Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Repeated dose toxicity: Oral

NOAEL for developmental toxicity: 450 mg/kg/day

NOAEL for systemic toxicity in males: 25 mg/kg/day

NOAEL for systemic toxicity in females: 450 mg/kg/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August 2015 - 14 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
The pre-study chemistry Control samples from Day 8 and Day 15 were not analyzed, in error.
Amendment 2 to the Protocol re-scheduled the post dose observation and not the detailed physical examination and arena observation occasions during lactation, in error. The post dose observations were recorded on Day 6 of lactation, as the animals were not dosed on Day 8 and the detailed physical examination and arena observations were performed on Day 8 of lactation and not on Day 6, for logistical reasons.
Detailed physical and arena observation of some animals on Day 6 after mating and Day 1 of lactation were performed on some animals (Group 1F 85, 89, 90; Group 2F 106, 108; Group 3F 71, 76, 77; Group 4F 51, 52, 56 and Group 1F 81, 87; Group 2F 102, 104, 109, 110; Group 3F 75, 79; Group 4F 57-60) after dose administration, in error.
Group 2; Dam/Litter No. 107 - offspring body weights were not recorded on Day 4 of age, in error.
Total leucocyte count and mean cell volume in males and hematocrit, erythrocyte count and mean cell volume in females were recorded in Week 2 of recovery, in error and are not reported.

The urinary values for protein, sodium, potassium and glucose concentration, measured in Recovery Week 2 are reported as actual values and not as urinary concentrations, as the volume of the urine samples were not recorded, in error. Urinary chloride concentration was not measured in Recovery Week 2 in error, as it was not listed as a parameter to be measured in the Study Protocol.
These unforeseen events were considered to have not affected the integrity or validity of the study.
Deviations:
yes
Remarks:
See remarks. These deviations were considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Appearance: Brown liquid
Storage conditions: Room temperature, under nitrogen
Lot number: 15B26
Expiry date 01 August 2016
Purity (by GLC) 81.7%
Species:
rat
Strain:
other: Crl:CD (SD)
Details on species / strain selection:
The (virgin) rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 77 days for male, approximately 70 days for females.
- Weight at study initiation: Males 352 to 426 g, Females 226 to 289 g.
- Housing: Solid (polycarbonate) bottom cages with a stainless steel mesh lid were used during the acclimatisation, gestation, littering and lactation periods. Grid (polycarbonate) bottomed cages were used during pairing. Solid bottom cages contained softwood based bark-free fiber bedding.
- Diet (e.g. ad libitum): Certified pelleted diet, non-restricted (removed overnight before blood sampling for haematology or blood chemistry and during the period of urine collection).
- Water (e.g. ad libitum): Potable water from the public supply, non-restricted (except during urine collection).
- Acclimation period: 12 days before commencement of treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark.
- Environmental Enrichment: Aspen chew block, throughout the study (except during pairing and lactation) and plastic shelter, throughout the study (except during pairing and lactation)
Route of administration:
oral: gavage
Details on route of administration:
Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Vehicle:
propylene glycol
Details on oral exposure:
Administration:
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.
Route Oral gavage
Treated at Constant doses in mg/kg/day.
Volume dose 5 mL/kg body weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.
Frequency Once daily.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision, calibratin standard stability and extracted
solution stability.

The homogeneity and stability was confirmed for test item in Propylene Glycol formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days.

The mean concentrations of test item in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation. Difference from mean values were within 5% confirming precise analysis.
Duration of treatment / exposure:
At least 5 weeks
Frequency of treatment:
Once daily at approximately the same time each day
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
5 mg/mL nominal concentration
6.1 mg/mL formulated concentration
Dose / conc.:
105 mg/kg bw/day (nominal)
Remarks:
21 mg/mL nominal concentration
25.62 mg/mL formulated concentration
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
90 mg/mL nominal concentration
109.8 mg/mL formulated concentration
No. of animals per sex per dose:
10 for main study phase.
5 for toxicity phase and recover phase (control and highest dose groups only)
Control animals:
yes, concurrent vehicle
Details on study design:
Preliminary study:
A preliminary study was carried out to assess the systemic toxic potential of the test substance, an
industrial chemical, in a 7 day oral gavage study in CD rats, to select a suitable high dose for
a subsequent OECD 422 screening study.

