Tar bases, coal, lutidine fraction

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral route

28-day oral repeated dose toxicity study, rat: NOAEL = 80 mg/kg bw/day

Inhalation route

No study available

Dermal route

No study available

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Oral (Gavage) Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: 30 March 2017 (study plan issued) Experimental start date: 06 April 2017 (animal arrival) 25 April 2017 (start of dosing) Experimental termination date: 03 October 2017 (pathology)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Version / remarks:

US Environmental Protection Agency, OPPTS Health Effects Test Guideline No. 870.3550, Reproduction/Developmental Toxicity Screening Test; adopted July 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The purpose of this study was to assess the potential toxicity of the test item and to provide information concerning the effects on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of the conceptus and parturition when administered by oral gavage to the rat.

Specific details on test material used for the study:

The test item was received on 05 January 2017 and on arrival it was given the Sequani log reference number TI/2017/003. Details of the consignment received were:

Batch number WC 2015.10533
Appearance Dark brown liquid
Re-test date 31 August 2017
Quantity supplied 1 kg
Purity 99.3 %

The test item was stored at room temperature and a certificate of analysis for it is presented in the attached APPENDIX 20.

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

The rat is a suitable rodent species, acceptable to regulatory authorities and for which extensive background data are available. The Han Wistar rat is commonly used in reproduction studies because of the good fertility and fecundity of the strain. Background data on the rate of spontaneous malformations have been accumulated. All animal work was conducted under authority of a Project Licence in compliance with the Animals (Scientific Procedures) Act 1986 (as amended).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Forty male and 48 female rats of the Crl:WI(Han) strain were supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England. The males were 8 to 9 weeks of age and the females were 11 to 12 weeks of age on arrival and on examination were found to be healthy. After at least 19 days acclimatisation, they were re-examined and confirmed to be suitable to use. On the first day of dosing, the males weighed 288 to 388g and the females weighed 201 to 238 g.

Animals were allocated to groups using a stratified body weight randomisation procedure based on individual body weights recorded on arrival. The cages were positioned in the battery using a randomised cage allocation procedure, starting at the top left-hand corner of the rack and then working from left to right, top to bottom. All groups were allocated to each rack. Each animal was uniquely identified by a subcutaneously implanted micro-identification device. To aid identification, animals were tail marked with their own unique identification numbers using an indelible pen.
The animals were housed in groups of up to 3 for males and in groups of up to 4 for females, until pairing and for males post-pairing, in solid-floor cages with appropriate bedding provided. Bedding was changed as often as was necessary to maintain hygiene. For pairing, 1 male and 1 female were housed together in grid-floor cages suspended over paper-lined trays. On confirmation of mating, the males were returned to the group cages and the females were housed individually and with their litter in solid-floor cages.
The study room was illuminated by fluorescent light set to give a cycle of 12 hours light and 12 hours dark and was air-conditioned. The target ranges for temperature was 19 °C to 23 °C, values were within these ranges with the exception of two occasions where a maximum temperature of to 25 °C was recorded. The target ranges for humidity were 40 % to 70 %, actual ranges were between 23 % and 67 % (see attached APPENDIX 21 for deviation).
A pelleted rodent diet, VRF1 (manufactured by SDS) supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England, and mains tap water (in bottles) were freely available (see attached APPENDIX 21 for deviation). A sample of each batch of diet used on the study was retained frozen and was discarded following finalisation of the report.
Certificates of analysis for diet and water are held centrally and retained within the Sequani archives. It was considered that none of the contaminants that were monitored was present at a level that might have prevented the study objective from being achieved.

Route of administration:

oral: gavage

Vehicle:

other: The test item was formulated on the day of dosing for each group separately, as a solution in 0.02 M phosphate buffer, pH 7.

Details on exposure:

The test item was formulated on the day of dosing for each group separately, as a solution in 0.02 M phosphate buffer, pH 7.

A weighed quantity of test item was added to a container and the required quantity of vehicle was added to make the formulation up to final weight. Formulations were mixed by inversion until homogeneous and clear and were protected from light.

Samples were taken from each test item formulation prepared for use on the first day of dosing and towards the end of the dosing period (where both sexes were dosed) and analysed to confirm achieved concentrations using a validated method (BFI063LC). Samples were also taken on the same occasions from the vehicle used to dose Controls and were analysed to confirm absence of test item.

All remaining samples were retained and discarded once the final formulation analysis results were accepted.

Details on mating procedure:

Oestrous cycling monitoring
From 14 days before the start of dosing and until the day of pairing, vaginal smears were taken daily by lavage. The smears were examined under light microscopy and the stage of the oestrous cycle was determined.
Before the start of dosing, 10 females with the lowest identification numbers in each group (from the 12 that started in each group) that were exhibiting typical 4-5 day cycles were selected to start the study based on pre-dosing oestrous cycle evaluation. On the day of necropsy, vaginal smears were taken, and the stage of the oestrous cycle was recorded.

Pairing and detection of mating
After the pre-pairing dosing period (14 days), each female was paired with a male from the same dose group for up to 14 days. On confirmation of mating, the males were returned to the group cages and the females were housed individually.
During the pairing period, vaginal smears were taken daily, by lavage, until mating was confirmed by sperm being found in the smears. The smears were examined under light microscopy and the stage of the oestrous cycle was determined by the type of cell present. The number of copulation plugs was recorded to give an assessment of the mating activity of the animals. The day on which sperm were detected was designated Day 0 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Individual achieved concentrations of the test itemused to dose animals during the first week and towards the end of the dosing period were within 5 % of their nominal values, which fulfilled the acceptance criteria for achieved concentration (± 10 %). No test item was detected in vehicle used to dose Control animals. It is considered, therefore, that formulations were accurately prepared. see attached appendix 18

Duration of treatment / exposure:

All selected animals were dosed once daily, using a rubber catheter and disposable syringe, for at least 14 days before pairing and then until the day before necropsy. Males were dosed before, during and after pairing; females were dosed before and during pairing, during gestation and until Day 12 of lactation. Females that were mid-parturition at the time of dosing were not dosed on that day. Individual dose volumes were based on the most recently recorded body weight using a dose volume of 10 mL/kg body weight.

Frequency of treatment:

All selected animals were dosed once daily, using a rubber catheter and disposable syringe, for at least 14 days before pairing and then until the day before necropsy. Males were dosed before, during and after pairing; females were dosed before and during pairing, during gestation and until Day 12 of lactation. Females that were mid-parturition at the time of dosing were not dosed on that day. Individual dose volumes were based on the most recently recorded body weight using a dose volume of 10 mL/kg body weight.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Four groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (Vehicle), 40, 80 or 200 mg/kg/day

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Remarks:

Four groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (Vehicle), 40, 80 or 200 mg/kg/day

Dose / conc.:

80 mg/kg bw/day (actual dose received)

Remarks:

Four groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (Vehicle), 40, 80 or 200 mg/kg/day

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Four groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (Vehicle), 40, 80 or 200 mg/kg/day

No. of animals per sex per dose:

Four groups of 10 male and 10 female rats

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered orally, by gavage, as the oral route had been defined by the Sponsor as a possible route of human exposure.

Dose levels of 0, 40, 80 and 200 mg/kg/day were selected for this study based on data generated in a preliminary study (1) in which groups of 2 males and 2 females were administered using water as the vehicle at dose levels ranging from 65 to 300 mg/kg/day. A high dose of 200 mg/kg/day was selected based on the presence of clinical signs of toxicity. A dose level of 300 mg/kg/day produced excessive toxicity leading to early termination. Dose levels of 40 and 80 mg/kg/day were selected to explore possible dose response and to identify a no observed adverse effect level (NOAEL).
The number of animals to be used on this study is the minimum number considered necessary to yield meaningful scientific results.
This study was conducted in accordance with the agreed study plan, 9 study plan amendments and 8 notes to file. Six deviations from the study plan were recorded and are detailed in the attached APPENDIX 21. None of the deviations was considered to have affected the outcome or integrity of this study.
A total of 80 animals were used in this study, the design of which was:
Group Animal
identification numbers Dose level Dose concentration
Males Females (mg/kg/day) (mg/mL)

1 1 - 10 41 - 50* Control 0
2 11 - 20 53 - 62* 40 4
3 21 - 30 65 - 74* 80 8
4 31 - 40 77 - 86* 200 20
* Pre-dose oestrous cycle monitoring was performed on Females 51, 52, 63, 64, 75, 76, 87 and 88, results are recorded in the raw data. Only the first 10 females in each group were selected for dosing on this study.

