[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 April 2018 to 13 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, reaction products with tetraethylenepentamine

Cas Number:

71799-54-7

Molecular formula:

Not applicable - UVCB substance

IUPAC Name:

Octadecanoic acid, reaction products with tetraethylenepentamine

Test material form:

solid

Details on test material:

- Appearance: Amber solid
- Storage: Room temperature (20 ± 5 °C); protected from light.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at arrival: 7 - 8 weeks
- Weight at arrival: 219 - 234 g for males, 184 - 209 g for females
- Housing: From arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material was provided as necessary and changed at least twice a week.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: An acclimatisation period of 28 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

DETAILS OF FOOD AND WATER QUALITY: There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test material was present in the drinking water or the diet.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 15 %
- Air changes: approximately 15 to 20 air changes per hour
- Photoperiod: the rooms were lit by artificial light for 12 hours each day

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test material was administered orally, by gavage. The oral route was selected as it is a possible route of human exposure to the test material.

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

VEHICLE
- The vehicle was 0.5 % aqueous solution of carboxymethylcellulose.
- The required amount of the test material was suspended in the vehicle. The formulations were prepared daily (concentrations of 10, 30 and 75 mg/mL). Concentrations were calculated and expressed in terms of test material as supplied.
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYTICAL METHOD
- The technique used was liquid chromatography (LC) with tandem mass spectrometric (MS/MS) detection of the analyte. The method was validated for the measurement of the test material in 0.5 % CMC (10 to 75 mg/mL).

FORMULATION PROCEDURE
Formulations were prepared at 10 and 75 mg/mL using the procedure described below:
1. Pre-heat the required amount of vehicle to 60 °C
2. Add the test material to the hot vehicle and leave at 60 °C for circa 2 h, under magnetic stirring
3. Reduce the temperature to 40 °C and keep under magnetic stirring prior to dosing or analysis
Aliquots were removed from each formulation (two from the top, two from the middle and two from the bottom), then diluted and analysed to assess the content and homogeneity.

REAGENTS
- Mobile Phases:
ELUENT-A: Water + 0.1 % HCO2H: add 1mL of HCO2H to some water in a 1 L volumetric flask and dilute to volume with water.
ELUENT-B: Methanol + 0.1 % HCO2H: add 1mL of HCO2H to some methanol in a 1 L volumetric flask and dilute to volume with CH3OH.

- Solutions:
SOL-1 Diluent: mix equal volumes of Eluent A + Eluent B
SOL-2 Weak wash (5 % Methanol- 95 % Water + 0.1 % formic acid): mix 50mL of Methanol with 950mL of water and add 1mL of formic acid.
SOL-3 Strong wash (acetonitrile-water-methanol-isopropanol (1:1:1:1)) : mix equal volumes of acetonitrile, water, methanol and isopropanol.

SAMPLING
Six 1 g samples (two from the top, two from the middle and two from the bottom) are required from the highest and lowest concentration formulations for the determination of content and homogeneity. Two 1 g samples are required from any intermediate concentrations for the determination of content. Sampling needs to be performed under vigorous magnetic stirring, with the formulation at +40 °C.

PROCEDURE
Preparation of Stock, Working Standard and Calibration Solutions
ODARP is used as the analytical standard and the weighings are not corrected for purity as specified in the protocol.
- Stock and Working Standard Solutions: Two independently prepared stock and standard solutions of ODARP are analysed by LC-MS/ MS to check the accuracy of the preparations prior to their use (acceptability criteria Δ < ± 5 %).
Stock: Transfer about 10mg of ODARP into a 10mL volumetric flask, dilute to volume withMeOH and sonicate for 10 minutes to give a 1000 μg/mL solution (Stock A/B).
SOL: Transfer 1.0mL of STOCK A/B into a 10mL volumetric flask and dilute to volume withMeOH to give an approximately 100 μg/mL solution (SOL A/B).
Standard: Transfer 1.0mL of SOL A/B into a 10mL volumetric flask and dilute to volume with diluent to give an approximately 10 μg/mL solution (STD A/B).

- Calibration Standards: a 5-point calibration curve was prepared by diluting samples of SOL A with diluent to give calibration standards with concentrations of 5, 7.5, 10, 15 and 20 µg/mL.

Accuracy and Precision Samples (for validation only)
- Low level (10 mg/mL): Six independent preparations are required: transfer 1 g of the formulation at 10 mg/mL into a 100 mL volumetric flask, dilute to volume with MeOH, sonicate for 10 minutes and then magnetically stir at room temperature for at least 30 minutes. Transfer 1 mL of the resulting solution into a 10 mL volumetric flask and dilute to volume with diluent (final concentration 10 μg/mL).
- High level (75 mg/mL): Six independent preparations are required: transfer 1 g of the formulation at 75 mg/mL into a 100 mL volumetric flask, dilute to volume with MeOH, sonicate for 10 minutes and then magnetically stir at room temperature for at least 30 minutes. Transfer 1.5 mL of the resulting solution into a 100 mL volumetric flask and dilute to volume with diluent (final concentration 11.25 μg/mL).