A cautious approach was adopted for this study, based on the information received from the
Sponsor from the acute toxicity study (Harlan Study Number 41300447). Initially,
two groups of animals were committed to the study at doses of 100 or 300 mg/kg/day. The
data from these groups was reviewed before treatment commenced to a third group of
animals at 600 mg/kg/day. Following review of the available data, a fourth group of animals
was subsequently allocated to the study and treated at 450 mg/kg/day.

During the study, clinical condition, body weight, food consumption, organ weight and
macropathology investigations were undertaken.

Results:
Animals at 450 or 600 mg/kg/day showed underactive behaviour and chin rubbing after
dosing; in addition, piloerection was evident in females receiving 600 mg/kg/day while
partially closed eyelids were evident in both sexes at 450 mg/kg/day. There were no signs
related to treatment at 100 or 300 mg/kg/day.

Animals treated at 450 or 600 mg/kg/day showed initial body weight losses and weight gain
was low in males treated at 100 mg/kg/day and both sexes treated at 300 mg/kg/day
(Days 1-4) and correlated with low food intake at all doses during this period. Body weight
gain was recorded and was markedly high in females treated at 600 mg/kg/day and food
intake was higher, during Days 4-8.

Adjusted mean liver weight appeared to be marginally high in males treated at 450 mg/kg/day
and males and females treated at 600 mg/kg/day.

Conclusion:
A high dose of 450 mg/kg/day and intermediate and low doses of 105 and 25 mg/kg/day,
respectively are considered suitable dose levels for the OECD 422 study (PLZ0086).

Main Study:
The study consisted of one control and three treated groups.
Some serial observations needed to be performed without the knowledge of the treatment
group; therefore the animal numbering system was such that it was not easy to identify a
treatment group from the animal number.

The F1 generation received no direct administration of the test item, PX-1E-PA-B. Any
exposure to the test item or metabolites was through the mother to the offspring in utero
and/or through the milk.

Positive control:
N/A
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: least twice daily

BODY WEIGHT: Yes
- Time schedule for examinations:
Main study males, recovery phase animals and toxicity phase females: Week -1 Before dosing on the day that treatment commenced (Week 0) and weekly thereafter (including recovery phase). On the day of necropsy.
Main study females: Week -1 Before dosing on the day that treatment commenced (Week 0) and weekly before pairing. Days 0, 6, 13 and 20 after mating. Day 1, 4, 7 and 8 of lactation. On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Main study males, recovery phase animals and toxicity phase females: Week -1 Weekly Food consumption was not recorded for main study phase males and females during the period when they were paired for mating (Week 3), but recommenced for males in Week 4. Food consumption was recorded continuously for toxicity phase females and recovery phase animals.
Main study females: Week -1, on the day that treatment commenced (Week 0). Weekly before pairing. Days 0-5, 6-12 and 13-19 after mating. Days 1-3 and 4-6 of lactation.
Sacrifice and pathology:
Necropsy
All adult animals were subject to a detailed necropsy.

After a review of the history of each animal, a full macroscopic examination of the tissues
was performed. All external features and orifices were examined visually. Any abnormality
in the appearance or size of any organ and tissue (external and cut surface) was recorded and
the required tissue samples preserved in appropriate fixative.

Time of Necropsy
Main study males and toxicity Week 6.
phase females

Main study females whose On or after day the last offspring died.
litter died before Day 7

Main study females Day 8 of lactation.

Recovery phase males and Following 14 days without treatment
females

F1 offspring Day 7 of age.

Method of Kill
All adult animals Carbon dioxide asphyxiation with subsequent
exsanguination.
Offspring Intraperitoneal injection of sodium pentobarbitone.
Sequence To allow satisfactory inter-group comparison.

Main study females
The following were recorded:
Each uterine horn Number of implantation sites.

Female whose Mammary tissue appearance.
litter died before
Day 7 of lactation

Premature deaths Where possible, a fresh macroscopic examination (external and internal)
with an assessment of stomach for milk content was performed.

Offspring at Examined externally (including palate), if found to be normal offspring
scheduled were discarded without further examination. Any externally abnormal
termination offspring were also examined internally. Abnormal tissues were retained
in an appropriate fixative.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above.
Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of
those detailed below:
Testes In modified Davidson’s fluid.
Eyes In Davidson’s fluid.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax
and sectioned at a nominal four to five micron thickness. For
bilateral organs, sections of both organs were prepared. A
single section was prepared from each of the remaining tissues
required.