Positive control:

No

Parental animals: Observations and examinations:

P Generation Clinical Observations and Measurements

Clinical observations
Animals were examined twice daily for mortality and morbidity and were given a detailed clinical examination weekly. From the start of dosing, animals were observed before and shortly after dosing. On weekdays during the dosing period, animals were also checked at 1 and 4 hours after dosing (see the attached APPENDIX 21 for deviation). On weekends during the dosing period, a final check was made at 1 hour after dosing or at the end of the working day (whichever was soonest). For males and females in the pre-pairing period and for males post-pairing, observations were based on group housed animals. For males and females during pairing and for females after pairing, observations were based on individual animals.

Body weight
Male body weights were recorded on the first day of dosing and then at weekly intervals throughout the study until necropsy.
Female body weights were recorded on the first day of dosing and then at weekly intervals until the day of mating. Females were also weighed on Days 0, 7, 14 and 20 of gestation and on Days 0 (if required for dose administration), 1, 4, 7, 10 and 13 of lactation.

Food intake
The amount of food consumed by the animals in each cage was recorded at weekly intervals for males and females during their pre-pairing dosing period. Individual food intake of the females was also recorded over Days 0 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 20 of gestation and over Days 1 to 4, 4 to 7, 7 to 10 and 10 to 13 of lactation. Two weeks after the start of the pairing period, food intake for males recommenced and was recorded weekly until necropsy.

Behavioural observations
All animals were observed once weekly, starting from the final week of the acclimatisation period, for their behaviour both within their cage and after placement in an open arena. Observations were made at approximately the same time of day on each occasion (afternoon). Observations were not conducted for any female that was mid-parturition.
Functional observation battery
Towards the end of the dosing period, sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli, grip strength and motor activity were recorded for 5 males and 5 females in each group.

Parturition observations
Females were observed from Day 21 of gestation until all females had littered or until the start of the working day when the last female was on Day 26 after mating.
Following completion of parturition, pups were designated as Day 0 of age with the next day classified as Day 1 of age. Dead offspring were not removed from the litter until parturition had been completed.

Observations of littering females
The females were allowed to rear their offspring to Day 13 of lactation. Abnormalities of nesting or nursing behaviour were recorded.

Clinical Laboratory Studies
Blood sample collection
Blood samples (1.5 mL) were taken under isoflurane anaesthesia from the sublingual vein of the 5 males and 5 females with the highest identification numbers in each group on Day 14 of dosing. Blood samples were taken into anticoagulant as follows:

0.5 mL into EDTA for haematology.
0.5 mL into 3.2 % w/v aqueous trisodium citrate for coagulation.
0.5 mL into lithium heparin for blood chemistry.

Sample analysis
The following parameters were measured.

Haematology
haemoglobin concentration (Hb) total leucocyte count (WBC)
red blood cell count (RBC) neutrophils (Neut)
packed cell volume (PCV) lymphocytes (Lymph)
mean cell volume (MCV) monocytes (Mono)
mean cell haemoglobin (MCH) eosinophils (Eosin)
mean cell haemoglobin concentration (MCHC) basophils (Baso)
red blood cell distribution width (RDW) large unstained cells (LUC)
platelet count (Plate) reticulocytes (Retics)
absolute reticulocyte counts (A retics)
All parameters were measured on the ADVIA 120 haematology analyser.

Coagulation
prothrombin time (PT) activated partial thromboplastin time (APTT)
fibrinogen (FIB)
Measured on the ACL Elite Pro.

Blood chemistry
Urea/BUN (Blood Urea Nitrogen) albumin/globulin ratio (A/G)
creatinine (Cren) total bilirubin (BiliT)
glucose (Gluc) cholesterol (Chol)
alkaline phosphatase (ALP) triglyceride (Trigs)
alanine aminotransferase (ALT) sodium (Na)
aspartate aminotransferase (AST) chloride (Cl)
gamma glutamyl transpeptidase (GGT) calcium (Ca)
total protein (T.Prot) potassium (K)
albumin (Alb) inorganic phosphate (I. Phos)
globulin (Glob)
All parameters were measured on the Roche Modular Evo (P800) clinical chemistry analyser.
All analyses except sodium, chloride and potassium were carried out at 37 °C. The analyses of sodium, chloride and potassium were carried out at 35 °C +/- 2 °C.
GGT results less than 3 U/L and total bilirubin results less than 0.15 mg/dL are reported as 3 U/L or 0.14 mg/dL, respectively.
Thyroid Hormone Assessments
Blood sampling
Blood samples were taken from all animals between 08.00 and 12.00 hours into tubes with no anticoagulant and allowed to clot for 30 minutes at room temperature. Samples were then centrifuged at 3000 g for 10 minutes at 4 ºC.

For P generation animals, the resultant serum was divided equally into 2 aliquots (Set 1 and Set 2), before being frozen (≤ -70 °C).

P generation animals
A blood sample (4 mL) was taken at necropsy (Day 13 of lactation for females – see the attached APPENDIX 21 for deviation), immediately after each animal was killed, from the vena cava before exsanguination. Analysis was conducted on samples taken from males; samples from females were retained frozen.

F1 pups - Day 4 of age
All culled pups were bled on Day 4 of age following decapitation; 1 pooled sample, of as much blood as possible, was collected per litter. Samples were retained for possible future analysis.

F1 pups - Day 13 of age
One pup/sex/litter where possible or two samples per litter (not from pups with gross external abnormalities) were sampled on Day 13 of age, following decapitation; as much blood as possible was taken. The litter of Female 83 (Group 4) contained two female pups only and therefore one pup was assigned for blood sampling and the remaining pup was examined.

Sample analysis
Only P generation male samples (Set 1) and the Day 13 pup samples were analysed; in the absence of a clear test item-related effect, all other serum samples were retained for possible future analysis. Samples were analysed for total thyroxine (T4) using a previously validated method (BMK023EL) and a Calbiotech Mouse/Rat Total T4 ELISA assay kit.
Each sample was analysed in duplicate, with the mean of the 2 results reported in the attached APPENDIX 15.
Any remaining serum samples were discarded following acceptance of the analytical results by the Study Director and the Sponsor.

Oestrous cyclicity (parental animals):

Pre-dose oestrous cycle monitoring was performed on Females 51, 52, 63, 64, 75, 76, 87 and 88, results are recorded in the raw data. Only the first 10 females in each group were selected for dosing on this study.

Sperm parameters (parental animals):

No

Litter observations:

F1a Generation
Litter size and sexes
The total litter size was recorded after completion of parturition and daily thereafter. Numbers of each sex were recorded daily from Day 1 of age. On Day 4 of age, the size of each litter was adjusted by eliminating extra pups to yield, as nearly as possible, 4 males and 4 females. Litters of fewer than 8 pups were not altered. Pups were selected on a total randomisation basis. Non-selected pups were killed using a suitable Schedule 1 method and discarded after blood sample collected.

Day 0 of age for the pups (equivalent to Day 0 of lactation for observations assigned to the dams) was defined as the day of completion of littering.

Identification
Pups were not individually identified. Identification numbers were assigned for the purposes of recording total T4 concentrations and organ weights, where the number of the parental female was used with an additional suffix (e.g. identification number 5551 to designate a pup from the litter of Female 55).
Clinical observations
Animals were examined twice daily for mortality and morbidity and were examined daily for clinical signs of toxicity or changes in behaviour and appearance.

Body weights
Pups were weighed individually on Days 1, 4, 7, 10 and 13 of age.

Ano-genital distance
The ano-genital distance of the F1 pups was measured on Day 1 of age.

Numbers of nipples/areolae
For each male pup, the number of nipples/areolae were counted on Day 12 of age.

Postmortem examinations (parental animals):

Post Mortem Investigations
P generation
Necropsy
Females with litters were killed and subjected to necropsy on Day 13 of lactation.
Females that were not selected to start the study following the pre-dosing oestrous cycle monitoring were killed and discarded without necropsy the day after the first dose administration.
Males were killed and subjected to necropsy after the females had littered.
All animals were killed by exposure to carbon dioxide gas in a rising concentration. Dead body weight was recorded and the cranial, thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs and uterus were examined. The number of implantation scars/sites for each female killed on Day 13 of lactation was recorded. Organs or tissues showing any macroscopic abnormalities were recorded and retained.
Organ weights
The following organs were weighed after trimming of fat and other contiguous tissue. Paired organs were weighed together:
adrenal glands prostate
brain seminal vesicles (incl. coagulating gland)
epididymides spleen
heart testes
kidneys thymus
liver thyroids (incl. parathyroids)(1)
ovaries uterus (incl. uterine cervix and oviducts)
(1) weighed after fixation

Macroscopic and microscopic pathology
For all animals, either whole organs or samples of the tissues listed below, with the exception of the testes, were preserved in neutral buffered formaldehyde. The testes were fixed in modified Davidson’s solution.
adrenal glands pituitary
aorta (thoracic) prostate & seminal vesicles (incl. coagulating gland)
bone marrow smear
brain (3 levels examined) rectum
caecum salivary gland (submandibular - 1 only)
colon sciatic nerve
duodenum skeletal muscle
epididymides spinal cord (cervical examined LS and TS)
eyes (including optic nerves) spleen
femur & joint (including marrow) sternum with bone marrow
heart stomach (glandular and non-glandular)
ileum submandibular lymph nodes
jejunum (including Peyer’s patch) testes
kidneys (one LS and one TS) thymus
liver thyroids (including parathyroids)
lungs (including mainstem bronchi) trachea
mammary gland (with inguinal skin) urinary bladder
mesenteric lymph nodes uterus (including uterine cervix and oviducts)
oesophagus vagina
ovaries all gross lesions
pancreas
For all animals, the tissues specified were wax embedded, cut at a nominal thickness of 4 µm to 5 µm and stained with haematoxylin and eosin. For all Control and high dose animals (including premature decedents), the tissues were examined microscopically. Due to potential test item-related findings, the liver from the low and intermediate group males were examined.