Blank and Selectivity Samples
- Blank: inject a sample of diluent as the blank sample.
- Blank + vehicle: transfer 1 g of the vehicle into a 100 mL volumetric flask, dilute to volume with MeOH, sonicate for 10 minutes and then magnetically stir at room temperature for at least 30 minutes. Transfer 1 mL of the resulting solution into a 10 mL volumetric flask and dilute to volume with diluent.

Unknown Samples
- Remove unknown samples, dilute to volume with MeOH, sonicate the resulting mixtures for 10 minutes and then magnetically stir at room temperature for at least 30 minutes. Perform subsequent dilutions with diluent, if necessary. Dilute any additional concentrations accordingly, to fall within the range of the calibration curve.

Density Determination
- Determine the density of each formulation by removing and weighing 1mL with a tared syringe. If it is not physically possible to measure the density of the formulation (due to the nature of the test material or vehicle), then estimate the density of the formulation using the displacement of the test material and the formulae. The result is recorded in the lab book.

Control Standard
- Inject a sample of Std 3 as a control sample before and after a maximum of six unknowns. The deviation of these controls from the theoretical concentration should be ≤ 3 %.

Assay Settings and Parameters
LC Settings
Set up the LC system as follows:
– Acquity BEH C8, 2.1 x 50 mm- 1.7 μm, (Waters, Part N°186002877)
Mobile phase:
– Eluent A:Water + 0.1% HCO2H
– Eluent B: CH3OH + 0.1% HCO2H
Elution:
– Isocratic: 5 % Eluent A – 95 % Eluent B
– Flow: 0.2 mL/min
– Injection volume: 10 μL
– Column temperature: +50 °C
– Autosampler temperature: +10 °C
– Run time: 4 minutes
– Retention time: ≈ 0.6 minutes

Detector parameters
Set up the MS system as follows:
– Ion Source: Turbo Ion Spray (API2000)
– Scan Type: MRM
– Ionisation polarity: Positive Ion Mode
– Axis: 6mm
The mass spectrometer is optimised by infusing pure standard solutions of ODARP. The following Q1 and Q3 masses have been selected to quantify ODARP:
ODARP
Q1 mass 666.7 amu… Q3 mass 648.7 amu/ 282.4 amu
Q3 mass 722.7 amu… Q3 mass 704.9 amu/ 310.5 amu
Retention Time ~ 0.6 minutes
Due to the calibration of the mass spectrometer, a ±0.5 amu variation in the exact mass may be observed. Chromatographic signal (TIC) for each analyte is obtained from the sum of each MRM transition extracted Ion Current (XIC).

- System Suitability Test (SST): As a test of system suitability, inject one of the standard solutions (Std A or Std B) and observe the retention time. To be acceptable, the retention time of the Test Material peak must fall within ± 0.2 minute of the time specified above (the isocratic mixture can be modified in order to comply with this requirement).
- Acceptance Criteria for Peak Retention Times: The chromatographic peak retention time for the Test Material must deviate by less than ± 10 % from the mean of all the retention times, for that Test Material, within the analytical session.

CALCULATIONS
Formulae
- Linearity: The LC-MS/MS system is calibrated using the chromatographic software which generates a linear fit calibration curve drawing the best fit of a line to the amounts of ODARP in μg/mL and the peak areas. The software uses linear least-squares fit formula without weighting. The result of the fitting is:
y = A + Bx
where:
y = Peak area
A = Intercept on the y-axis
B = Slope of the calibration curve
x = ODARP concentration in μg/mL
The correlation coefficient is a rough indicator of the goodness of fit. The equations used to determine the correlation coefficient are as follows:
R² = 1 – [ (Sy)² / σ²y ]
R = √R²
where:
R² = Determination coefficient
R = Correlation coefficient
Sy = Standard error of estimate of y on x
σ²y = Variance

Determination of the Test Material Concentration
- Unknown samples are analysed after the LC-MS/MS system has been calibrated. The ODARP amount, in μg/mL, is obtained directly from the software report. The software calculates the result as:
x = (y – A)/ B
The content of ODARP in formulations, in μg/mL, is obtained as follows:
C = (x.DF.d)/ (w.CF)
where:
C = Content of ODARP in 0.5 % CMC in μg/mL
x = ODARP amounts in μg/mL as read in the result table
d = Density of the formulation
w = Weight of sample taken
DF = Dilution factor
CF = Conversion factor ng/mL to μg/mL (= 1000)

Estimating the Density of a Formulation using the Displacement of the Test Material
- If it is not possible to physically measure the density of a formulation (due to the nature of the test material or the vehicle) use the following formulae to estimate the density:
dt = 1 / Disp
where:
dt = Density of test material
Disp = Displacement of test material

