Phosphoric acid, decyl octyl ester

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 29, 2011 to August 21, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, mono- and di-C6-10-alkyl esters

EC Number:

269-616-7

EC Name:

Phosphoric acid, mono- and di-C6-10-alkyl esters

Cas Number:

68307-94-8

Molecular formula:

No information available

IUPAC Name:

Esterification Products of Phosphorus Pentoxide and Alcohols C6-C10 (even numbered)

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST

Details on test animals or test system and environmental conditions:

Test animals
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: 12 weeks
- Weight at study initiation: males weighed 300 to 345 g, females weighed 189 to 220 g
- Housing: all animals were housed in groups of 5 in solid floor polypropylene cages with stainless mesh lids and softwood flake bedding. During the mating phase, 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.
- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 d

Environmental conditions
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15
- Air changes (per h): 15
- Photoperiod (h dark / h light): 12/12
- Life dates: From: December 07, 2011 (first day of treatment) to January 28, 2012 (final necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Arachis oil BP

Details on exposure:

Preparation of dosing solutions:
For the purpose of this study the test substance was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test substance formulations were determined by Harlan laboratories ltd., Shardlow, UK, Analytical services. Results showed the formulations to be stable for 2 h. Formulations were therefore prepared daily. The test substance 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 8 mL/kg bw/day of Arachis oil BP. The volume of test and control read across substance administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even Numbered) in the test substance formulations was determined by high performance liquid chromatography mass selective (HPLC/MS) using an external standard technique. The test substance formulations were sampled and analysed initially and then after storage at ambient conditions for two hour. This was performed as the low level formulations were found to be unstable over eight days and therefore the dose formulations for the study were to be performed prior to each dosing occasion, and dosed within two hour of preparation. The results indicate that the prepared formulations were within acceptable ranges for the purpose of the study.

Duration of treatment / exposure:

8 weeks

Frequency of treatment:

Twice daily

Details on study schedule:

Chronological Sequence of Study
i) Groups of ten male and ten female animals were treated twice daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) 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.
v) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post-partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
vii) At Day 4 post-partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post-partum. At Day 5 post-partum, all females and surviving offspring were killed and examined macroscopically.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 160, 500 mg/kg bw/day, Basis: actual ingested

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
The dose levels were chosen based on the results of previous toxicity work (7-Day dose range-finding study in the han wistar rat). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test substance.

Examinations

Parental animals: Observations and examinations:

Detailed clinical observations: Yes, all animals were examined for overt signs of toxicity, ill-health and behavioural change
- Time schedule: Immediately before the first dose, up to 30 min after each dosing occasion, and 1 and 3 h after each dosing occasion, during the working week. Animals were observed immediately before the first dose, soon after each dosing occasion, and 1 h after each dosing occasion at weekends and bank holidays

Body weight: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post-partum.

Food consumption:
During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post-partum.

Food efficiency:
Food efficiency was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

Water consumption and compound intake (if drinking water study): Yes
- Time schedule for examinations: daily (with the exception of the mating phase)

Laboratory investigations: Yes
Haematological and blood chemical investigations were performed on 5 males and 5 females selected from each test and control group prior to termination (Day 42 for males and Day 4 post-partum for females. Blood samples were obtained after the first dosing occasion from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

Haematology:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: haemoglobin (Hb), erythrocyte count (RBC), haematocrit (Hct), erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)), total leucocyte count (WBC), differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), platelet count (PLT), reticulocyte count ((Retic) methylene blue staining slides were prepared but reticulocytes were not assessed). Prothombin 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, glucose, total protein (Tot. Prot.), albumin, albumin/ globulin (A/G) ration (by calculation), sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca++), inorganic phosphorus (P), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine (Creat), total cholesterol (Chol), total bilirubin (Bili), bile acids (Bile).

Other: functional observations:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/ behavioural toxicity at least 1 h after the second dose. Functional performance tests were also performed on the 5 selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioural assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: gait, tremors, twitches, hyper/ hypothermia, skin colour, respiration, convulsions, bizarre/ abnormal/ stereotypic behaviour/ salivation/ piloerection, exophthalmia, lachrymation, palpebral closure, urination, defecation, transfer arousal, tail elevation.

Functional performance tests
Motor activity: purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was 30 min for each animal. The percentage of time each animal was active and mobile was recorded for the overall 30 min period and also during the 20 % of the period. Forelimb/ hind limb grip strength: an automated meter was used. Each 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 trails were performed for each animal.