Full List All animals killed or dying prematurely.
The five lowest main study males and females from all groups
and toxicity phase females and recovery phase animals
(Groups 1 and 4) at scheduled termination.

Short list All remaining main study animals.

Skin (Study Plan) The five lowest main study males and females from Groups 2
and 3 and recovery phase animals (Groups 1 and 4) at
scheduled termination.

Routine staining Sections were stained with hematoxylin and eosin; in addition
samples of the testes were stained using a standard periodic
acid/Schiff (PAS) method.

Light Microscopy
Findings were either reported as "present" or assigned a severity grade. In the latter case one
of the following five grades was used - minimal, slight, moderate, marked or severe. A
reviewing pathologist undertook a peer review of the microscopic findings.

For the assessment of the testes, a detailed qualitative examination was made, 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, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the
lumen. Any cell- or stage-specificity of testicular findings was noted.

For the assessment of the ovaries a qualitative evaluation of one section from each ovary was
made.

Statistics:
Statistical analyses were performed on the majority of data presented. For some parameters, including pre-coital interval, gestation length, gestation index, stage of estrous at termination and mating performance, the similarity of the data was such that analyses were not considered to be necessary.

All statistical analyses were carried out separately for males and females. For all other adult
parameters, the analyses were carried out using the individual animal as the basic
experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis
for statistical analysis and biological significance was assessed with relevance to the severity
of the anomaly and the incidence of the finding within the background control population.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment at 450 mg/kg/day elicited, during the first week of treatment in all or the majority of males and females, underactive behavior, with or without piloerection and/or partially closed eyelids that persisted in some at the end of the day. In addition, all females were noted to have low body temperature on Day 1.

Transient and isolated incidences of salivation were evident in some males and non-mated females at 450 mg/kg/day and in pregnant females at 105 or 450 mg/kg/day and in females with a litter at 25, 105 or 450 mg/kg/day, in a dose-related manner and one female with a
litter at 0 or 450 mg/kg/day had chin rubbing on one day. These minor incidences of transient salivation and chin rubbing was considered to be attributed to the palatability of the test item, or the dosing procedure.

At 450 mg/kg/day, one male (No. 24) had transient noisy respiration and piloerection on Day 21 of treatment and showed a full recovery. On Days 36-40 of treatment, one male (No. 25) had limited use of the right hind limb that was considered to be attributed to trauma and not treatment related.

When compared with Control, hindlimb grip strength in Week 5 of treatment was statistically significantly high in females treated at 450 mg/kg/day (0.42v 0.38 kg) and forelimb grip strength was low in females treated at 450 mg/kg/day at Day 4-6 of lactation (0.94v1.03 kg). However, these were attributed to normal biological variation, as the differences showed the opposite direction of effect.

There was no other effect of treatment on sensory reactivity and grip strength.

It is considered that motor activity was unaffected by treatment.

Motor activity (high beam) in Week 5 were statistically significantly low, when compared with Control; in males treated at 25, 105 or 450 mg/kg/day; however there was no relationship with dose, high intra-group variability and there was no consistent intergroup difference over time. Low beam activity was low in females treated at 450 mg/kg/day and was low in females in the same animals at the end of the off-dose period. However, as the activity of these animals was within the Historical Control Data (HCD) and Control values were at the upper end of the HCD and females investigated at Day 4-6 of lactation were comparable with Control, no effect of treatment is inferred.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (No. 80) receiving 105 mg/kg/day gave birth to a healthy litter, consisting of 17 offspring) and showed evidence of suckling. Approximately five hours later, after treatment, the animal was found in respiratory distress (gasping) and had a red stained muzzle; one pup had been cannibalised. The female was considered to be in poor clinical condition and was euthanized on welfare grounds at pre-Day 1 of lactation. The remaining offspring were killed at the same time, on welfare grounds. Macroscopic examination revealed fetal material or dark contents in the mouth, oesophagus, stomach, ileum, jejunum, lungs and bronchi and clear aerated fluid in the nasal turbinates and trachea; two placentas were found in the uterus. The cause of death was considered due to aspiration of fetal material and therefore not related to administration of the test item.