Vt = m.Disp
where:
Vt = Volume occupied by test material
m = Mass of test material used (in grams)
Disp = Displacement of test material

Vv = Vt ot – Vt
where:
Vv = Volume occupied by vehicle
Vt ot = Total volume of formulation
Vt = Volume occupied by test material

d = [(Vt.dt/Vtot) + (Vv.dv/Vtot)]
where:
d = Density of the formulation
Vt = Volume occupied by test material
dt = Density of test material
Vtot = Total volume of formulation
Vv = Volume occupied by vehicle
dv = Density of vehicle

RESULTS
- Specificity: Samples of the diluent and diluent + vehicle (prepared using the lowest dilution factor applied to the formulations) were injected to assess specificity. No signal was seen at the retention time or mass of the test material in either sample.
- Linearity: In order to assess linearity, calibration samples of the test material were prepared at five levels ranging from 4.860 to 19.44 μg/mL. Each sample was injected once and processed as described in the analytical method. The following correlation was found:
Equation: Response = (-1.15e+004) + (8.95e+003) × the test material concentration in μg/mL
r: 0.9978
Response type: Peak area
Fit type: Linear
Weighting: None
- Accuracy and Precision: Accuracy and precision were assessed at 10 and 75mg/mL by preparing formulations at each level and analysing six samples from each. The results met the acceptability criteria for accuracy (80 and 120 % of the theoretical values) and precision (CV< 10 %) outlined in the protocol. The results at each level were also used as the content at time-zero for the stability study.
- Formulation Stability: The stability of the formulations at 10 and 75 mg/mL was assessed after 6 hours at +40 °C. The results fulfilled the acceptability criteria for content and homogeneity (80 to 120 % of the theoretical concentration, with a CV < 10 %), indicating that the formulations were stable for up to 6 hours under these conditions.

CONCLUSIONS
- The validation of the method for the determination of the test material was successful, since linearity, accuracy and precision were all within the limits stated in the Study Protocol.
- Linearity was successfully assessed in the range from 4.860 to 19.44 μg/mL of the test material, since the correlation coefficient, r, was found to be 0.9978 (acceptability criterion > 0.98).
- Specificity was assessed by injecting samples of the diluent and diluent + vehicle (prepared using the lowest dilution factor applied to the formulations). No signal was seen at the retention time or mass of the test material in either sample.
- The accuracy and precision of the method were determined at 10 and 75 mg/mL by preparing formulations at these levels and analysing six independent samples from each (two from the top, two from the middle and two from the bottom). At the lower level, accuracy was found to be 102.55 %, with a precision, expressed as the percentage coefficient of variation (CV %), of 5.69 %, while at the higher level, accuracy was 102.06 %, with a precision of 6.25 %. These values were within the acceptability criteria outlined in the protocol (accuracy 80 to 120 % of the theoretical concentration, precision < 10 %). The results at each level were also used as the time-zero content of the formulations for the stability studies.
- The stability of the formulations was assessed after 6 hours at +40 °C. The results met the acceptability criteria outlined in the protocol (recovery 80 to 120 % of the theoretical value, with a homogeneity < 10 %), indicating that the formulations were stable under these conditions for up to 6 hours.

Duration of treatment / exposure:

Males: Animals were dosed once a day, 7 days a week, for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 34/35 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females: Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a total of 42 to 62 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels of 100, 300 and 750 mg/kg/day were selected by the Sponsor based on information from preliminary non GLP study
- Rationale for animal assignment: On the day of allocation all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. The rats were allocated to groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet, and ear notch and housed 5 of one sex per cage. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each groupwere distributed to minimise possible environmental effects. No replacements occurred after the first dose was administered.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Severely debilitated animals were observed carefully.

DETAILED CLINICAL OBSERVATIONS: Yes
- Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day (approximately 1-1.5 hour post-dose).
- Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). All observations were recorded for individual animals.

BODY WEIGHT: Yes
- Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7 and 13 post partum, and just prior to necropsy.

FOOD CONSUMPTION:
- The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

CLINICAL PATHOLOGY:
- Blood collection was performed for hormone determination from all animals at termination under condition of food deprivation. Blood samples for haematology, clinical chemistry and coagulation were collected by random selection from 5 males and 5 females (females with viable litters).
- Males: Blood samples for haematological and biochemical investigations and hormone determination were collected under isoflurane anaesthesia from the retro-orbital sinus. Blood samples for coagulation test (food available) were collected at necropsy from the vena cava under isoflurane anaesthesia. The order of collection was equalised between groups.
- Females: As a part of the sacrificial procedure, blood samples for all determinations were withdrawn from the abdominal vena cava under isoflurane anaesthesia. The order of collection was equalised between groups. The blood samples collected were divided into tubes as follows: EDTA anticoagulant for haematological investigations, Heparin anticoagulant for biochemical tests, Citrate anticoagulant for coagulation tests and No anticoagulant for hormone assay.

