Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 24 August 2016 and 25 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Identification: X-19574- for Amines, C16-18 and C18-unsatd. alkyl, O,O-di-Bu phosphorothioates (CASRN 97808-07-6)
Physical State/Appearance: Amber colored viscous liquid
Purity: 100%
Batch Number: X-19574-00-00
Label: X-19574 X 19574 00 00
Date Received: 31 March 2016
Storage Conditions: Room temperature in the dark
Expiry Date: 01 June 2017
No correction for purity was made.

Test animals

Species:
rat
Strain:
other: Wistar Han™:RccHan™:WIST
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not to be showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. A total of one hundred and sixteen animals (fifty eight males and fifty eight females) were accepted into the study. At the start of treatment the males weighed 291 to 346 g and were approximately eleven weeks old. The females weighed 191 to 237 g and were approximately fourteen weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of three (non-recovery) or five (recovery) in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, non-recovery animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 6. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during late gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least thirteen days. Formulations were therefore prepared weekly and stored at approximately 4 ºC in the dark.
Samples of test item formulation were taken on four occasions and analyzed for concentration of X-19574- for Amines, C16-18 and C18-unsatd. alkyl, O,O-di-Bu phosphorothioates (CASRN 97808-07-6) at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.


The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity was confirmed for test item in Arachis Oil BP formulations at nominal concentrations of 1.25 mg/ml and 25 mg/ml. The stability was confirmed for test item in Arachis Oil BP formulations at nominal concentrations of 2.5 mg/ml and 18.75 mg/mL when stored refrigerated for 13 days.
The mean concentrations of test item in test formulations analysed for the study were within ± 10% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
Approximately six weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females).
Two recovery groups were treated with the high dose level (75 mg/kg bw/day) or the vehicle alone (Arachis oil BP) for forty-three days and then maintained without treatment for a further fourteen days.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 males and 12 females per group, except the recovery groups which consisted of 5 males and 5 females.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were chosen in collaboration with the Sponsor and based on the results of previous toxicity work including a 14 Day range-finding toxicity study in the rat (Envigo Study Number: VR76VD). During this range-finder study, treatment at 1000 and 500 mg/kg bw/day was associated with clinical signs of toxicity, an effect on body weight performance and food consumption and macroscopic findings in either sex. Treatment at 250 mg/kg bw/day was also associated with an effect on body weight performance and food consumption and treatment at 100 mg/kg bw/day was associated with a slight effect on body weight gain and food consumption.

Chronological Sequence of Study
Non-Recovery Dose Groups
i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.
ii. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). During the pre-pairing period, vaginal smears were performed for females. The first day of dosing was designated as Day 1 of the study.
iii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iv. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
v. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
vi. On completion of the pairing phase five selected males per dose group were evaluated for functional/sensory responses to various stimuli during Week 6.
vii. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance and visible nipple counts (male offspring) and clinical signs were also recorded during this period.
viii. At Day 12 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
ix. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 43. The male dose groups were killed and examined macroscopically on Day 44 or 45.
x. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 13 post partum. At Day 13 post partum, all surviving offspring were killed and examined macroscopically. All females were killed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for all females in the morning of the day of necropsy. Any female which did not produce a pregnancy was also killed and examined macroscopically around the same time as littering females.
xi. Where possible, blood samples were taken from two offspring on Day 4 post partum and one male and one female offspring on Day 13 post partum. In addition, blood samples were taken from all adult males and females at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Recovery Dose Groups
i. Groups of five male and five female rats were dosed according to dose group continuously up to Day 43 at which time treatment was discontinued.
ii. The males and females were maintained without treatment for a further fourteen days.
iii. Blood samples were taken for hematological and blood chemical assessment on Day 57.
iv. After fourteen days of recovery, males and females were killed and examined macroscopically.
v. In addition, blood samples were taken from all animals at termination for possible thyroid hormone analysis.