An internal peer review was performed in accordance with current standard operating procedures. An external peer review was also conducted by the Sponsor’s pathologist, Jayne Wright.

Postmortem examinations (offspring):

F1a necropsy
Pups culled Day 4 of age
Culled pups were killed by decapitation and discarded without necropsy following blood sampling.
Pups - Day 13 of age
Pups were killed by an intraperitoneal injection of sodium pentobarbitone solution or by decapitation for those subjected to blood sampling.
Any pups killed or found dead during lactation and all pups killed on Day 13 of lactation, were examined externally for gross abnormalities only, with particular attention paid to the external reproductive genitals. In cases where gross abnormalities were identified, the pups were retained in neutral buffered formaldehyde.

On Day 13 of age, the thyroid from 1 male and 1 female pup/litter where possible (from pups that had not been sampled for thyroid hormone assessments or killed by decapitation) were preserved in neutral buffered formaldehyde and weighed (after fixation). A dead body weight was recorded for these pups (see attached APPENDIX 21 for deviation). Due to the age and size of pups, and to prevent damage, the thyroid was left in situ on the larynx and retained in neutral buffered formaldehyde. Following fixation of the carcass, the thyroid was removed and weighed. All remaining pups, including those sampled for thyroid hormone assessment, were discarded without further examination.

Statistics:

Due to lack of character space, please see any other information section

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Excessive salivation was seen in the groups given test item; the incidence of the observations increased with increasing dose level (this information is retained in the raw data) and on a single occasion, associated ploughing was seen for 2 females given 200 mg/kg/day.

On an isolated occasion, piloerection was seen for 3 females at 200 mg/kg/day and 1 female at 40 mg/kg/day. One male given 200 mg/kg/day also had partially closed eyes on one occasion.

For males, there were no deaths or clinical observations.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

There were 3 deaths during the study; one female given 200 mg/kg/day was killed due to poor clinical condition and one animal was killed in each of the groups given 80 or 200 mg/kg/day due to total litter loss as detailed below:

Female 84 given 200 mg/kg/day was killed on Day 7 of gestation (Day 23 of dosing) due to a deterioration in clinical condition including: decreased activity, a cold body surface, unsteady gait, slow breathing, piloerection and hair staining. Food intake was low over Days 4 to 7 of gestation; however, there was no effect on body weight before the animal was killed. Macroscopic findings included distension of the stomach with red areas in the non-glandular region, abnormal contents and brown fluid in the duodenum and oesophagus and incomplete collapse of the lungs. Microscopically, a marked ulceration of the non-glandular stomach with slight peritonitis, mural haemorrhage in the duodenum and luminal debris and plant material in the oesophagus were seen. The kidneys showed moderate tubular necrosis with focal mineralisation in the cortex and medulla and there was minimal centrilobular hepatocyte necrosis in the liver. The marked stomach ulceration would account for the poor clinical condition of the animal. The relationship to treatment of these findings in a single female in this dose group is unclear and can not be discounted as treatement-related.

Female 66 given 80 mg/kg/day and Female 85 given 200 mg/kg/day were killed on Day 1 and 0 of lactation respectively, due to total litter loss. There were no adverse effects on body weight gain or food intake of either dam before loss of the litter and no macroscopic and/or microscopic findings.

P Generation Litters
Mortality and clinical observations
(See attached APPENDIX 1)

There was an increased incidence of pup mortality at a maternal dose level of 200 mg/kg/day; with total litter loss for Female 85. Furthermore, at this dose level there was an increased incidence in the number of pups with little or no milk in the stomach (6 pups across 3 litters).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(See attached TABLE 1, TABLE 2, FIGURE 1, APPENDIX 3 and APPENDIX 4)

During the pre-pairing and lactation periods, female body weight gain was slightly higher than Controls at 80 and 200 mg/kg/day; however, during gestation, mean body weight gain was lower (p≤0.01) than Controls for females given 200 mg/kg/day.

There was no effect on body weight gain for females given 40 mg/kg/day or for males at any dose level.

P Generation Litters
Body weights
(See attached TABLE 1 and APPENDIX 3)

On Day 1 of age, combined male and female pups weight was less than Controls (p<0.05) at 200 mg/kg/day. Thereafter, combined male and female body weight gain was lower than Controls at 200 (p≤ 0.05 to p≤ 0.01) and 80 mg/kg/day resulting in a reduction in overall pup weight gain at both dose levels (p≤0.01 at 200 mg/kg/day).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

(see attached TABLE 3 and APPENDIX 5)

During lactation, mean food intake for females given 200 mg/kg/day was lower than Controls (p≤0.05 to p≤0.01).

Female mean food intake was similar to Controls during the pre-pairing and gestation periods at all dose levels and at 40 and 80 mg/kg/day during lactation.

Male mean food intake was similar to Controls throughout the study.

Food efficiency:

not specified

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):

Haematology
(See attached TABLE 6 and APPENDIX 7)

There were no test item-related effects on the haematological parameters.

The activated partial thromboplastin time was shorter than Controls for females given 200 mg/kg/day; however, as the change was in the wrong direction for diagnostic significance this finding is considered not to be related to administration of the test item.

Blood chemistry
(See attached TABLE 7 and APPENDIX 8)

There was an increase in mean alanine aminotransferase level for females administered 200 mg/kg/day (p ≤0.01 compared with Controls), with all individual values above background ranges (see attached APPENDIX 19), most notably that for Female 85. This female also had elevated levels of aspartate transaminase, but all other individual concentrations were within background range. Males administered 200 mg/kg/day also showed a statistically significant increased in alanine aminotransferase (p ≤0.05); however values were within the background range and this slight change was considered not to be related to treatment.

There was a slight, but statistically lower mean chloride ion concentration observed in males administered 200 mg/kg/day compared with Controls (p ≤0.01). This difference was considered not to be treatment-related.

All other blood chemistry parameters were similar across all groups for both males and females.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis
(See attached TABLE 15 and APPENDIX 15)

There were no effects on the T4 levels of the paternal males.

P Generation Litters
Thyroid hormone analysis
(See attached TABLE 15 and APPENDIX 15)

There were no effects of the test item on T4 levels of male and female pups killed on Day 13 of age.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Behavioural and functional observations battery
(See attached TABLE 4)

On Days 10, 17, 24 and 31, incidences of excessive salivation were seen in males and/or females at 80 or 200 mg/kg/day with a maximum score of 2 (marked). This correlated with observations of excessive salivation seen throughout the study.

At 200 mg/kg/day an unusual posture (flattened) was seen for a single female on Day 17 and abnormal gait (slight) was seen on Days 10, 17 and 45 in 1 to 3 females on each occasion. Two females at 80 mg/kg/day also had abnormal gait (slight) on Day 45.


Motor activity
(See attached TABLE 5 and APPENDIX 6 )

There was no test item-related effect on motor activity. The values that attained statistical significance are considered to be due to large inter-individual variation and were not consistant across the sexes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Other effects:

no effects observed

Description (incidence and severity):

P Generation Litters
Ano-genital distance
(See attached TABLE 12 and APPENDIX 12)

There was no effect on the ano-genital distance of the pups.

P Generation Litters
Nipple counts
(See attached TABLE 13 and APPENDIX 13)

There was no effect on the nipple count of male pups.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycles
(See attached TABLE 8 and APPENDIX 9)

There was no test item-related effect on the number of oestrous cycles or on the mean cycle length during the pre-pairing period.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Fertility and mating performance
(See attached TABLE 9, TABLE 10 and APPENDIX 10)

There was no test item-related effect on the time course of mating: all animals mated within 4 days.

1, 1, 2 and 3 females in groups given 0, 40, 80, 200 mg/kg/day test item respectively, were not pregnant. This resulted in a fertility index of 70 % at 200 mg/kg/day compared with 90 % for the Controls.

Gestation and parturition
(See attached TABLE 11)

There was no effect of the test item on the mean duration of gestation or on parturition. For Female 85, given 200 mg/kg/day, there were no live pups; however, as signs of parturition were identified in the cage, the animal was considered to have given birth. It was not possible to determine if the pups were born live.