One female (No. 60) receiving 450 mg/kg/day was humanely killed, following the death of her litter on Day 5 of age. The litter was cold to touch and all offspring had a small amount of milk in the stomach on Days 1 and 2 and were found dead during Days 2-5. Microscopic examination of the mammary tissue of the adult revealed reduced secretory activity.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
When compared with Control, overall body weight gain (Week 0-5) was marginally low, or low, in males treated at 105 or 450 mg/kg/day (91 or 80%, respectively). The body weight gain of males previously treated at 450 mg/kg/day was higher (162%) than Control, during the two week off-dose period.

Body weight gain in un-mated females and females during gestation and lactation were unaffected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption of males was considered to be unaffected by treatment.

Food intake of unmated females at 450 mg/kg/day was low (85%) during the first week of treatment, when compared with Control; however there was no adverse effect on body weight gain.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Investigation of the cellular constituents of the blood in Week 5 and when compared with Control, indicated mild anaemia in treated males, but not in non-mated females treated at 450 mg/kg/day. Haematocrit, haemoglobin concentration and red cell count at 25, 105 or 450 mg/kg/day were all marginally low (94, 95 or 91%; 95, 94 or 90% and 92, 95 or 90%, respectively) and reticulocyte count at 105 or 450 mg/kg/day was marginally high (114 or 128%, respectively). In females, haemoglobin concentration was marginally low (92%), on Day 8 of lactation. Activated partial thromboplastin time was slightly short in males and non-mated females receiving 450 mg/kg/day (88% and 92%, respectively) and was short (87%) on Day 8 of lactation.

All other differences were minor, confined to one sex and included high lymphocyte count in males at 450 mg/kg/day (134%) and low mean cell volume in females at the same dose (92%).

Following two weeks respite from treatment, all parameters affected during treatment were comparable with Control and therefore showed full recovery.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma in males and non-mated females in Week 5 and on Day 8 of lactation did not identify any toxicologically significant differences from Control that were clearly attributable to treatment.

All differences were confined to males. Such differences included the low triglyceride concentration at 105 or 450 mg/kg/day (72 or 56%) and low phosphorous concentration (80%), in which all individual values were lower than the Control range. Further investigation of these parameters, following two weeks respite from treatment, showed that the values were comparable with Control.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Investigation of the composition of the urine in Week 5 and when compared with Control, revealed high protein, glucose, sodium, potassium and chloride concentrations in males treated at 105 or 450 mg/kg/day and in females at 450 mg/kg/day.

Urinary volume was high in males and females treated at 450 mg/kg/day (165% or 193%, respectively) and pH was low in males treated at 25, 105 or 450 mg/kg/day (85, 85 or 77%, respectively).

Unfortunately, as the urinary volume was not recorded at the recovery phase investigation; the concentration of these parameters, relative to urinary output cannot be presented.
However, the data shows that concentrations of protein, glucose, sodium and potassium were comparable with Control, following two weeks respite from treatment. It was considered that chloride concentration would show similar recovery.
Behaviour (functional findings):
not specified
Description (incidence and severity):
At 450 mg/kg/day: in all or the majority of males and females, underactive behavior, with or without piloerection and/or partially closed eyelids that persisted in some at the end of the day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared with Control and at 450 mg/kg/day, adjusted liver weight was marginally high in males and non-mated females (109% or 113%, respectively) following five weeks of treatment and was marginally high in females at this dose on Day 8 of lactation (113%). Adjusted liver weight remained high in these animals (120% or 108%, respectively) following two weeks respite from treatment.

Brain weight was marginally high in males only; following five weeks of treatment at 25, 105 or 450 mg/kg/day (105, 105% or 106% of Control, respectively) and epididymis weight was marginally low at 450 mg/kg/day (90%). Following two weeks respite from treatment, brain and epididymis weights were similar to Control. Adjusted adrenal weight was statistically significantly high in females treated at 450 mg/kg/day (119%) and uterus weight was statistically significantly low (49%), when compared with Control; however this was considered fortuitous, as there was no difference to Control at the end of the treatment period.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Animals killed after 5 weeks of treatment:
The macroscopic examination performed after 5 weeks of treatment revealed the following changes in the skin:

Skin
Scabs were seen in two males treated at 450 mg/kg/day. Depressions were seen in the skin of another two males treated at 450 mg/kg day and one male treated at 105 mg/kg/day.

The incidence and distribution of all other findings were consistent with the common background of changes seen in CD rats at these laboratories.

