toxicity Study in Rodents, 1995

- OPPTS 870.3050, Repeated dose 28 -day oral toxicity study in rodents

The test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Treatment of Wistar rats with the substance for 28 consecutive days by oral gavage at dose levels up to 1000 mg/kg/day resulted in the premature sacrifice or death of all high dose animals. Stomach effects (forestomach hyperplasia and ulceration) were considered to be contributory to death or the retarded health condition of these animals, and point to corrosive/irritating properties of the test substance. Corroborative signs of ill-health were noted during treatment together with reduced food intake and food conversion efficiency, and a marked reduction of weight gain.

Transient lower body weight gain was also obtained for animals dosed at 150 mg/kg/day, but without supportive changes in food intake. In this dose group, piloerection was noted among all females with hunched posture, leanness and general swelling of the skin observed less frequently. However, no stomach abnormalities were noted at 150 mg/kd/day. Decreased epididymis weights in high dose males was considered to have occurred secondary to the markedly reduced body weight gain and the overall condition of these animals. There were no changes in performance of neurological functional observations that were considered to be an effect of treatment. At 150 mg/kg/day, clinical signs (in the absence of histopathological changes), the LOAEL is considered to be 150 mg/kg/day and the NOAEL to be 50 mg/kg/day. However, on the basis of the very slight and/or transient body weight effects at 150 mg/kg/day, and the sporadic appearance of clinical signs throughout the study that were in the absence of any histopathological findings, it is considered that 150 mg/kg/day is the NOAEL for this study.

Chase (2011) performed a subchronic 90-day oral toxicity study with the substance by daily gavage in the rat. The rats were administered for 90 consecutive days by oral gavage at dose levels of 15, 50 and 150 mg/kg/day. This study also included enhanced sperm mobility and histopathology evaluations to clarify the results of the sperm evaluations from the two-generation reproductive toxicity study. There were no deaths to the study animals during the study. The appearance and behaviour of the animals were unaffected by treatment. Bodyweight gain, food consumption and organ weights were unaffected by treatment. There were no macroscopic or microscopic findings that were associated with treatment. Sensory reactivity, grip strength and motor activity were unaffected by treatment. There were no changes in the performance of neurological functional observations that were considered to be an effect of treatment. There were no effects of treatment on sperm number or morphology. The NOAEL was considered to be 150 mg/kg/day. This study supports the results of the 28 day repeated dose test with a NOAEL of 150 mg/kg/day.

The results of this study indicate that there are no effects on sperm number, motility or morphology at 150 mg/kg/day and support the conclusion that the substance is not a reproductive toxicant.

Repeated dose toxicity: inhalation and dermal

Neither the properties of the substance nor exposure considerations trigger the need for a repeated dose toxicity study via inhalation or the dermal route.

Justification for classification or non-classification

The NOAEL for systemic toxicity was 150 mg/kg in the 90-day oral toxicity study. Because the Guidance Value Range to assist in potential STOT RE classification (specific target organ toxicity repeated exposure) is 10 - 100 mg/kg/day for the oral route of exposure, no classification is justified for the substance according to the CLP Regulation.