Pregnancy and litter data
(See attached TABLE 11 and APPENDIX 11)

Females given 200 mg/kg/day had an increased incidence of post-implantation loss (26.6 %) compared with Controls (9.5 %), resulting in a reduction in the mean number of pups born. In addition, females given 200 mg/kg/day had an increased incidence of pup deaths between Days 0 and 4 of lactation compared with Controls and Female 85 at this dose level also had total litter loss on Day 0 of lactation. There was no effect on pup survival thereafter; however, the cumulative survival index was reduced at 200 mg/kg/day (64.31 %) compared with Controls (82.86 %).

At 200 mg/kg/day, the percentage of male pups born was statistically significantly lower
(p ≤0.01) than Controls (26.53 % vs 63.93 %). However, the apparent effect on pup sex ratio is considered to be a result of the reduced group size (3 females that were not pregnant and 1 female with total litter loss) and effects on pup survival at 200 mg/kg/day, rather than an effect of the test item.

At 40 and 80 mg/kg/day, there was no effect on the incidence of post-implantation loss, mean number of pups born alive or on the postnatal survival of the pups to Day 13 of age. There was no effect on the mean pup sex ratio.

(See attached APPENDIX 1)

There was an increased incidence of pup mortality at a maternal dose level of 200 mg/kg/day; with total litter loss for Female 85. Furthermore, at this dose level there was an increased incidence in the number of pups with little or no milk in the stomach (6 pups across 3 litters).

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

body weight and weight gain

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

reproductive performance

Key result

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No adverse effects were reported.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was an increased incidence of pup mortality at a dose of 200 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Combined body weights of female and male pups was statistically significantly lower on Day 1 of age at 200 mg/kg/bw compared to controls. Thereafter, body weights in female and male pups individually and combined and combined female and male body weight gain was statistically significantly lower than in the control group at 200 mg/kg/day.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There was no effect on absolute or adjusted thyroid weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were not test item related macroscopic findings. At 200 mg/kg/day, one pup had an irregularly shaped head; as the pup was found dead the abnormality may have been due to autolysis.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no effects on anogenital distance, nipple count of male pups or on absolute and adjusted thyroid weights. No effect on T4 thyroid hormone levels of male and female pups killed on Day 13 of age was found.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

The no observed adverse effect level (NOAEL) was 200 mg/kg/day for males and 80 mg/kg/day for females. The NOAEL for reproductive performance and foetal/developmental toxicity was considered to be 80 mg/kg/day.

Executive summary:

The reproductive toxicity of the substance in the rat was studied under GLP in a combined 28-day repeated oral dose toxicity/reproductive and developmental toxicity in accordance with OECD TG 422. Four groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (Vehicle), 40, 80 or 200 mg/kg/day test item. Males were dosed for 14 days before and during pairing and until the day before necropsy and the females were dosed for 14 days before pairing, during pairing and gestation and until Day 12 of lactation.

Before the start of the dosing period, the oestrous cycles of 12 females per group were monitored and the first 10 in each group, which were cycling normally, were selected to be dosed on the study. Oestrous cycle monitoring continued through the pre-pairing phase of the study. All selected animals were examined for effects on general condition, body weight and food intake. Functional observations were monitored throughout the study and on Day 14 of dosing (before pairing), blood samples were taken for assessment of clinical pathology parameters. During the pairing period, vaginal smears were taken daily until sperm were detected. The females were allowed to litter and rear their offspring to Day 13 of age. All parental males and females, all culled pups on Day 4 of age and 1 pup/sex/litter on Day 13 of age, where possible, had blood samples taken for thyroid hormone analysis; only those from the parental males and Day 13 of age pups were analysed.

A necropsy was performed on all parental animals and a selection of organs were weighed, fixed and examined microscopically. All Day 13 of age pups had a gross external examination and the thyroids were removed and weighed from 1 male and 1 female per litter, where possible.

There were 3 female deaths during the study. One female given 200 mg/kg/day, was killed on Day 7 of gestation due to a deterioration in clinical condition. Two females receiving 40 mg/kg/day and 200 mg/kg/day were killed on Days 1 and 0 of lactation, respectively, due to total litter loss. There was an increased incidence of excessive salivation in groups given test item, with associated ploughing seen on sporadic occasions at 200 mg/kg/day. An increased incidence of excessive salivation was also seen during the functional observation assessments, with sporadic incidences of unusual posture and/or abnormal gait. There were no effects on motor activity for either sex. 

During pre-pairing and lactation, female body weight gains were slightly higher than Controls at 80 and 200 mg/kg/day; however, during gestation, mean body weight gain was lower than Controls at 200 mg/kg/day. There were no effects on body weights for males or for females given 40 mg/kg/day. Females given 200 mg/kg/day also had reduced food intake during lactation, compared with Controls. Food intake during all other periods and for all other animals was similar in all groups.

There were no test item-related effects on the haematological parameters. At 200 mg/kg/day alanine aminotransferase activity was increased in females.

There was no test item-related effect on oestrous cycles, the time course of mating or on the length of gestation or parturition. 1, 1, 2 and 3 females in groups given 0, 40, 80, 200 mg/kg/day test item, respectively, were not pregnant and at 200 mg/kg/day there were increases in the incidence of post-implantation loss and the number of pup deaths between Days 0 and 4 of lactation. At 40 and 80 mg/kg/day, both fertility and pregnancy parameters were similar to Controls.

There was an increase in liver weights for males and females given the test item. In males, there was an increase in kidney weight and in females a reduction in spleen weight in all groups given the test item compared with Control.

There were no effects on T4 levels for the parental males or the F1a males and females at Day 13 of age.

Centrilobular hepatocyte hypertrophy (minimal) was found in most males given 200 mg/kg/day but not in males given 40 or 80 mg/kg/day or in females at any dose level. 

There was an increased incidence of pup mortality at a maternal dose level of 200 mg/kg/day and a reduction in overall mean pup body weight gain. The ano-genital distance, nipple count and thyroid organ weights of the F1a animals were unaffected by maternal administration of the test item.

In conclusion the oral administration of 200 mg/kg/day test item to the female rat, resulted in the death of one female and an increase in the number of non-pregnant females. Furthermore, there was an increased incidence of post-implantation loss resulting in a reduction in the number of pups born and an increased incidence of pup deaths between Days 0 and 4 of lactation. Dose levels of 40 and 80 mg/kg/day were better tolerated resulting in non-adverse changes that included liver and spleen weight changes at both dose levels and increases in dam body weight gain and slightly lower pup weight gain at 80 mg/kg/day only. 

The test item was well tolerated by the male rat, resulting in non-adverse increases in kidney and liver weight at all dose levels and non-adverse microscopic findings in the liver (minimal hypertrophy, hepatocyte, centrilobular) at 200 mg/kg/day only. 

The no observed adverse effect level (NOAEL) was 200 mg/kg/day for males and 80 mg/kg/day for females. The NOAEL for reproductive performance and foetal/developmental toxicity was considered to be 80 mg/kg/day.

  

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

80 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The NOAEL was established in a GLP study conducted to the OECD TG 422, and the generated information on the toxicity on fertility via the oral route is considered as relevant, adequate and reliable.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Oral route

28-day oral repeated dose toxicity study, rat: NOAEL = 80 mg/kg bw/day

Inhalation route

No study available

Dermal route

No study available

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

Oral (Gavage) Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: 30 March 2017 (study plan issued) Experimental start date: 06 April 2017 (animal arrival) 25 April 2017 (start of dosing) Experimental termination date: 03 October 2017 (pathology)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals, No. 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test; adopted 29th July 2016.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US Environmental Protection Agency, OPPTS Health Effects Test Guideline No. 870.3550, Reproduction/Developmental Toxicity Screening Test; adopted July 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

The test item was received on 05 January 2017 and on arrival it was given the Sequani log reference number TI/2017/003. Details of the consignment received were:

Batch number WC 2015.10533
Appearance Dark brown liquid
Re-test date 31 August 2017
Quantity supplied 1 kg
Purity 99.3 %

The test item was stored at room temperature and a certificate of analysis for it is presented in the attached APPENDIX 20.

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

The rat is a suitable rodent species, acceptable to regulatory authorities and for which extensive background data are available. The Han Wistar rat is commonly used in reproduction studies because of the good fertility and fecundity of the strain. Background data on the rate of spontaneous malformations have been accumulated. All animal work was conducted under authority of a Project Licence in compliance with the Animals (Scientific Procedures) Act 1986 (as amended).

Route of administration:

oral: gavage

Vehicle:

other: The test item was formulated on the day of dosing for each group separately, as a solution in 0.02 M phosphate buffer, pH 7.

Details on exposure:

The test item was formulated on the day of dosing for each group separately, as a solution in 0.02 M phosphate buffer, pH 7.