HAEMATOLOGY
– The following measurements were performed: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count: Neutrophils, Lymphocites, Eosinophils, Basophils, Monocytes and Large unstained cells, Platelets. These parameters were analysed by Siemens Advia 120.
- Coagulation: Prothrombin time. This parameter was analysed by Instrumentation Laboratory ACL Elite PRO.

CLINICAL CHEMISTRY
- The following measurements were performed: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride and Inorganic phosphorus. These parameters were analysed by Siemens Advia 1200.

THYROID HORMONE DETERMINATION
- Blood collection from pups on Days 4 and 14 post partum: Blood samples were withdrawn under light ether anaesthesia from the heart (by intracardiac puncture). On Day 4 post partum, approximately 0.2mL of blood samples were taken from at least two pups (1 sample/sex, when possible) per litter. On Day 14 post partum, approximately 0.5mL of blood samples were taken from at least two pups (1 sample/sex, when possible) per litter.
- Parental animals: As a part of the necropsy procedure, approximately 0.8mL of blood was taken from all parental males and females.
- All samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature. The serum obtained was divided into two aliquots and stored at -80 °C pending analysis.
- Bioanalysis - Thyroid hormone determination (T3, T4 and TSH): Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using LuminexMagpix system and the MILLIPLEX MAP Rat Thyroid Hormone Magnetic Bead Panel kit (MerkMillipore, cat. no. RTHYMAG-30K). The determination was restricted as detailed below: Samples from all parental males from all groups and Samples from pups on Day 14 post partum. No analysis was carried out on serum samples collected from selected pups on Day 4 post partum and from females.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Once during the study, towards the end of treatment (Day 12 post partum for females with viable litters), 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurementswere performed using a computer generated random order.
- Once during the study, towards the end of treatment (Day 12 post partum for females with viable litters), 5 males and 5 females were randomly selected from each group and the motor activity measured (for approximately 5 minutes) by an automated activity recording device.

OTHER:
Vaginal smears and oestrous cycle:
- Stock females: Oestrous cycle was monitored by vaginal smears in all females received for the study for at least 2 weeks before allocation in order to exclude from the study those with irregular cycle.
- Females allocated to groups: Vaginal smears were taken in the morning from Day 1 of dosing up to positive identification of mating. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Vaginal smears were also taken from all females, before despatch to necropsy. No vaginal smears were taken from females sacrificed for humane reasons.

Mating
- Matings were monogamous (one male to one female). Vaginal smears were taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plug found on the cage tray).
- The female was paired with the same male until positive identification of copulation occurred or 14 days had elapsed.

Parturition and gestation length
- A parturition check was performed from Day 20 to Day 25 post coitum. Females which did not give birth after 25 days of post coitum period were sacrificed shortly after. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth. The day of birth (when parturition was complete) was defined as Day 0 post partum.

Pups identification, weight and observations
- As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
- Live pups were individually weighed on Days 1, 4 and 13 post partum.
- Observations were performed once daily for all litters.
- Pups killed or dying during the lactation period were weighed before despatch to necropsy.
- After culling all pups were sacrificed with the dams on Day 14 post partum.

Culling and pups selection for blood collection (serum hormones) at necropsy
- On Day 4 post partum, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment (for example, 6 males and 2 females) was performed when necessary.
- At least two pups (males or females, selected for culling) were sacrificed to collect blood for possible determination of serum T3, T4 and TSH levels.

Anogenital distance (AGD)
- The AGD of each pup was measured on Day 1 post partum. The AGD was normalised to the cube root of body weight collected on Day 1 post partum.

Nipple count
- Male pups were observed for the presence of nipples/areolae on Day 13 post partum (no nipples were observed, therefore these data were not reported).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Euthanasia:
- Parental animals and those that had completed the scheduled test period were killed by exsanguination under isoflurane anaesthesia. Animals sacrificed for humane reasons were killed with carbon dioxide. Pups killed for humane reasons or those that had completed the scheduled test period (Day 4 or Day 14 post partum) were euthanised by intraperitoneal injection of Thiopenthal.
- Parental males: The males were killed after the mating of all females or after at least 28 days of treatment.
- Parental females: The females with live pups were killed on Day 14 post partum. The female with total litter loss was killed one day after the occurrence of total litter loss. The females showing no evidence of copulation were killed 26 days after the last day of the mating session.

Necropsy
- The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.
- Females: All females were examined also for the following: number of visible implantation sites (pregnant animals) and number of corpora lutea (pregnant animals). Uteri of females with no visible implantations were immersed in a 20 % solution of ammonium sulphide to reveal evidence of implantation.
- Pups: All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonad inspection. All live pups sacrificed at Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonad inspection. All pups with abnormalities were retained in a 10 % neutral buffered formalin.
- Nipples retention at Day 14 post partum: No nipples/areolae were observed in male pups.