Examinations

Observations and examinations performed and frequency:
Serial Observations
General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, up to thirty minutes after dosing, and one hour after dosing (except for females during parturition where applicable). During the treatment-free period, recovery animals were observed daily. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for non-recovery males until termination and weekly for non-recovery females until pairing. During pairing phase non-recovery females were weighed daily until mating was confirmed. Body weights were then recorded for non-recovery females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal kill. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of non-recovery adults. This was continued for males after the mating phase. For non-recovery females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1, 4, 7 and 14 post partum. Weekly food consumptions were performed for each cage of recovery group animals throughout the study period.
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for non-recovery males and recovery animals throughout the study period (with the exception of the mating phase for non-recovery males) and for non-recovery females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Estrous Cycle Assessment
Vaginal smears were taken daily for non-recovery females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at approximately weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected non-recovery males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each non-recovery animal using a purpose built arena. The following parameters were observed:
Gait
Hyper/Hypothermia
Tremors
Skin color
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behavior
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Non-recovery animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).
Forelimb/Hindlimb Grip Strength. An automated meter was used. Each non-recovery animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each non-recovery animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
The following parameters were observed:
Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

Reproductive Performance
Mating
Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4, 7 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group prior to termination (Day 43 for males and Day 13 post partum for females). In addition hematological and blood chemical investigations were performed on all recovery group animals after the fourteen day treatment-free period (Day 57). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Inorganic phosphorus (P)
Glucose
Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.)
Alanine aminotransferase (ALAT)
Albumin
Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Creatinine (Creat)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)

Thyroid Hormone Analysis
Where possible, blood samples were taken, allowed to clot, centrifuged and the serum from each blood sample was stored frozen at lower than -60 °C for possible evaluation of thyroid hormones from the following:
Two offspring from each litter at Day 4 post partum.
One male and one female offspring from each litter at Day 13 post partum.
All adult males (non-recovery and recovery) at termination.
All adult females (non-recovery and recovery) at termination.
All samples were dispatched to the Test Site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) where the serum from adult non-recovery males and Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigators (T Borsos/A Peard).
Sacrifice and pathology:
Terminal Investigations
Necropsy
Adult non-recovery males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult non-recovery females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 14 post partum. Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Any females which failed to achieve pregnancy were killed around the same time as littering females.
For all non-recovery females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
Recovery group animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 58.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. For offspring used for blood sampling, a more limited gross necropsy, including internal confirmation of the sex of the offspring, was performed following blood sampling.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each non-recovery dose group and from all recovery group animals. Tissues indicated with the symbol ~ were weighed from all remaining non-recovery animals:
Adrenals
Liver
Brain
~Ovaries
~Cowpers Glands
~Pituitary (post-fixation)
~Epididymides
~Prostate and Seminal Vesicles (with Coagulating Gland)
~Glans Penis
Spleen
Heart
~Testes
Kidneys
Thymus
~LABC (levator ani-bulbocavernous)
~Thyroid (weighed post-fixation with Parathyroid)
~Muscle
~Uterus (weighed with Cervix and Oviducts)
On Day 13 of age, where possible, for one male and one female offspring per litter, the whole or samples of thyroid/parathyroid were retained in 10% Buffered Formalin.

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each non-recovery dose group and from all recovery group animals and preserved in buffered 10% formalin, except where stated. Tissues marked with ~ were preserved from all remaining non-recovery animals:
Adrenals
~Mammary gland
Aorta (thoracic)
Muscle (skeletal)
Bone & bone marrow (femur including stifle joint)
~Ovaries
Bone & bone marrow (sternum)
Pancreas
Brain (including cerebrum, cerebellum and pons)
~Pituitary
Caecum
~Prostate
~Coagulating gland
Rectum
Colon
Salivary glands (submaxillary)
~Cowpers glands ●
Sciatic nerve
Duodenum
~Seminal vesicles
~Epididymides ♦
Skin
Esophagus
Spinal cord (cervical, mid thoracic and lumbar)
Eyes *
~Glans penis ●
Spleen
~Gross lesions
Stomach
Heart
~Testes ♦
Ileum (including peyer’s patches)
~Thyroid/Parathyroid
Jejunum
Trachea
Kidneys
Thymus
~LABC (levator ani-bulbocavernous) muscle ●
Urinary bladder
Liver
~Uterus & Cervix
Lungs (with bronchi)#
~Vagina
Lymph nodes (mandibular and mesenteric)

● Retained only
* Eyes fixed in Davidson’s fluid
♦ Preserved in Modified Davidsons fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