A weighed quantity of test item was added to a container and the required quantity of vehicle was added to make the formulation up to final weight. Formulations were mixed by inversion until homogeneous and clear and were protected from light.

Samples were taken from each test item formulation prepared for use on the first day of dosing and towards the end of the dosing period (where both sexes were dosed) and analysed to confirm achieved concentrations using a validated method (BFI063LC). Samples were also taken on the same occasions from the vehicle used to dose Controls and were analysed to confirm absence of test item.

All remaining samples were retained and discarded once the final formulation analysis results were accepted.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Individual achieved concentrations of the test itemused to dose animals during the first week and towards the end of the dosing period were within 5 % of their nominal values, which fulfilled the acceptance criteria for achieved concentration (± 10 %). No test item was detected in vehicle used to dose Control animals. It is considered, therefore, that formulations were accurately prepared. see attached appendix 18

Details on mating procedure:

Oestrous cycling monitoring
From 14 days before the start of dosing and until the day of pairing, vaginal smears were taken daily by lavage. The smears were examined under light microscopy and the stage of the oestrous cycle was determined.
Before the start of dosing, 10 females with the lowest identification numbers in each group (from the 12 that started in each group) that were exhibiting typical 4-5 day cycles were selected to start the study based on pre-dosing oestrous cycle evaluation. On the day of necropsy, vaginal smears were taken, and the stage of the oestrous cycle was recorded.

Pairing and detection of mating
After the pre-pairing dosing period (14 days), each female was paired with a male from the same dose group for up to 14 days. On confirmation of mating, the males were returned to the group cages and the females were housed individually.
During the pairing period, vaginal smears were taken daily, by lavage, until mating was confirmed by sperm being found in the smears. The smears were examined under light microscopy and the stage of the oestrous cycle was determined by the type of cell present. The number of copulation plugs was recorded to give an assessment of the mating activity of the animals. The day on which sperm were detected was designated Day 0 of gestation.

Duration of treatment / exposure:

All selected animals were dosed once daily, using a rubber catheter and disposable syringe, for at least 14 days before pairing and then until the day before necropsy. Males were dosed before, during and after pairing; females were dosed before and during pairing, during gestation and until Day 12 of lactation. Females that were mid-parturition at the time of dosing were not dosed on that day. Individual dose volumes were based on the most recently recorded body weight using a dose volume of 10 mL/kg body weight.

Frequency of treatment:

All selected animals were dosed once daily, using a rubber catheter and disposable syringe, for at least 14 days before pairing and then until the day before necropsy. Males were dosed before, during and after pairing; females were dosed before and during pairing, during gestation and until Day 12 of lactation. Females that were mid-parturition at the time of dosing were not dosed on that day. Individual dose volumes were based on the most recently recorded body weight using a dose volume of 10 mL/kg body weight.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Four groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (Vehicle), 40, 80 or 200 mg/kg/day

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Remarks:

Four groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (Vehicle), 40, 80 or 200 mg/kg/day

Dose / conc.:

80 mg/kg bw/day (actual dose received)

Remarks:

Four groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (Vehicle), 40, 80 or 200 mg/kg/day

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Four groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (Vehicle), 40, 80 or 200 mg/kg/day

No. of animals per sex per dose:

Four groups of 10 male and 10 female rats

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered orally, by gavage, as the oral route had been defined by the Sponsor as a possible route of human exposure.

Dose levels of 0, 40, 80 and 200 mg/kg/day were selected for this study based on data generated in a preliminary study (1) in which groups of 2 males and 2 females were administered using water as the vehicle at dose levels ranging from 65 to 300 mg/kg/day. A high dose of 200 mg/kg/day was selected based on the presence of clinical signs of toxicity. A dose level of 300 mg/kg/day produced excessive toxicity leading to early termination. Dose levels of 40 and 80 mg/kg/day were selected to explore possible dose response and to identify a no observed adverse effect level (NOAEL).
The number of animals to be used on this study is the minimum number considered necessary to yield meaningful scientific results.
This study was conducted in accordance with the agreed study plan, 9 study plan amendments and 8 notes to file. Six deviations from the study plan were recorded and are detailed in the attached APPENDIX 21. None of the deviations was considered to have affected the outcome or integrity of this study.
A total of 80 animals were used in this study, the design of which was:
Group Animal
identification numbers Dose level Dose concentration
Males Females (mg/kg/day) (mg/mL)

1 1 - 10 41 - 50* Control 0
2 11 - 20 53 - 62* 40 4
3 21 - 30 65 - 74* 80 8
4 31 - 40 77 - 86* 200 20
* Pre-dose oestrous cycle monitoring was performed on Females 51, 52, 63, 64, 75, 76, 87 and 88, results are recorded in the raw data. Only the first 10 females in each group were selected for dosing on this study.

Maternal examinations:

P Generation Clinical Observations and Measurements

Clinical observations
Animals were examined twice daily for mortality and morbidity and were given a detailed clinical examination weekly. From the start of dosing, animals were observed before and shortly after dosing. On weekdays during the dosing period, animals were also checked at 1 and 4 hours after dosing (see the attached APPENDIX 21 for deviation). On weekends during the dosing period, a final check was made at 1 hour after dosing or at the end of the working day (whichever was soonest). For males and females in the pre-pairing period and for males post-pairing, observations were based on group housed animals. For males and females during pairing and for females after pairing, observations were based on individual animals.

Body weight
Male body weights were recorded on the first day of dosing and then at weekly intervals throughout the study until necropsy.
Female body weights were recorded on the first day of dosing and then at weekly intervals until the day of mating. Females were also weighed on Days 0, 7, 14 and 20 of gestation and on Days 0 (if required for dose administration), 1, 4, 7, 10 and 13 of lactation.

Food intake
The amount of food consumed by the animals in each cage was recorded at weekly intervals for males and females during their pre-pairing dosing period. Individual food intake of the females was also recorded over Days 0 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 20 of gestation and over Days 1 to 4, 4 to 7, 7 to 10 and 10 to 13 of lactation. Two weeks after the start of the pairing period, food intake for males recommenced and was recorded weekly until necropsy.

Behavioural observations
All animals were observed once weekly, starting from the final week of the acclimatisation period, for their behaviour both within their cage and after placement in an open arena. Observations were made at approximately the same time of day on each occasion (afternoon). Observations were not conducted for any female that was mid-parturition.
Functional observation battery
Towards the end of the dosing period, sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli, grip strength and motor activity were recorded for 5 males and 5 females in each group.

Parturition observations
Females were observed from Day 21 of gestation until all females had littered or until the start of the working day when the last female was on Day 26 after mating.
Following completion of parturition, pups were designated as Day 0 of age with the next day classified as Day 1 of age. Dead offspring were not removed from the litter until parturition had been completed.

Observations of littering females
The females were allowed to rear their offspring to Day 13 of lactation. Abnormalities of nesting or nursing behaviour were recorded.

Clinical Laboratory Studies
Blood sample collection
Blood samples (1.5 mL) were taken under isoflurane anaesthesia from the sublingual vein of the 5 males and 5 females with the highest identification numbers in each group on Day 14 of dosing. Blood samples were taken into anticoagulant as follows:

0.5 mL into EDTA for haematology.
0.5 mL into 3.2 % w/v aqueous trisodium citrate for coagulation.
0.5 mL into lithium heparin for blood chemistry.

Sample analysis
The following parameters were measured.

Haematology
haemoglobin concentration (Hb) total leucocyte count (WBC)
red blood cell count (RBC) neutrophils (Neut)
packed cell volume (PCV) lymphocytes (Lymph)
mean cell volume (MCV) monocytes (Mono)
mean cell haemoglobin (MCH) eosinophils (Eosin)
mean cell haemoglobin concentration (MCHC) basophils (Baso)
red blood cell distribution width (RDW) large unstained cells (LUC)
platelet count (Plate) reticulocytes (Retics)
absolute reticulocyte counts (A retics)
All parameters were measured on the ADVIA 120 haematology analyser.

Coagulation
prothrombin time (PT) activated partial thromboplastin time (APTT)
fibrinogen (FIB)
Measured on the ACL Elite Pro.

Blood chemistry
Urea/BUN (Blood Urea Nitrogen) albumin/globulin ratio (A/G)
creatinine (Cren) total bilirubin (BiliT)
glucose (Gluc) cholesterol (Chol)
alkaline phosphatase (ALP) triglyceride (Trigs)
alanine aminotransferase (ALT) sodium (Na)
aspartate aminotransferase (AST) chloride (Cl)
gamma glutamyl transpeptidase (GGT) calcium (Ca)
total protein (T.Prot) potassium (K)
albumin (Alb) inorganic phosphate (I. Phos)
globulin (Glob)
All parameters were measured on the Roche Modular Evo (P800) clinical chemistry analyser.
All analyses except sodium, chloride and potassium were carried out at 37 °C. The analyses of sodium, chloride and potassium were carried out at 35 °C +/- 2 °C.
GGT results less than 3 U/L and total bilirubin results less than 0.15 mg/dL are reported as 3 U/L or 0.14 mg/dL, respectively.
Thyroid Hormone Assessments
Blood sampling
Blood samples were taken from all animals between 08.00 and 12.00 hours into tubes with no anticoagulant and allowed to clot for 30 minutes at room temperature. Samples were then centrifuged at 3000 g for 10 minutes at 4 ºC.