Sensory reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: grasp response, vocalisation, toe pinch, touch escape, pupil reflex, blink reflex, tail pinch, finger approach, startle reflex.

Pregnancy and Parturition
Each pregnant female was observed at approximately 0830, 1230 and 1630 h and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 h 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

Oestrous cyclicity (parental animals):

During mating a vaginal smear was prepared for each female and the stage of oestrus 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).

Sperm parameters (parental animals):

During histopathology examination, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Litter observations:

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 and 4 postpartum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights werecalculated retrospectively from this data)
Physical Development: Live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

Sacrifice and pathology:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post-partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum. For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. The procedure was enhanced as necessary, by staining the uteri with a 0.5 % ammonium polysulphide solution. All adult and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ weights:
The following organs, removed from animals were killed at the end of the study, were dissected free from fat and weighed before fixation: adrenals, brain, epididymides, heart, prostate, seminal vesicles, spleen, testes, kidneys, liver, ovaries, pituitary (post fixation), thymus, thyroid (weighed post-fixation with parathyroid), uterus (weighed with cervix).

Histopathology:
Samples of the following tissues were removed from all animals and preserved in buffered 10 % formalin, except where stated: adrenals, aorta (thoracic), bone and bone marrow (femur including stifle joint), bone and bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland, colon, duodenum, epididymides**, eyes*, gross lesions, heart, ileum (including peyer’s patches) jejunum, kidneys, liver, lungs (with bronchi)#, lymph nodes (cervical and mesenteric), mammary gland, muscle (skeletal), ovaries, pancreas, pituitary, prostate, oesophagus, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, thyroid/ parathyroid, trachea, testes**, thymus, urinary bladder, uterus/ cervix, vagina. *= eyes fixed in Davidson’s fluid**= preserved in Bouin’s fluid then transferred to 70 % Industrial Methylated Spirits (IMS) approximately 48-h later#= lungs were inflated to approximately normal inspiratory volume with buffered 10 % formalin before immersion in fixative The tissues from 5 selected control and 500 mg/kg bw/day dose group animals and any animals which did not achieve pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. The following tissues from the remaining control and 500 mg/kg bw/day animals were also processed: coagulating gland, epididymides, mammary gland, ovaries, pituitary, prostate, seminal vesicles, testes, uterus/ cervix, vagina. In addition, sections of testes and epididymides from all control and 500 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined. Since there were indications of treatment-related liver, stomach, thyroid and adrenal changes, examination was subsequently extended to include similarly prepared sections of the liver, stomach, thyroid and adrenals from 5 animals per sex form the low and intermediate groups. Microscopic examination was conducted by the Study Pathologist. A peer review of the histopathology examinations was also performed in this phase of the study.

Postmortem examinations (offspring):

Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

The following parameters were subjected to statistical analysis: quantative functional performance data, body weight and body weight change, food and water consumption during gestation and lactation, pre-coital interval and gestation length, litter size and litter weights, sex ratio, corpora lutea and implantation sites, implantation losses and viability indices, offspring body weight and body weight change, offspring surface righting, haematology, blood chemistry, adult absolute and body weight-relative organ weights.
Probability values (P) were calculated as follows: P<0.001***P<0.01**P<0.05*P≥0.05 (not significant)

Reproductive indices:

Mating performance and fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
1) Pre-coital intervalCalculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
2) Fertility indices: For each group the following were calculated:
Mating index (%) = (Number of animals mated/ Number of animals paired) x 100Pregnancy index (%) = (Number of pregnant females/ Number of animals mated) x 100Gestation and Parturition data

The following parameters were calculated for individual data during the gestation and parturition period of the parental generation:
1) Gestation lengthCalculated as the number of days of gestation including the day for observation of mating and the start of parturition.
2) Parturition index: The following was calculated for each group:
Parturition index (%) = (Number of females delivering live offspring/ Number of pregnant females) x 100

Offspring viability indices:

Litter responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using the individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

1) Implantation losses (%)
Group mean percentile pre-implantation and post-implantation losses were calculated for each female/ litter as follows:
Pre-implantation loss= ((Number of corpora lutea – Number of implantation sites)/ Number of corpora lutea) x 100
Post-implantation loss= ((Number of implantation sites – Total number of offspring born)/ Number of implantation sites) x 100