Tissues were dispatched to the Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: J Schofield). The tissues from five selected non-recovery control and 75 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining non-recovery control and 75 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all non-recovery control and 75 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Since there were indications of treatment-related mesenteric lymph nodes and stomach changes, examination was subsequently extended to include similarly prepared sections of the mesenteric lymph nodes and stomach from five animals per sex in the non-recovery low and intermediate dose groups and in all animals from the recovery dose groups.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 75 mg/kg bw/day showed instances of increased salivation from Day 5 to Day 44 (males) and from Day 7 to Day 50 (females). Incidences of noisy respiration was also evident in six males and two females from this treatment group between Days 7 and 44 (males) and Days 9 and 38 (females).
Nine males treated with 25 mg/kg bw/day showed episodes of increased salivation from Day 9 to Day 44 and four males from this treatment group also showed noisy respiration from Day 9 to Day 44.
No such effects were evident in females treated with 25 mg/kg bw/day or in animals of either sex treated with 10 mg/kg bw/day.
One female treated with 75 mg/kg bw/day had a stained snout on Day 31 and a stained mouth on Day 49. One female treated with 10 mg/kg bw/day had increased salivation on Day 19 and fur loss between Days 28 and 36. In isolation, these findings were considered to be of no toxicological significance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no unscheduled deaths that were related to treatment.
One male treated with 75 mg/kg bw/day was killed in extremis on Day 23 due to a physical injury to the foot.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 75 mg/kg bw/day showed a reduction in body weight gain throughout the treatment period. Statistical significance (p<0.05-0.01) was achieved during Weeks 1, 2 and 6 and overall gain was 58% lower than controls. Recovery was evident during the fourteen day treatment free period, with body weight gains for males previously given 75 mg/kg bw/day exceeding control animals during this period (statistical significance was achieved during the first week of the recovery period). A slight reduction (11%) in overall body weight gain was also evident in males treated with 25 mg/kg bw/day although no statistically significant differences in body weight gain were evident in these males throughout the treatment period.
No adverse effects were detected in females treated with 75 or 25 mg/kg bw/day or in animals of either sex treated with 10 mg/kg bw/day.
Females treated with 75 mg/kg bw/day that were designated to the recovery dose group showed a statistically significant increase (p<0.05) in body weight gain during Week 4 and then a statistically significant reduction (p<0.01) in body weight gain during Week 6. These females also showed a reduction in body weight gain during the second week of the recovery period. Overall, body weight gain during the study for these females was comparable to controls and therefore the intergroup differences were considered not to be of toxicological importance.
Females treated with 25 mg/kg bw/day showed a statistically significant reduction (p<0.01) in body weight gain during the second week of maturation. In the absence of a similar effect detected in females treated with 75 mg/kg bw/day during this week, the intergroup difference was considered to be of no toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males treated with 75 mg/kg bw/day showed a reduction in food consumption and food conversion efficiency throughout the treatment period. Recovery was evident during the fourteen day treatment free period.
No such effects were evident in males treated with 25 or 10 mg/kg bw/day. No adverse effects were evident in treated females during maturation, gestation or lactation.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Minor fluctuations in food conversion efficiency were evident in treated females however these were considered to reflect the slight changes in body weight gain seen in these females and were considered not to be of toxicological significance.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not indicate any intergroup differences in water intake for animals of either sex given the test item in relation to controls.
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematology parameters examined.
Males treated with 75 mg/kg bw/day showed statistically significant reductions in mean corpuscular hemoglobin (p<0.05), mean corpuscular hemoglobin concentration (p<0.01) and prothrombin time (p<0.05) and a statistically significant increase in neutrophil count (p<0.05). The effect on mean corpuscular hemoglobin concentration and prothrombin time also extended to males treated with 25 mg/kg bw/day and the effect on mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration was also evident in recovery males that were previously given 75 mg/kg bw/day. Males from all treatment groups and recovery males that were previously given 75 mg/kg bw/day showed a statistically significant increase in reticulocyte count (p<0.01). Females from all treatment groups showed a statistically significant increase in erythrocyte count (p<0.05) and a statistically significant reduction in mean corpuscular hemoglobin concentration (p<0.05-0.01). Females treated with 75 and 25 mg/kg bw/day also showed statistically significant increases in hematocrit (p<0.05) and reticulocyte count (p<0.05-0.01) and a statistically significant reduction in mean corpuscular hemoglobin (p<0.05-0.01). The majority of the individual values were within background control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Following fourteen days without treatment, males that were previously given 75 mg/kg bw/day showed a statistically significant increase (p<0.05) in prothrombin time and statistically significant reductions (p<0.05) in hemoglobin, mean corpuscular volume, total leucocyte count and lymphocyte count whilst females showed statistically significant reductions (p<0.05) in hemoglobin, hematocrit and lymphocyte count and a statistically significant increase (p<0.05) in neutrophil count. The majority of the individual values were within background control ranges and in the absence of any associated histopathological correlates or any similar effects in animals at the end of the treatment period, the intergroup differences were considered not to be of toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined.