ORGAN WEIGHTS
- Parental animals: From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed: Adrenal glands, Brain (cerebrum, cerebellum, medulla/pons), Duodenum, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Prostate gland (dorsolateral and ventral), Seminal vesicles with coagulating glands, Spleen, Testes, Thymus (where present), Thyroid and Uterus – cervix. The ratios of organ weight to body weight were calculated for each animal.
- Pups at Day 14 post partum: Thyroid was weighed from one male and female from each litter and preserved for possible histopathological examination. The thyroid weight was determined after fixation.

HISTOPATHOLOGY:
The tissues required for histopathological examination were: Abnormalities, Adrenal glands, Bone marrow (from sternum), Brain (cerebrum, cerebellum, medulla/pons), Caecum, Clitoral gland, Colon, Duodenum, Epididymides, Eyes, Femur with joint, Heart, Ileum, Jejunum (including Peyer’s patches), Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes – cervical, Lymph nodes – mesenteric, Mammary area – Females, Mammary area - Males, Oesophagus, Ovaries with oviducts, Parathyroid glands, Pituitary gland, Penis, Prostate gland (dorsolateral and ventral), Rectum, Sciatic nerve, Seminal vesicles with coagulating glands, Skeletal muscle, Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach, Testes, Thymus (where present), Thyroid, Trachea, Urinary bladder, Uterus – cervix and Vagina. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed.
The examination was restricted as detailed below:
i Tissues specified above from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term.
ii Tissues specified above from all animals killed or dying during the treatment period.
iii All abnormalities in all groups
The examination was then extended to include the examination of the stomach in the remaining 5 males and 5 females (animals not evaluated for clinical pathology) of the other dose groups and the heart in the remaining 5 males of the control and high dose groups, since treatment-related changes were observed in the high dose group animals.

Statistics:

Standard deviations were calculated as appropriate. For variables such as body weight, food consumption, clinical pathology parameters and organ weight, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Hunched posture and piloerection were occasionally observed in 2 males (nos. X0900074 and X0900080) dosed at 750 mg/kg/day during the pre-mating and mating phases. Swollen abdomen was also seen during the pre-mating phase in a single female of the same group (no. X0900071).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- One male and one female from the control group (nos. X0900006 and X0900007) and one male and one female from the high dose group (nos. X0900062 and X0900079), died during the study.
- The control male (no. X0900006) died due to accidental trauma on Day 21 of the mating phase of the study. No clinical signs were observed during the study. At macroscopic observations, multiple dark areas of lungs and thymus were observed, while at microscopic observations, the most relevant findings were haemorrhage of brain (cortex, thalamus and hypothalamus) and lungs (alveolar) and congestion of thymus. The cause of death of this animal is traumatic in nature.
- Female no. X0900007 (control) was sacrificed for humane reasons on Day 6 post partum. Dyspnoea, salivation and rales were seen in this animal on Days 5 and 6. Macroscopic findings observed in this animal were dark red colour of cervical lymph nodes and pale colour of lungs, whereas the major findings observed at microscopic observations were moderate chronic inflammation of trachea (submucosal) and bronchioles and alveoles in the lungs (neutrophils and few multinucleated giant cells with macrophages), associated with presence of foreign material (bronchial, bronchiolar and alveolar space). Mis-dosing could be considered as a factor contributory to the illness status of the animal sacrificed for humane reasons.
- Male no. X0900062 (high dose) was found dead on Day 16 of the mating phase. No clinical signs were oberved during the study. Macroscopically, the most relevant findings included red fluid in the thoracic cavity, dark colour and pale creamy adhesions of lungs, enlarged adrenals and dark colour of thymus. At histopathology, the most relevant findings observed were moderate acute inflammation of pericardium (heart) and pleura, associated with pigmentation of amber solid colour with oedema and congestion of lungs. The cause of death of this animal was mis-dosing.
- Female no. X0900079 (high dose) was sacrificed for humane reasons on Day 15 post coitum. Emaciated appearance, hunched posture, piloerection, dyspnoea, pallor, rales, hard and swollen abdomen were observed in this animal during gestation and post partum periods. Macroscopic findings observed in this animal included caecum, duodenum, ileum, jejunum and stomach distended with gas, swollen and firm abdominal cavity, enlarged adrenals, thin spleen and small thymus. At histopathology, the major findings observed were moderate mucosal ulceration, associated with moderate epithelial hyperplasia of the adjacent epithelium and acute submucosal inflammation of glandular region of the stomach, atrophy of thymus and spleen. Gastric tract disease and stress related effects (atrophy of thymus and spleen) could be considered the factors contributing to the illness status of this animal.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Slight reductions in body weight (-7 %), compared to controls were observed in the males dosed at 750 mg/kg/day at the end of mating phase.
- Body weight gain was also reduced in these animals during pre-mating (-70 %) and mating (-63 %) phases. These reductions were essentially due to slight body weight losses occasionally observed in individual animals.
- Due to the episodical nature of these losses/reductions and to the slight impact on the body weight group mean values, this change was not considered to be adverse.
- No changes were observed in the females. The statistically significant increases occasionally observed in the treated females during post coitum and post partum periods were considered to be incidental, due to the absence of dose-relation and the direction of the variation (they were ascribed to body weight losses observed in some control females).
- A statistically significant decrease in terminal bodyweight was observed in high dose treated males (8 %), when compared to the controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- No changes of toxicological significance were observed in food consumption during the study in either males or females.
- The slight but statistically significant increase observed in the females dosed at 750 mg/kg/day at Day 20 post coitum, was considered of no toxicological significance, due to the direction of the change (increase).