2) Live birth and viability indices
The following indices were calculated for each litter as follows:
Live birth index (%)= (Number of offspring alive on Day 1/ Number of offspring born) x 100
Viability index (%)= (Number of offspring alive on Day 4/ Number of offspring alive on Day 1) x 100

3) Sex ratio (% males)
Sex ratio was calculated for each litter at birth on Day 1 and Day 4 post partum, using the following formula:
(Number of male offspring/ Total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Clinical signs and mortality
Animals of either sex treated with 500 mg/kg bw/day showed increased salivation and noisy respiration throughout the treatment period. One male treated with 500 mg/kg bw/day showed laboured respiration, a decreased respiratory rate and lethargy on Day 2. A further male from this treatment group also showed laboured respiration and piloerection on Days 1 and 3 respectively. One female treated with 500 mg/kg bw/day showed laboured respiration on Day 1 and a further female from this treatment group showed laboured and gasping respiration on Day 3. Isolated incidences of staining around the mouth or snout were evident in animals of either sex treated with 500 mg/kg bw/day later in the treatment period (Days 10-11 females and Days 34-35 males). No such effects were detected in animals or either sex treated with 160 or 50 mg/kg bw/day.

Body weight and weight gain
Males treated with 500 mg/kg bw/day showed a statistically significant reduction in body weight gain during Weeks 1, 3, 4 and 5 compared to controls. Males treated with 160 mg/kg bw/day also showed a statistically significant reduction in body weight gain during weeks 3, 4 and 5. Subsequent overall body weight gain for these males was reduced. Males treated with 50 mg/kg bw/day showed a statistically significant reduction in body weight gain during Week 3 only. In isolation and in view of the fact that overall body weight gain was comparable to control animals the intergroup difference was considered not to be of toxicological importance. No toxicologically significant effects were detected in females treated with 500 or 160 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day.

Food consumption and food efficiency
Males treated with 500 mg/kg bw/day showed a reduction in overall food consumption. Food efficiency in these males was also affected. A slight reduction in food consumption was evident in males treated with 160 mg/kg bw/day during Week 2 only. No such effects were detected in males treated with 50 mg/kg bw/day. No adverse effect on food consumption was detected for females during the pre-mating, gestation or lactation phases of the study. No adverse effect on food efficiency was detected for females during pre-mating. Statistical analysis of gestation and lactation data revealed no significant differences.

Water consumption
Females treated with 500 mg/kg bw/day showed an increase in water consumption during the maturation phase of the study. No adverse effect on water consumption was detected in males treated with 500 mg/kg bw/day or animals of either sex treated with 160 or 50 mg/kg bw/day.

Haematology
There were no toxicologically significant effects detected in the haematological parameters examined. Males from all treatment groups showed statistically significant reductions in haemoglobin, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration whilst females from all treatment groups showed a statistically significant reduction in activated partial thromboplastin time. In the absence of a true dose related response the intergroup differences were considered not to be of toxicological importance. Males treated with 500 mg/kg bw/day also showed a statistically significant reduction in total leucocyte count, lymphocyte count and activated partial thromboplastin time. The majority of individual values were within the normal ranges for rats of the strain and age used, therefore the intergroup differences were considered not to be of toxicological importance.

Clinical chemistry
Animals of either sex treated with 500 mg/kg bw/day or females treated with 160 mg/kg bw/day showed a statistically significant increase in albumin/globulin ratio with all the individual values being outside the normal ranges for rats of the strain and age used. No such effects were detected in males treated with 160 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day. Males from all treatment groups showed statistically significant increases in creatinine and calcium concentration and a statistically significant reduction in bilirubin values. Males treated with 50 mg/kg bw/day also showed a statistically significant reduction in phosphorus. Females treated with 500 and 160 mg/kg bw/day showed a statistically significant increase in glucose levels. The majority of individual values within the normal ranges for rats of the strain and age used, and in the absence of any true dose related responses the intergroup differences were considered not to be of toxicological importance.

Organ weights
Animals of either sex treated with 500 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weight. Males treated with 500 mg/kg bw/day also showed an increase in absolute and relative adrenal weight. Females from this treatment group also showed an increase in absolute and relative thyroid weight, however a true dose related response was not evident. No such effects were detected in animals of either sex treated with 160 or 50 mg/kg bw/day. Males treated with 500 mg/kg bw/day showed a statistically significant reduction in absolute kidney weight and a statistically significant increase in kidney weight relative to terminal body weight. Males treated with 500 and 160 mg/kg bw/day also showed a statistically significant reduction in prostate weight both absolute and relative to terminal body weight and statistically significant increases in absolute and relative thyroid and pituitary weight. In the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance. Females treated with 160 or 50 mg/kg bw/day showed a statistically significant increase in thyroid weight both absolute and relative to terminal body weight. In the absence of any associated histology correlates at these levels the intergroup differences were considered of no toxicological importance.