Males treated with 75 mg/kg bw/day showed statistically significant reductions in urea (p<0.05), cholesterol (p<0.01) and alkaline phosphatase (p<0.01). The effect on alkaline phosphatase also extended to males treated with 25 mg/kg bw/day (p<0.05). All of the individual values were within background control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Following fourteen days without treatment, males that were previously given 75 mg/kg bw/day showed statistically significant reductions (p<0.05) in phosphorus and bilirubin whilst females showed statistically significant reductions (p<0.05) in total protein and alanine aminotransferase. The majority of the individual values were within background control ranges and in the absence of any associated histopathological correlates or any similar effects in animals at the end of the treatment period, the intergroup differences were considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
No toxicologically significant effects in behaviour were detected.
One female treated with 75 mg/kg bw/day had increased salivation during Day 4 of gestation behavioral observations and one male treated with 25 mg/kg bw/day showed noisy respiration during Week 6 behavioral assessments. These types of observations were also evident during the clinical observations performed following dosing.
No such effects were evident in the remaining treated animals of either sex.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the organ weights measured.
Males treated with 75 mg/kg bw/day showed a statistically significant reduction (p<0.05) in absolute kidney weight and a statistically significant increase (p<0.05) in relative kidney weight. Males treated with 25 mg/kg bw/day showed a statistically significant increase (p<0.05) in absolute and relative kidney weights. With the exception of one absolute value at 25 mg/kg bw/day, all remaining individual values were within historical control ranges and in the absence of a true dose related response or any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.
Males treated with 75 mg/kg bw/day also showed statistically significant reductions (p<0.05) in levator ani-bulbocavernous muscle, thyroid/parathyroid, prostate and seminal vesicle weights: both absolute and relative to terminal body weight. The effect on seminal vesicle weights also extended to males treated with 25 mg/kg bw/day. The majority of the individual values for prostate and seminal vesicle weights were within historical control ranges and no associated histopathological correlates were evident in these tissues. There was no effect on fertility in treated animals nor were there any effects in the testes of treated males. Therefore, the intergroup differences were considered not to be of toxicological significance.
Following fourteen days without treatment, males that were previously given 75 mg/kg bw/day showed a statistically significant reduction (p<0.01) in glans penis weight; both absolute and relative to terminal body weight and females showed statistically significant increases in kidney, thyroid/parathyroid and uterus and cervix weight; both absolute and relative to terminal body weight. In the absence of any associated histopathological correlates or any similar effects in animals at the end of the treatment period, the intergroup differences were considered not to be of toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related macroscopic findings detected in adult animals.
Incidental findings that were not associated with either a true dose related response or any histopathological correlates and were considered to be unrelated to treatment included fluid filled left kidney (one recovery control male), small left seminal vesicle (one non-recovery control male), small thymus (one non-recovery control female), reddened lungs (one recovery control female and one non-recovery and one recovery female treated with 75 mg/kg bw/day), mottled liver (one female treated with 25 mg/kg bw/day), increased pelvic space in the left kidney (one non-recovery male treated with 75 mg/kg bw/day) and a raised white spot on the non-glandular region of the stomach (one recovery male treated with 75 mg/kg bw/day).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following treatment-related microscopic abnormalities were detected:
Mesenteric Lymph Node: Foamy macrophage accumulation, minimal to moderate was present in all animals of either sex treated with 75 mg/kg bw/day. It was also present in three males and two females treated with 25 mg/kg bw/day at a minimal level. Following the two week treatment free period this finding was evident in all animals that were previously treated with 75 mg/kg bw/day, at a minimal or mild level. There was however, a minor reduction in severity in both sexes.
Stomach: Mild hyperplasia of the non-glandular region was evident in two males treated with 75 mg/kg bw/day. No such effect was evident in males treated with 25 or 10 mg/kg bw/day and following the two week treatment free period, this finding had completely resolved in males that were previously given 75 mg/kg bw/day.
There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Functional Performance Tests
There were no intergroup differences at any dose level considered to be related to treatment with the test item.
Males treated with 75 mg/kg bw/day showed a statistically significant increase in hindlimb grip strength (p<0.05). Statistical significance was minimal and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup difference was considered not to be of toxicological significance. During motor activity evaluations, males from all treatment groups showed a statistically significant reduction during the final 20% of activity monitoring time (p<0.05) when compared to controls. In the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered not to be of toxicological significance.