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):

- Minimal increase in erythrocytes, haemoglobin and haematocrit were recorded in males dosed at 300 and/or 750 mg/kg/day. Compared with controls, changes were 5 to 9 %, with some dose relation. Reticulocytes were decreased in only one male receiving the highest dosage (no. X0900080). In this animal, reticulocytes were decreased by 75 %. Due to the limited severity, the above changes were considered to be of no toxicological relevance.
- Animal no. X0900080 showed additional leucocytosis. Compared with controls, the increase was 2.2 fold.
- Coagulation: No changes were recorded.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Compared with controls, animal no. X0900080 (750 mg/kg/day ) showed moderate increases of alanine aminotransferase (10 fold), aspartate aminotransferase (2.4 fold), urea (1.8 fold) and creatinine (1.7 fold). Alanine aminotransferase and/or aspartate aminotransferase were also increased in two males dosed at 750 mg/kg/day (nos. X0900066 and X0900068) and in almost all males receiving 300 mg/kg/day. Compared with controls, the increments were 2.6 to 7 fold for alanine aminotransferase and 1.3 to 1.8 fold for aspartate aminotransferase, with no clear dose-relation. Two females dosed at 750 mg/kg/day (nos. X0900063 and X0900067) and two females receiving 300 mg/kg/day (nos. X0900043 and X0900057) showed increases of alanine aminotransferase (1.5 to 3.2 fold), with no relevant increase of aspartate aminotransferase.
- In addition, triglycerides were increased in one male and two females dosed at 100 mg/kg/day (nos. X900026, X900031 and X900035, 2.2, 2.9 and 3.5 fold, respectively), one female dosed at 300 mg/kg/day (no. X900043, 4.1 fold), one male and one female receiving 750 mg/kg/day (nos. X900068 and X0900069, 2.0 and 5.4 fold, respectively).
- The statistically significant differences between controls and males dosed at 300 mg/kg/day (creatinine and chloride) and females receiving 100 and 750 mg/kg/day (urea) were not dose-related, therefore they were considered to be incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test material.
- No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment. The slight reduction observed in the treated males, when compared to controls (not significant at statistical analysis due to the high individual variability) was not considered to be toxicologically significant, since it was not associated to any other toxic or neurotoxic effect.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- No relevant changes were observed in absolute and relative organ weights of treated animals, when compared to the controls. The sporadic statistically significant changes observed in some organs: i.e. increased relative male brain weight (11 %) or decreased relative female heart weight (11 %) or kidney weight (8 %) of the high dose treated group were considered toxicologically irrelevant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- No treatment-related changes were noted following gross pathology examination in the animals. The sporadic changes such as small thymus in one high dose male (no. X0900080), enlarged adrenals of one low (no. X0900036) and one high dose male (no. X0900064), small prostate in one low (no. X0900038), one mid- (no. X0900050) and two high dose males (nos. X0900070 and X0900078) or small seminal vesicle in one high dose male (no. X0900078), single or multiple dark and/or red area/s depressed in the glandular region of the stomach of control and treated males and females, were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were seen in the stomach of the high dose group of both sexes.

Stomach (non glandular)
- Hyperplasia of the non glandular epithelium was only observed in high dose treated males (5 out of 9) with minimal or moderate degree while, in females, an increased incidence of minimal to mild degree was observed in high dose treated females (2 out of 9) when compared to controls. The above mentioned gastric lesion was occasionally associated with submucosal oedema and acute inflammation. One high dose treated animal (male no. X0900080) showed moderate atrophy of thymus and spleen, together with hyperplasia of the non glandular epithelium.
- The apparently increased incidence of inflammatory reaction in the heart noted in the five randomly high dose treated males, after the extension of histopathological evaluation to the remaining control and high dose group males, was seen comparable to control animals.
- All other observed changes had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age, under our experimental conditions. In a single male rat from the high dose group, moderate atrophy of the thymus and spleen was observed. As this change was seen only in a single male (no. X0900080) of this group, it was considered as secondary to the stress induced by the gastric lesion and therefore not related to treatment. In one male and one female of the low dose group (nos. X0900032 and X0900029), hepatocytic necrosis was noted in the liver. As this change was seen only once in the low dose group for each sex and as such a change is sporadically seen in untreated animals, the present cases are considered as incidental findings.