For P generation animals, the resultant serum was divided equally into 2 aliquots (Set 1 and Set 2), before being frozen (≤ -70 °C).

P generation animals
A blood sample (4 mL) was taken at necropsy (Day 13 of lactation for females – see the attached APPENDIX 21 for deviation), immediately after each animal was killed, from the vena cava before exsanguination. Analysis was conducted on samples taken from males; samples from females were retained frozen.

F1 pups - Day 4 of age
All culled pups were bled on Day 4 of age following decapitation; 1 pooled sample, of as much blood as possible, was collected per litter. Samples were retained for possible future analysis.

F1 pups - Day 13 of age
One pup/sex/litter where possible or two samples per litter (not from pups with gross external abnormalities) were sampled on Day 13 of age, following decapitation; as much blood as possible was taken. The litter of Female 83 (Group 4) contained two female pups only and therefore one pup was assigned for blood sampling and the remaining pup was examined.

Sample analysis
Only P generation male samples (Set 1) and the Day 13 pup samples were analysed; in the absence of a clear test item-related effect, all other serum samples were retained for possible future analysis. Samples were analysed for total thyroxine (T4) using a previously validated method (BMK023EL) and a Calbiotech Mouse/Rat Total T4 ELISA assay kit.
Each sample was analysed in duplicate, with the mean of the 2 results reported in the attached APPENDIX 15.
Any remaining serum samples were discarded following acceptance of the analytical results by the Study Director and the Sponsor.

Ovaries and uterine content:

Macroscopic and microscopic pathology & Organ weights

Fetal examinations:

F1a Generation
Litter size and sexes
The total litter size was recorded after completion of parturition and daily thereafter. Numbers of each sex were recorded daily from Day 1 of age. On Day 4 of age, the size of each litter was adjusted by eliminating extra pups to yield, as nearly as possible, 4 males and 4 females. Litters of fewer than 8 pups were not altered. Pups were selected on a total randomisation basis. Non-selected pups were killed using a suitable Schedule 1 method and discarded after blood sample collected.

Day 0 of age for the pups (equivalent to Day 0 of lactation for observations assigned to the dams) was defined as the day of completion of littering.

Identification
Pups were not individually identified. Identification numbers were assigned for the purposes of recording total T4 concentrations and organ weights, where the number of the parental female was used with an additional suffix (e.g. identification number 5551 to designate a pup from the litter of Female 55).
Clinical observations
Animals were examined twice daily for mortality and morbidity and were examined daily for clinical signs of toxicity or changes in behaviour and appearance.

Body weights
Pups were weighed individually on Days 1, 4, 7, 10 and 13 of age.

Ano-genital distance
The ano-genital distance of the F1 pups was measured on Day 1 of age.

Numbers of nipples/areolae
For each male pup, the number of nipples/areolae were counted on Day 12 of age.

Statistics:

Due to lack of character space, please see any other information section

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Excessive salivation was seen in the groups given test item; the incidence of the observations increased with increasing dose level (this information is retained in the raw data) and on a single occasion, associated ploughing was seen for 2 females given 200 mg/kg/day.

On an isolated occasion, piloerection was seen for 3 females at 200 mg/kg/day and 1 female at 40 mg/kg/day. One male given 200 mg/kg/day also had partially closed eyes on one occasion.

For males, there were no deaths or clinical observations.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were 3 deaths during the study; one female given 200 mg/kg/day was killed due to poor clinical condition and one animal was killed in each of the groups given 80 or 200 mg/kg/day due to total litter loss as detailed below:

Female 84 given 200 mg/kg/day was killed on Day 7 of gestation (Day 23 of dosing) due to a deterioration in clinical condition including: decreased activity, a cold body surface, unsteady gait, slow breathing, piloerection and hair staining. Food intake was low over Days 4 to 7 of gestation; however, there was no effect on body weight before the animal was killed. Macroscopic findings included distension of the stomach with red areas in the non-glandular region, abnormal contents and brown fluid in the duodenum and oesophagus and incomplete collapse of the lungs. Microscopically, a marked ulceration of the non-glandular stomach with slight peritonitis, mural haemorrhage in the duodenum and luminal debris and plant material in the oesophagus were seen. The kidneys showed moderate tubular necrosis with focal mineralisation in the cortex and medulla and there was minimal centrilobular hepatocyte necrosis in the liver. The marked stomach ulceration would account for the poor clinical condition of the animal. The relationship to treatment of these findings in a single female in this dose group is unclear and can not be discounted as treatement-related.

Female 66 given 80 mg/kg/day and Female 85 given 200 mg/kg/day were killed on Day 1 and 0 of lactation respectively, due to total litter loss. There were no adverse effects on body weight gain or food intake of either dam before loss of the litter and no macroscopic and/or microscopic findings.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(See attached TABLE 1, TABLE 2, FIGURE 1, APPENDIX 3 and APPENDIX 4)

During the pre-pairing and lactation periods, female body weight gain was slightly higher than Controls at 80 and 200 mg/kg/day; however, during gestation, mean body weight gain was lower (p≤0.01) than Controls for females given 200 mg/kg/day.

There was no effect on body weight gain for females given 40 mg/kg/day or for males at any dose level.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

(see attached TABLE 3 and APPENDIX 5)

During lactation, mean food intake for females given 200 mg/kg/day was lower than Controls (p≤0.05 to p≤0.01).

Female mean food intake was similar to Controls during the pre-pairing and gestation periods at all dose levels and at 40 and 80 mg/kg/day during lactation.

Male mean food intake was similar to Controls throughout the study.

Food efficiency:

not specified

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):

Haematology
(See attached TABLE 6 and APPENDIX 7)

There were no test item-related effects on the haematological parameters.

The activated partial thromboplastin time was shorter than Controls for females given 200 mg/kg/day; however, as the change was in the wrong direction for diagnostic significance this finding is considered not to be related to administration of the test item.

Blood chemistry
(See attached TABLE 7 and APPENDIX 8)

There was an increase in mean alanine aminotransferase level for females administered 200 mg/kg/day (p ≤0.01 compared with Controls), with all individual values above background ranges (see attached APPENDIX 19), most notably that for Female 85. This female also had elevated levels of aspartate transaminase, but all other individual concentrations were within background range. Males administered 200 mg/kg/day also showed a statistically significant increased in alanine aminotransferase (p ≤0.05); however values were within the background range and this slight change was considered not to be related to treatment.

There was a slight, but statistically lower mean chloride ion concentration observed in males administered 200 mg/kg/day compared with Controls (p ≤0.01). This difference was considered not to be treatment-related.

All other blood chemistry parameters were similar across all groups for both males and females.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis
(See attached TABLE 15 and APPENDIX 15)

There were no effects on the T4 levels of the paternal males.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Behavioural and functional observations battery
(see attached TABLE 4)

On Days 10, 17, 24 and 31, incidences of excessive salivation were seen in males and/or females at 80 or 200 mg/kg/day with a maximum score of 2 (marked). This correlated with observations of excessive salivation seen throughout the study.

At 200 mg/kg/day an unusual posture (flattened) was seen for a single female on Day 17 and abnormal gait (slight) was seen on Days 10, 17 and 45 in 1 to 3 females on each occasion. Two females at 80 mg/kg/day also had abnormal gait (slight) on Day 45.

Motor activity
(see attached TABLE 5 and APPENDIX 6 )

There was no test item-related effect on motor activity. The values that attained statistical significance are considered to be due to large inter-individual variation and were not consistant across the sexes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weights
(see attached TABLE 14 and APPENDIX 14)

In males and females, group mean liver weight adjusted for body weight was higher than Controls with statistical significance achieved at 80 and 200 mg/kg/day (p ≤0.05 to p ≤0.01). A microscopic correlate (hepatocellular hypertrophy) was seen in males only at 200 mg/kg/day.

In males, group mean kidney weight adjusted for body weight was significantly higher than Controls in all groups given the test item (p ≤0.01). In females, group mean absolute spleen weight (p ≤0.05 to p≤0.01) and spleen weight adjusted for body weight (p<0.05 at 80 and 200 mg/kg/day) were lower than Controls at all dose levels in a dose-related manner. There were no microscopic correlates for these findings.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic pathology
(See attached APPENDIX 17)

There were no test-item related findings at necropsy.