Sensory Reactivity Assessments
Sensory reactivity scores across all test item-treated dose groups were similar to controls.

Reproductive Performance
Estrous Cycle
There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with the majority of females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy.

Mating
There was no adverse effect of treatment on mating performance. With the exception of one female treated with 10 mg/kg bw/day and two females treated with 25 mg/kg bw/day, all remaining animals mated within the first four days after pairing.

Fertility
Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level.
One female treated with 25 mg/kg bw/day was found to be non-pregnant following positive evidence of mating. Histopathological examinations of the female and the respective male partner did not reveal any significant microscopic changes which could account for the lack of pregnancy therefore this was considered incidental and unrelated to treatment.

Gestation Length
Gestation lengths were generally between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.
Statistical analysis of gestation lengths did not reveal any statistically significant intergroup differences.
One female treated with 25 mg/kg bw/day that had a total litter loss at birth had a gestation length of 24½ days. It is not unusual for females with extended gestation periods to fail to maintain their litter. In the absence of any such effects at the high dose group, the intergroup difference was considered to be incidental.

Thyroid Hormone Analysis
An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.
Statistical analysis of the data did reveal a statistically significant reduction (p<0.05) in T4 values in adult males treated with 75 and 25 mg/kg bw/day. However, in the absence of a true dose related response, or any histopathological correlates in the thyroid, the intergroup differences were considered not to be of toxicological significance.

Effect levels

open allclose all
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
water consumption and compound intake
haematology
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
reproductive and developmental toxicity
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mating performance and fertility was unaffected by treatment and there were no treatment-related effects detected in the offspring parameters observed.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral administration of X-19574- for Amines, C16-18 and C18-unsatd. alkyl, O,O-di-Bu phosphorothioates (CASRN 97808-07-6) to rats by gavage, at dose levels of 10, 25 and 75 mg/kg bw/day, resulted in reduced body weight gains in males treated with 75 and 25 mg/kg bw/day and microscopic effects detected in the stomach of males treated with 75 mg/kg bw/day and in the mesenteric lymph nodes of animals of either sex treated with 75 and 25 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 10 mg/kg bw/day.
The microscopic changes evident in the mesenteric lymph nodes were considered likely to represent the accumulation of material as part of the process of clearance of the test item or its metabolites. The reduction in severity of the change in recovery animals in both sexes indicated reversibility and at the severities observed in this study along with the absence of necrosis in the lymph node, it was considered that this finding was non-adverse. The reduced body weight development and microscopic changes evident in the stomach were considered to be the result of an irritant effect of the test item rather than a true systemic effect and as such was considered adaptive and non-adverse. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 75 mg/kg bw/day for animals of either sex.
The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 75 mg/kg bw/day.
Executive summary:

SUMMARY

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development), to evaluate some endocrine disruptor relevant endpoints and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 28 July 2015).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, forapproximately 6 weeks (males) and up to eight weeks (females)(including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 10, 25 and 75 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (75 mg/kg bw/day) or the vehicle alone for forty-three consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. 

Pairing of non-recovery animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of ano-genital distance and visible nipple count (male offspring only).

Extensive functional observations were performed on five selected non-recovery males from each dose group after the completion of the pairing phase, and for five selected non-recovery parental females from each dose group on Day 12post partum. Hematology and blood chemistry were evaluated prior to termination on five selected non-recovery males and females from each dose group. Additionally, blood samples were taken at termination from all non-recovery adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13post partum, for thyroid hormone analysis; samples from all non-recovery adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all non-recovery treated females including controls through pre-pairing, pairing and up to confirmation of mating.

Vaginal smears were also performed in the morning on the day of termination for all non-recovery treated females.

Non-recovery adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and non-recovery adult females on Days 13 and 14post partum, respectively. Any non-recovery female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Following forty-three days of treatment, recovery group animals were maintained without treatment for a further fourteen days. Hematological and blood chemistry were evaluated on all recovery group animals at the end of the treatment-free period. These animals were then subjected to a gross necropsy and histopathological examinations of selected tissues was performed. Additionally, blood samples were taken at termination from all recovery adult animals for possible thyroid hormone analysis.

Results…….

Adult Responses

Mortality

There were no treatment-related deaths.

One male treated with 75 mg/kg bw/day was killedin extremison Day 23 due to a physical injury to the foot.