Necropsy
Offspring
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test substance. Adult nine males and 2 females treated with 500 mg/kg bw/day had sloughing on the non-glandular region of the stomach. One female treated with 160 mg/kg bw/day had sloughing on the glandular region of the stomach. No toxicologically significant effects were detected in males treated with 160 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day. One male treated with 500 mg/kg bw/day had small testes at necropsy. One control male had small and flaccid testes and small epididymides at necropsy. Histopathological examination revealed microscopic changes that were considered to be incidental and in the absence of treatment in the case of the control male or histology correlates in the remaining treated groups the macroscopic findings were considered to be within the range of normal background alterations. One female treated with 500 mg/kg bw/day and 1 male treated with 50 mg/kg bw/day had increased pelvic space in both or left kidney(s) (respectively). In the absence of any histology correlates the intergroup differences were considered of no toxicological importance.

Histopathology: non-neoplastic
The following treatment related microscopic abnormalities were detected:

Liver: Minimal to slight centrilobular hepatocellular hypertrophy was evident in animals of either sex treated with 500 mg/kg bw/day. Minimal to slight glycogen depletion was also evident in 3 males from this treatment group. At 160 mg/kg bw/day, 3 males showed minimal centrilobular hepatocellular hypertrophy.

Thyroid: Minimal diffuse follicular cell hypertrophy was evident in 2 females treated with 500 mg/kg bw/day.

Stomach: Diffuse minimal to moderate squamous cell hyperplasia was evident in the forestomach (non-glandular region) in 4 males and 3 females treated with 50 mg/kg bw/day, in 4 males and 4 females treated with 160 mg/kg bw/day and in 9 males and 6 females treated with 500 mg/kg bw/day. This finding was associated in all animals with minimal to marked diffuse hyperkeratosis and in animals or either sex treated with 500 mg/kg bw/day and males treated with 160 and 50 mg/kg bw/day with minimal to moderate multifocal parakeratosis and minimal to moderate multifocal epithelial vesicles. Minimal to moderate submucosal inflammation was also evident in animals of either sex from all treated groups. This was associated in one 500 mg/kg bw/day female with minimal focal lymphoid cell infiltration.

Adrenals: Diffuse fatty change, mainly affecting the zona fasciculata, was slightly increased in males treated with 500 mg/kg bw/day. All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence of severity between control and treatment groups that were considered to be of toxicological significance.

Other findings:
Functional observations behavioural assessments
Open field arena observations during Week 5 of treatment confirmed the observation of noisy respiration in 3 males treated with 500 mg/kg bw/day. The remaining open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls. All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.

Functional performance tests
There were no treatment related changes in functional performance. Statistical analysis of the data did not reveal any significant intergroup differences.

Sensory reactivity assessments
There were no treatment-related changes in sensory reactivity.

Effect levels (P0)

1

Key result

Dose descriptor:

NOEL

Remarks:

reproductive/developmental toxicity

Effect level:

ca. 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects were detected in the reproductive parameters examined

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

ca. 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects were detected

Target system / organ toxicity (P0)

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

no

Dose response relationship:

yes

Relevant for humans:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

endocrine system

Organ:

thyroid gland

Treatment related:

no

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

Offspring Litter Size and Viability
Of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum were comparable to controls. There were no significant intergroup differences in sex ratio.

Offspring Growth and Development
Offspring body weight gain and litter weights at birth and subsequently on Day 1 and 4 post partum were comparable to controls.