Microscopic pathology
(See attached APPENDIX 17)

Centrilobular hepatocyte hypertrophy (minimal) was detected in 9 of 10 males given 200 mg/kg/day but not in males given 40 or 80 mg/kg/day or in females at any dose level.

The incidence and severity of the test item-related change is shown in the following table.

Males
Group 1 2 3 4
Dose Level (mg/kg/day) 0 40 80 200
Number of rats examined 10 10 10 10
Liver
Hypertrophy, hepatocyte, centrilobular Minimal 0 0 0 9

Centrilobular hepatocyte hypertrophy correlated with the increased liver weights in males from this dose group when compared with the Controls.

Neuropathological findings:

not examined

Details on results:

Mortality and clinical observations
(See attached TABLE 1, TABLE 2, TABLE 3, APPENDIX 1, APPENDIX 2, APPENDIX 3,
APPENDIX 4, APPENDIX 5 and APPENDIX 17).

There were 3 deaths during the study; one female given 200 mg/kg/day was killed due to poor clinical condition and one animal was killed in each of the groups given 80 or 200 mg/kg/day due to total litter loss as detailed below:

Female 84 given 200 mg/kg/day was killed on Day 7 of gestation (Day 23 of dosing) due to a deterioration in clinical condition including: decreased activity, a cold body surface, unsteady gait, slow breathing, piloerection and hair staining. Food intake was low over Days 4 to 7 of gestation; however, there was no effect on body weight before the animal was killed. Macroscopic findings included distension of the stomach with red areas in the non-glandular region, abnormal contents and brown fluid in the duodenum and oesophagus and incomplete collapse of the lungs. Microscopically, a marked ulceration of the non-glandular stomach with slight peritonitis, mural haemorrhage in the duodenum and luminal debris and plant material in the oesophagus were seen. The kidneys showed moderate tubular necrosis with focal mineralisation in the cortex and medulla and there was minimal centrilobular hepatocyte necrosis in the liver. The marked stomach ulceration would account for the poor clinical condition of the animal. The relationship to treatment of these findings in a single female in this dose group is unclear and can not be discounted as treatement-related.

Female 66 given 80 mg/kg/day and Female 85 given 200 mg/kg/day were killed on Day 1 and 0 of lactation respectively, due to total litter loss. There were no adverse effects on body weight gain or food intake of either dam before loss of the litter and no macroscopic and/or microscopic findings.

On an isolated occasion, piloerection was seen for 3 females at 200 mg/kg/day and 1 female at 40 mg/kg/day. One male given 200 mg/kg/day also had partially closed eyes on one occasion.

Excessive salivation was seen in the groups given test item; the incidence of the observations increased with increasing dose level (this information is retained in the raw data) and on a single occasion, associated ploughing was seen for 2 females given 200 mg/kg/day.

For males, there were no deaths or clinical observations.

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Pregnancy and litter data
(See attached TABLE 11 and APPENDIX 11)

Females given 200 mg/kg/day had an increased incidence of post-implantation loss (26.6 %) compared with Controls (9.5 %), resulting in a reduction in the mean number of pups born. In addition, females given 200 mg/kg/day had an increased incidence of pup deaths between Days 0 and 4 of lactation compared with Controls and Female 85 at this dose level also had total litter loss on Day 0 of lactation. There was no effect on pup survival thereafter; however, the cumulative survival index was reduced at 200 mg/kg/day (64.31 %) compared with Controls (82.86 %).

At 200 mg/kg/day, the percentage of male pups born was statistically significantly lower
(p ≤0.01) than Controls (26.53 % vs 63.93 %). However, the apparent effect on pup sex ratio is considered to be a result of the reduced group size (3 females that were not pregnant and 1 female with total litter loss) and effects on pup survival at 200 mg/kg/day, rather than an effect of the test item.

At 40 and 80 mg/kg/day, there was no effect on the incidence of post-implantation loss, mean number of pups born alive or on the postnatal survival of the pups to Day 13 of age. There was no effect on the mean pup sex ratio.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Gestation and parturition
(See attached TABLE 11)

There was no effect of the test item on the mean duration of gestation or on parturition. For Female 85, given 200 mg/kg/day, there were no live pups; however, as signs of parturition were identified in the cage, the animal was considered to have given birth. It was not possible to determine if the pups were born live.

At 200 mg/kg/day, the percentage of male pups born was statistically significantly lower
(p ≤0.01) than Controls (26.53 % vs 63.93 %). However, the apparent effect on pup sex ratio is considered to be a result of the reduced group size (3 females that were not pregnant and 1 female with total litter loss) and effects on pup survival at 200 mg/kg/day, rather than an effect of the test item.

At 40 and 80 mg/kg/day, there was no effect on the incidence of post-implantation loss, mean number of pups born alive or on the postnatal survival of the pups to Day 13 of age. There was no effect on the mean pup sex ratio.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Gestation and parturition
(See attached TABLE 11)

There was no effect of the test item on the mean duration of gestation or on parturition. For Female 85, given 200 mg/kg/day, there were no live pups; however, as signs of parturition were identified in the cage, the animal was considered to have given birth. It was not possible to determine if the pups were born live.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation and parturition
(See attached TABLE 11)

There was no effect of the test item on the mean duration of gestation or on parturition. For Female 85, given 200 mg/kg/day, there were no live pups; however, as signs of parturition were identified in the cage, the animal was considered to have given birth. It was not possible to determine if the pups were born live.

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

Pregnancy and litter data
(See attached TABLE 11 and APPENDIX 11)

Females given 200 mg/kg/day had an increased incidence of post-implantation loss (26.6 %) compared with Controls (9.5 %), resulting in a reduction in the mean number of pups born.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Oestrous cycles
(See attached TABLE 8 and APPENDIX 9)

There was no test item-related effect on the number of oestrous cycles or on the mean cycle length during the pre-pairing period.

Fertility and mating performance
(See attached TABLE 9, TABLE 10 and APPENDIX 10)

There was no test item-related effect on the time course of mating: all animals mated within 4 days.
Pregnancy and litter data
(See attached TABLE 11 and APPENDIX 11)

Females given 200 mg/kg/day had an increased incidence of post-implantation loss (26.6 %) compared with Controls (9.5 %), resulting in a reduction in the mean number of pups born. In addition, females given 200 mg/kg/day had an increased incidence of pup deaths between Days 0 and 4 of lactation compared with Controls and Female 85 at this dose level also had total litter loss on Day 0 of lactation. There was no effect on pup survival thereafter; however, the cumulative survival index was reduced at 200 mg/kg/day (64.31 %) compared with Controls (82.86 %).

At 200 mg/kg/day, the percentage of male pups born was statistically significantly lower
(p ≤0.01) than Controls (26.53 % vs 63.93 %). However, the apparent effect on pup sex ratio is considered to be a result of the reduced group size (3 females that were not pregnant and 1 female with total litter loss) and effects on pup survival at 200 mg/kg/day, rather than an effect of the test item.

At 40 and 80 mg/kg/day, there was no effect on the incidence of post-implantation loss, mean number of pups born alive or on the postnatal survival of the pups to Day 13 of age. There was no effect on the mean pup sex ratio.

Details on maternal toxic effects:

Mortality and clinical observations
(See attached TABLE 1, TABLE 2, TABLE 3, APPENDIX 1, APPENDIX 2, APPENDIX 3,
APPENDIX 4, APPENDIX 5 and APPENDIX 17).

There were 3 deaths during the study; one female given 200 mg/kg/day was killed due to poor clinical condition and one animal was killed in each of the groups given 80 or 200 mg/kg/day due to total litter loss as detailed below:

Female 84 given 200 mg/kg/day was killed on Day 7 of gestation (Day 23 of dosing) due to a deterioration in clinical condition including: decreased activity, a cold body surface, unsteady gait, slow breathing, piloerection and hair staining. Food intake was low over Days 4 to 7 of gestation; however, there was no effect on body weight before the animal was killed. Macroscopic findings included distension of the stomach with red areas in the non-glandular region, abnormal contents and brown fluid in the duodenum and oesophagus and incomplete collapse of the lungs. Microscopically, a marked ulceration of the non-glandular stomach with slight peritonitis, mural haemorrhage in the duodenum and luminal debris and plant material in the oesophagus were seen. The kidneys showed moderate tubular necrosis with focal mineralisation in the cortex and medulla and there was minimal centrilobular hepatocyte necrosis in the liver. The marked stomach ulceration would account for the poor clinical condition of the animal. The relationship to treatment of these findings in a single female in this dose group is unclear and can not be discounted as treatement-related.

Female 66 given 80 mg/kg/day and Female 85 given 200 mg/kg/day were killed on Day 1 and 0 of lactation respectively, due to total litter loss. There were no adverse effects on body weight gain or food intake of either dam before loss of the litter and no macroscopic and/or microscopic findings.