Clinical Observations

Animals of either sex treated with 75 mg/kg bw/day and to a lesser extent, males treated with 25 mg/kg bw/day showed increased salivation and noisy respiration during the treatment period. No such effects were evident in females treated with 25 mg/kg bw/day or in animals of either sex treated with 10 mg/kg bw/day.

Behavioral Assessment

One female treated with 75 mg/kg bw/day had increased salivation during Day 4 of gestation behavioral observations and one male treated with 25 mg/kg bw/day showed noisy respiration during Week 6 behavioral assessments. No such effects were evident in the remaining treated animals of either sex.


Functional Performance Tests

There was no effect of treatment with the test item at any dose level on functional performance in animals of either sex.

Sensory Reactivity Assessments

Sensory reactivity scores across all test item-treated dose groups were similar to controls.

Body Weight

Males treated with 75 mg/kg bw/day showed a reduction in body weight gain throughout the treatment period. Recovery was evident however during the fourteen day treatment free period. A slight reduction in overall body weight gain was also evident in males treated with 25 mg/kg bw/day. No such effects were evident in males treated with 10 mg/kg bw/day.

No adverse effects were evident in treated females during maturation, gestation or lactation.

Food Consumption

Males treated with 75 mg/kg bw/day showed a reduction in food consumption and food conversion efficiency throughout the treatment period. Recovery was evident however during the fourteen day treatment free period. No such effects were evident in males treated with 25 or 10 mg/kg bw/day. 

No adverse effects were evident in treated females during maturation, gestation or lactation.

Water Consumption

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Reproductive Performance

Estrous Cycle

There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy.

Mating

There was no effect of treatment on mating performance. With the exception of three animals, all remaining animals mated within four days of pairing.

Fertility

There were no treatment-related effects in conception rates for test item-treated animals in relation to controls.

Gestation Lengths

There were no differences in gestation lengths in animals receiving the test item when compared with controls.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no detrimental effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 10, 25 or 75 mg/kg bw/day.

Offspring Growth and Development

There was no detrimental effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights, ano-genital distance on Day 1post partum, visible nipple count in male offspring on Day 13post partumor clinical signs up to Day 13 of age at 10, 25 or 75 mg/kg bw/day.

Laboratory Investigations

Hematology

There were no toxicologically significant effects detected in the hematological parameters examined.

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters examined.

Pathology

Necropsy

Neither the type, incidence or distribution of macroscopic observations in surviving adult animals or offspring indicated any treatment-related effect up to a dose level of 75 mg/kg bw/day.

Organ Weights

No toxicologically significant changes were evident in animals of either sex treated up to a dose level of 75 mg/kg bw/day.


Histopathology

The following treatment-related microscopic abnormalities were detected:

Mesenteric Lymph Node:Foamy macrophage accumulation, minimal to moderate was present in all animals of either sex treated with 75 mg/kg bw/day. It was also present in three males and two females treated with 25 mg/kg bw/day at a minimal level. Following the two week treatment free period this finding was evident in all animals that were previously treated with 75 mg/kg bw/day, at a minimal or mild level. There was however, a minor reduction in severity in both sexes.

Stomach:Mild hyperplasia of the non-glandular region was evident in two males treated with 75 mg/kg bw/day. No such effect was evident in males treated with 25 or 10 mg/kg bw/day and following the two week treatment free period, this finding had completely resolved in males that were previously given 75 mg/kg bw/day.

Thyroid Hormone Analysis

An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Conclusion

The oral administration of X-19574- for Amines, C16-18 and C18-unsatd. alkyl, O,O-di-Bu phosphorothioates (CASRN 97808-07-6) to rats by gavage, at dose levels of 10, 25 and 75 mg/kg bw/day, resulted in reduced body weight gains in males treated with 75 and 25 mg/kg bw/day and microscopic effects detected in the stomach of males treated with 75 mg/kg bw/day and in the mesenteric lymph nodes of animals of either sex treated with 75 and 25 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 10 mg/kg bw/day.

The microscopic changes evident in the mesenteric lymph nodes were considered likely to represent the accumulation of material as part of the process of clearance of the test item or its metabolites. The reduction in severity of the change in recovery animals in both sexes indicated reversibility and at the severities observed in this study along with the absence of necrosis in the lymph node, it was considered that this finding was non-adverse. The reduced body weight development and microscopic changes evident in the stomach were considered to be the result of an irritant effect of the test item rather than a true systemic effect and as such was considered adaptive and non-adverse. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 75 mg/kg bw/day for animals of either sex.

The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 75 mg/kg bw/day.