Offspring Observations
No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: No treatment related effects

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Lowest effective dose / conc.:

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

System:

other: Overall observations

Organ:

other: No treatment related effects

Results: F2 generation

Effect levels (F2)

Remarks on result:

not measured/tested

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

no

Relation to other toxic effects:

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

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Relevant findings in liver:

Groups	1		2		3		4
Tissues examined	5 M	5 F	5 M	5 F	5 M	5 F	5 M	5 F
Hepatocellular hypertrophy: Mean severity	0 0	0 0	0 0	0 0	3 1.0	0 0	5 1.4	5 1.8
Total Affected	0	0	0	0	3	0	5	5
Glycogen depletion: Mean severity	0 0	0 0	0 0	0 0	0 0	0 0	3 1.3	0 0
Total Affected	0	0	0	0	0	0	3	0

 

Relevant findings in thyroid gland:

Groups	1		2		3		4
Tissues examined	5 M	5 F	5 M	5 F	5 M	5 F	5 M	5 F
Follicular cell hypertrophy: Mean severity	0 0	0 0	0 0	0 0	0 0	0 0	0 0	2 1.0
Total Affected	0	0	0	0	0	0	0	2

 

Relevant findings in forestomach:

Groups	1		2		3		4
Tissues examined	5 M	5 F	5 M	5 F	5 M	5 F	9 M	6 F
Squamous cell hyperplasia: Mean severity	0 0	0 0	4 1.3	3 1.3	4 1.5	4 1.3	9 2.2	6 1.8
Total Affected	0	0	4	3	4	4	9	6
Hyperkeratosis: Mean severity	0 0	0 0	4 1.5	3 1.0	4 1.5	4 1.3	9 2.6	6 2.2
Total Affected	0	0	4	3	4	4	9	6
Parakeratosis: Mean severity	0 0	0 0	1 1.0	0 0	2 1.0	0 0	8 1.9	2 1.5
Total Affected	0	0	1	0	2	0	8	2
Epithelial vesicles: Mean severity	0 0	0 0	1 1.0	0 0	2 1.5	0 0	8 2.5	4 1.3
Total Affected	0	0	1	0	2	0	8	4
Submucosal inflammation: Mean severity	0 0	0 0	3 1.3	2 1.0	4 1.8	4 1.3	9 2.4	6 1.7
Total Affected	0	0	3	2	4	4	9	6

 

Findings in adrenal cortex:

Groups	1		2		3		4
Tissues examined	5 M	5 F	5 M	5 F	5 M	5 F	5 M	5 F
Diffuse fatty change: Mean severity	2 1.0	0 0	3 1.0	0 0	1 1.0	0 0	5 1.2	0 0
Total Affected	2	0	3	0	1	0	5	0

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the NOAEL for systemic toxicity and NOEL for reproductive/developmental toxicity for the test substance, mono-, di- and tri- C6-10 PSE were considered to be 500 mg/kg bw/day.

Executive summary:

A study was conducted to determine the systemic toxicity and potential adverse effects of the test substance, mono-, di- and tri- C6-10 PSE, on reproduction (including offspring development), according to the OECD Guideline 422, in compliance with GLP. The test substance was administered twice daily by oral gavage to 3 groups, each of 10 male and 10 female Wistar Han™ RccHam™ WIST strain rats, for up to 8 weeks (including a 2 week maturation phase, pairing, gestation and early lactation for females), at dose levels of 50, 160 and 500 mg/kg bw/day. A control group of 10 males and 10 females was dosed with vehicle alone (Arachis oil BP). Clinical signs, behavioural assessments, body weight change, food and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a 1 male: 1 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 5 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 surface righting reflex. Extensive functional observations were performed on 5 selected males from each dose group after the completion of the mating phase, and for 5 selected parental females from each dose group on Day 4 post-partum. Haematology and blood chemistry were evaluated prior to termination on 5 selected males and females from each dose group. Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post-partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