Animals killed after 2 weeks of recovery:
The macroscopic examination performed after 2 weeks of recovery revealed no test item-related lesions.

The incidence and distribution of all findings were consistent with the common background of changes seen in CD rats at these laboratories.

Animals killed on day 8 of lactation:
The macroscopic examination performed on Day 8 of lactation revealed no test item-related lesions.

The incidence and distribution of all findings were consistent with the common background of changes seen in CD rats at these laboratories.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Animals killed after 5 weeks of treatment:
Treatment related findings
Findings considered to be related to treatment with PX-1E-PA-B were seen in the skin.

Skin
Slight to moderate epidermal ulceration and/or necrosis and dermal inflammation were seen in four males receiving 450 mg/kg/day and one receiving 105 mg/kg/day, all of which correlated with findings seen at necropsy. In all cases, these changes were associated with one or more inflammatory and/or degenerative changes, including scabs, subcutaneous inflammation, epidermal hyperplasia, myofibre degeneration/necrosis of the panniculus carnosus, and dermal/subcutaneous oedema.

The toxicological significance of these changes is unclear.

Incidental findings
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Animals killed after 2 weeks of recovery:
Treatment related findings
There were no findings seen in the tissues presented for histopathological evaluation which were considered to be related to previous treatment with the test substance.

Incidental findings
Minimal dermal inflammation was seen in one recovery phase female previously receiving 450 mg/kg/day. This finding was, however, associated with apparent traumatic damage to the skin and was considered not to be of a similar nature to dermal changes seen in some
male animals at the end of the treatment period.

Animals killed on Day 8 of lactation:
The microscopic examination performed on Day 8 of lactation revealed no test item-related lesions.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
It was considered that estrous cycles were not affected by treatment.

One animal receiving 25 mg/kg/day (No. 106) and two animals receiving 450 mg/kg/day (No’s 53 and 58) had irregular oestrus cycles and all other animals showed regular 4 or 5 day cycles; however, it was considered that there was no relationship with treatment

Similar proportions of toxicity phase females in the Control and 450 mg/kg/day groups showed oestrus at the end of the treatment period and the pattern of recorded cycle stages suggested that animals were cycling normally.

Pre-coital interval was not affected by treatment.

One Control female (No. 82) and one female receiving 450 mg/kg/day (No. 53) mated at the second oestrus occasion after pairing, all other females mated at the first oestrus occasion.

Mating performance and fertility was not affected by treatment.

The gestation length of all animals was within acceptable limits (22-23.5 days). All animals were pregnant.

Body temperature was noted to be low, following the first dose in females and the food intake of females only was low during the first week of treatment.
Details on results:
F1 Litter Responses:
Offspring Clinical Signs
There were no signs considered to be related to treatment.

Litter Size, Sex Ratio and Survival Indices:
All animals were pregnant. All litters were raised to Day 7 of age, with the exception of on
litter at 105 mg/kg/day, killed following the euthanasia of the dam (No. 80) and one litter at 450 mg/kg/day (Dam No. 60) that were not sufficiently suckled and died at five days of age and, in the absence of increased pup mortality in other study litters, no effect of parental treatment was inferred.

Litter size, sex ratio and survival indices were unaffected by treatment.

Offspring Body Weight:
There was no effect of treatment on mean offspring body weight on Day 1 of age.
Offspring body weight gain from birth to Day 7 of age was slightly low, with parental treatment at 25, 105 or 450 mg/kg/day (male 89, 84 or 87%; female 85, 87 or 83%, respectively).

Offspring Macropathology:
Macroscopic observation of offspring that were humanely killed or found dead before Day 7 and those terminated on Day 7 of age revealed no findings considered to be related with treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
4 550 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
dermal irritation
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: systemic toxicity
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
25 mg/kg bw/day (nominal)
System:
other: Skin
Organ:
skin
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
It was concluded that the No-Adverse-Effect-Level (NOAEL) for reproductive and developmental toxicity was 450 mg/kg/day.
The NOAEL for systemic toxicity in males was set at 25 mg/kg/day, since one male at 105 mg/kg/day and four males at 450 mg/kg/day showed macroscopic and microscopic changes in the skin of unclear aetiology, but which appeared to be related to treatment.
The NOAEL for systemic toxicity in females was 450 mg/kg/day.
Executive summary:

The purpose of this study was to assess the systemic toxic potential of the test item, an industrial chemical, in Sprague-Dawley rats, including a screen for reproductive/ developmental effects, with administration of the test item by oral gavage administration for at least five weeks.