Spermatogenic cycle
- A detailed qualitative examination of the testes was performed on 5 randomly selected animals in control and high dose groups respectively. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germcells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952. The PAS-stained sections were used to identify the spermatogenic stages.
- Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Fate of females
- All females did mate. One female from the low dose group (no. X0900025, dosed at 100 mg/kg/day) and 1 female from the high dose group (no.X0900061, dosed at 750 mg/kg/day) were found not pregnant, while 1 female from the high dose group (no. X0900079, dosed at 750 mg/kg/day) and 1 from the control group (no. X0900007) were humanely killed during post coitum and post partum phases. In addition, 1 female from the mid-dose group (no. X0900049 dosed at 300 mg/kg/day) showed total litter loss on Day 3 post partum. One female dosed at 100 mg/kg/day conceived after Day 5 of mating (Day 12, no. X0900033).
- The number of females with live pups on Day 14 post partum was: 9 in each of the control, low and mid-dose groups (0, 100 and 300 mg/kg/day), 8 in the high dose group (750 mg/kg/day).

Thyroid hormone determination
- Parental males: Thyroxine (T4) was decreased in a number of animals dosed at 750 mg/kg/day (19 % below controls). Due to the slight severity and the absence of changes of the other hormones, this finding was considered to be of no toxicological importance.
- Pups - Day 14 post partum: A statistically significant increase of triiodothyronine (T3) was recorded in some male pups of Group 4 (750 mg/kg/day). Compared with mean control data, the increment was 2.6 fold. Since high value of triiodothyronine was also recorded in one control animal (no. X0900002), and no relevant changes of the other hormones were recorded, the above finding was considered not to be indicative of hypothyroidism.

Oestrous cycle, reproductive parameters, pairing combination and mating performance
- No treatment-related anomalies were noted in the oestrous cycle and pre-coital interval of the treated females, when compared to controls.
- All females mated. However, 1 female in the low and 1 in the high dose groups (nos. X0900025 and X0900061, respectively) were found not pregnant. The number of copulation plugs was similar between control and treated groups. All conceivings were within 5 days of mating, with the exception of 1 low dose female (no. X0900033) that was found to be spermpositive on Day 12 of mating.
- Males and females: The copulatory indices were 100 % for controls and all treated groups. The fertility indices were 100 % for controls and animals dosed at 300 mg/kg/day, 90 % for animals dosed at 100 and 750 mg/kg/day.

Implantation, pre-natal loss data and gestation length of females
- Gestation periods were similar in treated groups and controls. All dams gave birth between Days 22 and 23 post coitum.
- Corpora lutea, implantations and pre-implantation loss, total litter size and pre-natal loss (percentage) did not show dose-related or treatment-related differences.

Litter data of females and sex ratio of pups
- No significant differences in total litter size, live litter size, mean pup loss, sex ratio and mean pup weights were observed among the treated dams and the controls at birth and on Days 1, 4 and 13 post partum.

Clinical signs of pups
- Pallor, apparent no food intake and small appearance were in general the clinical signs noted in control and treated pups.

Anogenital distance and nipple count
- No differences in the anogenital distance (normalised value), performed on Day 1 post partum, were seen between control and treated groups both for male and female pups.
- No nipples were observed in male pups.

Pups thyroid weights
- No significant differences were observed in the weight of thyroid of treated pups, when compared to controls.

Necropsy findings in pups
- Autolysed abdominal and/or thoracic organs or no milk in the stomach were generally observed in pups found dead at birth, in pups which died during the lactation period and in a single pup killed on Day 4 post partum. No findings were seen in the remaining pups killed on Day 4. Tip of tail missing was observed in 2 pups killed on Day 14 post partum.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Microscopic Observations in the Stomach

Diagnoses	No. of Animals Affected
	Males				Females
	Control (n=8)	100 mg/kg (n=10)	300 mg/kg (n=10)	750 mg/kg (n=9)	Control (n=9)	100 mg/kg (n=10)	300 mg/kg (n=10)	750 mg/kg (n=9)
Epithelial hyperplasia	0	0	0	5	1	0	0	2
Mucosal erosion	0	1	0	2	2	1	2	2
Acute inflammation	0	0	0	2	0	0	0	0
Mucosal haemorrhage	0	0	0	1	0	0	0	0
Oedema	0	0	0	1	0	0	0	0
Glandular dilation	0	0	0	0	2	3	1	1
Mucosal ulceration	0	0	0	0	0	0	0	0

n = number of animals examined in group

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg/day for males and females.

Executive summary:

The repeated dose toxicity of the test material was investigated in accordance with the standardised guideline OECD 422, under GLP conditions.