There was no unscheduled mortality observed. Animals of either sex treated with 500 mg/kg bw/day showed increased salivation and noisy respiration throughout the treatment period. Incidences of laboured respiration, decreased respiratory rate, gasping respiration, lethargy or piloerection were also evident in these animals during the first 3 days of treatment and isolated incidences of staining around the mouth or snout were evident later in the study. No such effects were detected in animals of either sex treated with 160 or 50 mg/kg bw/day. Three males treated with 500 mg/kg bw/day showed noisy respiration during week 5. There were no toxicologically significant changes in the behavioural parameters measured for females treated with 500 mg/kg bw/day or for animals of either sex treated with 160 or 50 mg/kg bw/day. There were no treatment-related changes in functional performance. There were no treatment-related changes in sensory reactivity. Males treated with 500 mg/kg bw/day showed a reduction in body weight gain between Weeks 1 and 5 compared to controls. Males treated with 160 mg/kg bw/day also showed a reduction in body weight gain during Weeks 3, 4 and 5. Subsequent overall body weight gain for these males was reduced. No toxicologically significant effects were detected in females treated with 500 160 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day. Males treated with 500 mg/kg bw/day showed a reduction in overall food consumption. Food efficiency in these males was also affected. No such effects were detected in females treated with 500 mg/kg bw/day or animals of either sex treated with 160 or 50 mg/kg bw/day. Females treated with 500 mg/kg bw/day showed a slight increase in water consumption during the maturation phase of the study. No adverse effect on water consumption was detected in males treated with 500 mg/kg bw/day or animals of either sex treated with 160 or 50 mg/kg bw/day. There were no toxicologically significant effects detected in the haematological parameters examined. Animals of either sex treated with 500 mg/kg bw/day or females treated with 160 mg/kg bw/day showed a statistically significant increase in albumin/ globulin ratio. No such effects were detected in males treated with 160 mg/kg bw/day or in animals or either sex treated with 50 mg/kg bw/day. Nine males and 2 females treated with 500 mg/kg bw/day had sloughing on the non-glandular region of the stomach. One female treated with 160 mg/kg bw/day had sloughing on the glandular region of the stomach. No such effects were detected in males treated with 160 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day. Animals of either sex treated with 500 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weight. Males treated with 500 mg/kg bw/day also showed an increase in absolute and relative adrenal weight whilst females also showed an increase in absolute and relative thyroid weight. No such effects were detected in animals of either sex treated with 160 or 50 mg/kg bw/day. Minimal to slight centrilobular hepatocellular hypertrophy was evident in animals of either sex treated with 500 mg/kg bw/day. Minimal to slight glycogen depletion was also evident in 3 males from this treatment group. At 160 mg/kg bw/day, 3 males showed minimal centrilobular hepatocellular hypertrophy. Minimal diffuse follicular cell hypertrophy was evident in 2 females treated with 500 mg/kg bw/day. Diffuse minimal to moderate squamous cell hyperplasia was evident in the forestomach (non-glandular region) in 4 males and 3 females treated with 50 mg/kg bw/day, in 4 males and 4 females treated with 160 mg/kg bw/day and in 9 males and 6 females treated with 500 mg/kg bw/day. This finding was associated in all animals with minimal to marked diffuse hyperkeratosis and in animals or of either sex treated with 500 mg/kg bw/day and males treated with 160 and 50 mg/kg bw/day with minimal to moderate multifocal parakeratosis and minimal to moderate multifocal epithelial vesicles. Minimal to moderate submucosal inflammation was also evident in animals of either sex from all treated groups. This was associated in one 500 mg/kg bw/day female with minimal focal lymphoid cell infiltration. Diffuse fatty change, mainly affecting the zona fasciculata, was slightly increased in males treated with 500 mg/kg bw/day.

There were no treatment-related effects on mating or conception rates for treated animals. There were no treatment-related differences in gestation lengths. The distribution for treated females was comparable to controls. Of the litters born, litter size at birth and subsequently on Day 1 and 4 post-partum were comparable to controls. There were no significant intergroup differences in sex ratio. Offspring body weight gain and litter weights at birth and subsequently on Day 1 and 4 post-partum were comparable to controls. No clinically observable signs of toxicity were detected for offspring from all treatment groups.

The oral administration of the test substance to rats by gavage,at dose levels 50, 160 and 500 mg/kg bw/day, resulted in treatment related effects detected in animals of either sex from all treatment groups. The study author concluded that, the microscopic changes identified in the liver and thyroid together with the associated blood chemical and organ weight changes seen at 500 or 160 mg/kg bw/day were considered to be an adaptive response rather than adverse effect of treatment. The stomach changes identified together with the associated reductions in body weight and food consumption at 500 mg/kg bw/day may be considered to be an adverse effect of treatment, however they are also considered to be a result of local irritation of the test substance rather than a true effect of systemic toxicity. The effects were sufficient to elicit an adaptive response in the adrenal glands. Based on these findings the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was therefore considered to be 500 mg/kg bw/day. No treatment related effects were detected in the reproductive parameters examined therefore the No Observed Effect Level (NOEL) for reproductive/ developmental toxicity was considered to be 500 mg/kg bw/day. Under the study conditions, the NOAEL for systemic toxicity and NOEL for reproductive/developmental toxicity for the test substance, mono-, di- and tri- C6-10 PSE, were established at 500 mg/kg bw/day (YXZK, 2012).