On an isolated occasion, piloerection was seen for 3 females at 200 mg/kg/day and 1 female at 40 mg/kg/day. One male given 200 mg/kg/day also had partially closed eyes on one occasion.

Excessive salivation was seen in the groups given test item; the incidence of the observations increased with increasing dose level (this information is retained in the raw data) and on a single occasion, associated ploughing was seen for 2 females given 200 mg/kg/day.

For males, there were no deaths or clinical observations.

Key result

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

pre and post implantation loss

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

P Generation Litters
Body weights
(See attached TABLE 1 and APPENDIX 3)

On Day 1 of age, combined male and female pups weight was less than Controls (p<0.05) at 200 mg/kg/day. Thereafter, combined male and female body weight gain was lower than Controls at 200 (p≤ 0.05 to p≤ 0.01) and 80 mg/kg/day resulting in a reduction in overall pup weight gain at both dose levels (p≤0.01 at 200 mg/kg/day).

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

There was an increased incidence of pup mortality at a maternal dose level of 200 mg/kg/day; with total litter loss for Female 85. Furthermore, at this dose level there was an increased incidence in the number of pups with little or no milk in the stomach (6 pups across 3 litters).

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

At 200 mg/kg/day, the percentage of male pups born was statistically significantly lower
(p ≤0.01) than Controls (26.53 % vs 63.93 %). However, the apparent effect on pup sex ratio is considered to be a result of the reduced group size (3 females that were not pregnant and 1 female with total litter loss) and effects on pup survival at 200 mg/kg/day, rather than an effect of the test item.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

(See attached TABLE 11 and APPENDIX 11)

Females given 200 mg/kg/day had an increased incidence of post-implantation loss (26.6 %) compared with Controls (9.5 %), resulting in a reduction in the mean number of pups born. In addition, females given 200 mg/kg/day had an increased incidence of pup deaths between Days 0 and 4 of lactation compared with Controls and Female 85 at this dose level also had total litter loss on Day 0 of lactation. There was no effect on pup survival thereafter; however, the cumulative survival index was reduced at 200 mg/kg/day (64.31 %) compared with Controls (82.86 %).

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There was no effect on pup survival, however, the cumulative survival index was reduced at 200 mg/kg/day (64.31 %) compared with Controls (82.86 %).

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

P Generation Litters
Macroscopic findings (external gross abnormalities)

There were no test item related macroscopic findings. At 200 mg/kg/day, one pup had an irregularly shaped head; as the pup was found dead the abnormality may have been due to autolysis.

Skeletal malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

P Generation Litters
Ano-genital distance
(See attached TABLE 12 and APPENDIX 12)

There was no effect on the ano-genital distance of the pups.

P Generation Litters
Nipple counts
(See attached TABLE 13 and APPENDIX 13)

There was no effect on the nipple count of male pups.

P Generation Litters
Organ weights
(TABLE 14 and APPENDIX 14)

There was no effect of the test item on absolute or adjusted thyroid weights.

P Generation Litters
Thyroid hormone analysis
(See attached TABLE 15 and APPENDIX 15)

There were no effects of the test item on T4 levels of male and female pups killed on Day 13 of age.

Key result

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: cranium

Description (incidence and severity):

At 200 mg/kg/day, one pup had an irregularly shaped head; as the pup was found dead the abnormality may have been due to autolysis.

Key result

Developmental effects observed:

no

Conclusions:

The NOAEL for reproductive performance and foetal toxicity was considered to be 80 mg/kg/day. No malformations were detected at gross necropsy in any dose group.

Executive summary:

The developmental toxicity of the substance in the rat was studied under GLP in a combined 28-day repeated oral dose toxicity/reproductive and developmental toxicity in accordance with OECD TG 422. Four groups of 10 male and 10 female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (Vehicle), 40, 80 or 200 mg/kg/day test item. Males were dosed for 14 days before and during pairing and until the day before necropsy and the females were dosed for 14 days before pairing, during pairing and gestation and until Day 12 of lactation.

Before the start of the dosing period, the oestrous cycles of 12 females per group were monitored and the first 10 in each group, which were cycling normally, were selected to be dosed on the study. Oestrous cycle monitoring continued through the pre-pairing phase of the study. All selected animals were examined for effects on general condition, body weight and food intake. Functional observations were monitored throughout the study and on Day 14 of dosing (before pairing), blood samples were taken for assessment of clinical pathology parameters. During the pairing period, vaginal smears were taken daily until sperm were detected. The females were allowed to litter and rear their offspring to Day 13 of age. All parental males and females, all culled pups on Day 4 of age and 1 pup/sex/litter on Day 13 of age, where possible, had blood samples taken for thyroid hormone analysis; only those from the parental males and Day 13 of age pups were analysed.

A necropsy was performed on all parental animals and a selection of organs were weighed, fixed and examined microscopically. All Day 13 of age pups had a gross external examination and the thyroids were removed and weighed from 1 male and 1 female per litter, where possible.

There were 3 female deaths during the study. Female 84 given 200 mg/kg/day, was killed on Day 7 of gestation due to a deterioration in clinical condition. Females 66 (40 mg/kg/day) and 85 (200 mg/kg/day) were killed on Days 1 and 0 of lactation, respectively, due to total litter loss. There was an increased incidence of excessive salivation in groups given test item, with associated ploughing seen on sporadic occasions at 200 mg/kg/day. An increased incidence of excessive salivation was also seen during the functional observation assessments, with sporadic incidences of unusual posture and/or abnormal gait. There were no effects on motor activity for either sex.

During pre-pairing and lactation, female body weight gains were slightly higher than Controls at 80 and 200 mg/kg/day; however, during gestation, mean body weight gain was lower than Controls at 200 mg/kg/day. There were no effects on body weights for males or for females given 40 mg/kg/day. Females given 200 mg/kg/day also had reduced food intake during lactation, compared with Controls. Food intake during all other periods and for all other animals was similar in all groups.

There were no test item-related effects on the haematological parameters. At 200 mg/kg/day alanine aminotransferase activity was increased in females.

There was no test item-related effect on oestrous cycles, the time course of mating or on the length of gestation or parturition. 1, 1, 2 and 3 females in groups given 0, 40, 80, 200 mg/kg/day test item, respectively, were not pregnant and at 200 mg/kg/day there were increases in the incidence of post-implantation loss and the number of pup deaths between Days 0 and 4 of lactation. At 40 and 80 mg/kg/day, both fertility and pregnancy parameters were similar to Controls.

There was an increase in liver weights for males and females given the test item. In males, there was an increase in kidney weight and in females a reduction in spleen weight in all groups given the test item compared with Control.

There were no effects on T4 levels for the parental males or the F1a males and females at Day 13 of age.

Centrilobular hepatocyte hypertrophy (minimal) was found in most males given 200 mg/kg/day but not in males given 40 or 80 mg/kg/day or in females at any dose level. 

There was an increased incidence of pup mortality at a maternal dose level of 200 mg/kg/day and a reduction in overall mean pup body weight gain. The ano-genital distance, nipple count and thyroid organ weights of the F1a animals were unaffected by maternal administration of the test item.

In conclusion the oral administration of 200 mg/kg/day test item to the female rat, resulted in the death of one female and an increase in the number of non-pregnant females. Furthermore, there was an increased incidence of post-implantation loss resulting in a reduction in the number of pups born and an increased incidence of pup deaths between Days 0 and 4 of lactation. Dose levels of 40 and 80 mg/kg/day were better tolerated resulting in non-adverse changes that included liver and spleen weight changes at both dose levels and increases in dam body weight gain and slightly lower pup weight gain at 80 mg/kg/day only.

The test item was well tolerated by the male rat, resulting in non-adverse increases in kidney and liver weight at all dose levels and non-adverse microscopic findings in the liver (minimal hypertrophy, hepatocyte, centrilobular) at 200 mg/kg/day only.

The no observed adverse effect level (NOAEL) was 80 mg/kg/day for females. The NOAEL for foetal/developmental toxicity was considered to be 80 mg/kg/day.

  

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

80 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The NOAEL was established in a GLP study conducted to the OECD TG 422, and the generated information on the repeated dose oral toxicity is considered as relevant, adequate and reliable.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Description of key information

No study available

Justification for classification or non-classification

The oral administration of the substance to the rat in a combined 28-day oral repeated dose toxicity/reproductive and developmental toxicity screening study in accordance with OECD TG 422 resulted in maternal toxicity at the highest dose level of 200 mg/kg bw/day, including the death of one female and an increase in the number of non-pregnant females. Furthermore, at this dose level there was an increased incidence of post-implantation loss resulting in a reduction in the number of pups born and an increased incidence of pup deaths between Days 0 and 4 of lactation. No adverse effects on oestrous cycle, mating behaviour, fertility, gestation and parturition were observed. A classification for reproductive or developmental toxicity on the basis of the effects detected in the OECD TG 422 study is not warranted.