The toxic effects on Sprague Dawley rats after repeated dosing by oral route with the test material, as well as any effects of the test material on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and early lactation of the offspring were investigated.

The vehicle was 0.5 % aqueous solution of carboxymethylcellulose. All doses (0, 100, 300 and 750 mg/kg/day) were administered at a constant volume of 10 mL/kg body weight. Males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 34/35 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a total of 42 to 62 days.

Three cases of not treatment-related premature deaths occurred during the study (1 control male, 1 control female and 1 high dose male). In addition, 1 female dosed at 750 mg/kg/day was sacrificed for humane reasons on Day 15 post coitum in the presence of a number of clinical signs (hunched posture, emaciated appearance, piloerection, dispnoea, pallor, rales, hard and swollen abdomen). The gastric tract disease and stress related effects could be considered the factors contributory to humane sacrifice.

Some clinical signs (hunched posture, piloerection and/or swollen abdomen) were occasionally observed in individual animals dosed at 750 mg/kg/day during the pre-mating and mating phases of the study. No signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reactivity to stimuli) were observed during the study in parental males and females.

Slight bodyweight losses/reduced bodyweight gain and mean body weight were observed in the males dosed at 750 mg/kg/day. Due to the episodical nature of these losses/reductions and to the slight impact on the body weight group mean values, this change was not considered to be adverse. No changes were observed in the females. No differences in food consumption were seen in treated animals, compared to the control group.

Decreased reticulocytes and leucocytosis were observed in only one male receiving 750 mg/kg/day. No other treatment-related changes in haematological parameters were observed (including coagulation). Increments in alanine aminotransferase and/or aspartate aminotransferase were observed in comparison with controls, in individual males and/or females dosed at 300 and 750 mg/kg/day, resulting in mean group increases not clearly dose-related. Similar changes were seen in triglycerides. In the absence of a clear dose-relation, these changes were not considered to be adverse. Compared with controls, thyroxine (T4) was increased in parental males dosed at 750 mg/kg/day. Due to the slight severity and the absence of changes in the other hormones or any pathological correlated lesion, this finding was considered to be of no toxicological importance.

At post mortem, no relevant changes were observed on absolute and relative organ weights of treated animals, when compared to the controls. No treatment-related changes were noted following gross pathology examination. Treatment-related findings (epithelial hyperplasia of the non glandular stomach) were seen in the stomach of males and females dosed at 750 mg/kg/day. Moderate atrophy of thymus and spleen was also seen in one high dose treated animal together with hyperplasia of the non glandular epithelium. This change was considered to be a local irritant effect in the stomach of the test material when administered by oral gavage. Although local, this treatment-related lesion was able to affect the general health status of the animals resulting in the poor health status observed in animal no. X0900079 killed for humane reasons or in the atrophy of thymus and spleen observed in a male animal of the same group. For this reason, this change was considered to be adverse. Nevertheless, since the observed lesions were localised in the epithelium of the non glandular stomach (not present in humans), this effect may not be relevant for human health. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

All females mated. The number of females with live pups on Day 14 post partum was: 9 in each of the control, low and mid-dose groups (0, 100 and 300 mg/kg/day), 8 in the high dose group (750 mg/kg/day). No treatment-related anomalies were noted in the oestrous cycle of the treated females, when compared to controls. Copulatory and fertility indices did not show any treatment related differences among treated and control groups. Parturition, lactation, implantation, litter data and sex ratio did not show any changes of toxicological relevance. No significant differences in the anogenital distance (normalised value), were seen between control and treated groups both for male and female pups. No nipples were observed in male pups. No changes of toxicological relevance were noted in thyroid hormone levels in pups killed on Day 14 post partum. Necropsy findings and thyroid weight in pups did not reveal any treatment-related effect.

In conclusion, signs of effects related to treatment with the test material, when administered to rats by oral route at dose levels of 100, 300 and 750 mg/kg/day, were mainly observed in animals dosed at 750 mg/kg/day. Most of these effects (occasional clinical signs, slight body weight losses/reduced body weight gain, slight increments in alanine aminotransferase and/or aspartate aminotransferaseincrease and/or triglycerides), observed at 750 mg/kg/day, were not considered to be adverse, due to the low magnitude and to their occasional and individual nature. These findings were associated with epithelial hyperplasia of the non glandular stomach in male and female animals dosed at 750 mg/kg/day, which was considered to be adverse for its capacity to affect the general health status of the animals but likely irrelevant for human health. No signs of treatment-related toxicity were observed at the dose levels of 100 and 300 mg/kg/day.

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg/day for males and females.

No effects of the test material on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and early lactation of the offspring were observed at any of the dose levels investigated. Therefore, the NOAEL (No Observed Adverse Effect Level) for fertility and reproduction parameters was considered to be 750 mg/kg/day for males and females.