Main study males were treated daily for two weeks before pairing, throughout pairing and up to necropsy after a minimum of five consecutive weeks.

Main study females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 7 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 8 of lactation. The F1 generation were killed on Day 7 of lactation, but received no direct administration of the test substance; any exposure was in utero or via the milk.

Toxicity phase females were treated daily for a minimum of five consecutive weeks.

Recovery phase male and female rats were treated daily for a minimum of six consecutive weeks and were retained, without treatment for 14 days (non-treatment period commenced from the first main study Dam necropsy on Day 8 of lactation), to assess the potential for any treatment-related change to recover. Recovery phase animals were not paired.

A similarly constituted Control group received the vehicle, propylene glycol, throughout the same relative treatment period.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, urinalysis, oestrous cycles, pre-coital interval, mating performance and fertility, gestation length, organ weight and macropathology were undertaken. Full histopathology investigations were undertaken for the first five Main study

males, Main study and Toxicity phase females from the Control and high dose groups and for skin only from the first five Main study males from the low and intermediate dose groups and all recovery phase males.

The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed.

Results

One female with a litter, receiving 105 mg/kg/day, was killed for welfare reasons due to aspiration of fetal material and female with a litter, receiving 450 mg/kg/day was killed following the death of her poorly suckled litter, due to poor mammary secretion. Neither was considered to be related to treatment.

During the first week of treatment at 450 mg/kg/day, the majority or all of the animals had underactive behavior, with or without piloerection and/or partially closed eyelids that persisted in some at the end of the day. All females also had low body temperature on Day 1.

Overall male body weight gain was low at 105 or 450 mg/kg/day and only partial recovery was evident during the off-dose period. Body weight gain in un-mated females and during gestation and lactation were unaffected.

Food intake of females receiving 450 mg/kg/day was low during the first week of treatment.

There was evidence of mild anaemia in treated males in Week 5; haematocrit, haemoglobin concentration and red cell count at 25, 105 or 450 mg/kg/day were marginally low and reticulocyte count was marginally high at 105 or 450 mg/kg/day; activated partial thromboplastin time was slightly short in males and in non-mated females and females at Day 8 of lactation at 450 mg/kg/day. Activated partial thromboplastin time was slightly short

in males and non-mated females and females on Day 8 of lactation. All affected parameters showed full recovery, following two weeks respite from treatment and there were no microscopic correlates.

Urinary protein, glucose, sodium, potassium and chloride concentrations were high in males and females treated at 450 mg/kg/day and in males only at 105 mg/kg/day; recovery was apparent after two weeks respite from treatment and there were no microscopic correlates.

Estrous cycles, pre-coital interval, mating performance and fertility, gestation length and gestation index were not affected by treatment.

At 450 mg/kg/day, adjusted liver weight was high in males and non-mated females and females with a litter and remained high following two weeks respite from treatment; however, there were no microscopic correlates.

All animals were pregnant. Litter size and sex ratio were unaffected by treatment.

Overall body weight gain of offspring (birth to Day 7 of age) was slightly low at 25, 105 or 450 mg/kg/day; however all remained in good clinical condition and survival indices were unaffected.

In males only, four at 450 mg/kg/day and one at 105 mg/kg/day had slight to moderate epidermal ulceration and/or necrosis and dermal inflammation in the skin and all were associated with one or more inflammatory and/or degenerative changes, including scabs, subcutaneous inflammation, epidermal hyperplasia, myofibre degeneration/necrosis of the panniculus carnosus, and dermal/subcutaneous oedema.

Conclusion

It was concluded that the No-Adverse-Effect-Level (NOAEL) for reproductive and developmental toxicity was 450 mg/kg/day. The NOAEL for systemic toxicity in males was set at 25 mg/kg/day, since one male at 105 mg/kg/day and four males at 450 mg/kg/day showed macroscopic and microscopic changes in the skin of unclear aetiology, but which appeared to be related to treatment. The NOAEL for systemic toxicity in females was 450 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
other: Hematological, urinal, goss pathology, histopathological (non-neoplastic)

Additional information

Justification for classification or non-classification

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