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Description of key information

In an oral OECD422 screening study with rats, the NOAEL was determined to be 150 mg/kg bw/day, based on adverse effects on kidney, liver and thyroid glands at 500 mg/ kg bw/ 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
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22JUN2015 - 14SEP2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(March 1996)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
(July 2000)
Deviations:
no
Guideline:
other: other guidance as listed under Principles of method if other than guideline
Principles of method if other than guideline:
In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 1995);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 11-12 weeks.
- Weight at study initiation: 301-344 gr (males) and 196-230 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except for max. 24 hours before sacrifice.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Temporary deviations from the daily mean relative humidity occurred, laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 22JUN2015 TO: 14SEP2015
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
Method of formulation: Formulations (w/w) were prepared daily within 1 hour prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance (1.29) and vehicle (1.125). No correction was made for the purity/composition of the test substance.

Storage conditions of formulations: At room temperature.

Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on two occasions during the treatment phase (13 and 24 August 2015), according to a validated method (WIL Project 508562). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
Stability in vehicle over 1 hour (lowest concentration) and 6 hours (highest and lowest concentration) at room temperature was also determined. The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 40-46 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
One control female, and two high dose females were not dosed on day 1 of lactation as these females were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
Frequency of treatment:
Once daily, 7 d/w
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 10-day dose range finding study (WIL Research project 508567). Female rats (3 per dose group) were exposed for 10 days to 500, 750 or 1000 mg/kg bw/ day (procedures identical to main study). At necropsy, all animals were subjected to an external, thoracic and abdominal
examination after the last observation of clinical signs (scheduled necropsy) or sooner (decedents). No organs were fixed. Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were recorded.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: Daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at least immediately after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT:
Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on days 1 and 4.

FOOD CONSUMPTION:
Yes
- Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on days 1 and 4 of lactation.

FOOD EFFICIENCY:
Yes.
- Average food consumption [per animal per day]/average body weight per cage x 1000

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours, water was provided)
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Yes (with a maximum of 24 hours, water was provided)
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: The selected males were tested during week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (from lactation day 4 onwards)
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: hearing ability, pupillary reflex and static righting reflex, fore- and hind-limb grip strength and locomoter activity (total movements and ambulations)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

All animals were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.

- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis:
According to test guidelines

- All remaining females which failed to deliver and the remaining males: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines

- All remaining males:
Epididymides and Testes

HISTOPATHOLOGY: Yes
According to test guidelines
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

References:
Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 3 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 4 Kruskal W.H. and Wallis W.A.. Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47 (260): 583-621, December (1952).
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were noted during daily detailed observations and weekly arena observations that were considered toxicologically relevant. Piloerection was noted in two males and two females of the high dose group for 2-5 days, but this was not considered a severe effect. Salivation was observed in several rats at 150 and 500 mg/kg bw/day, which was considered a physiological response to exposure to the test substance. Incidental findings that were noted included exophthalmos, alopecia and scabbing. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted up to 500 mg/kg bw/ day. The slightly reduced food consumption during the first week of treatment for males and females was not considered toxicologically relevant as it already recovered to normal values during the second week of treatment. In addition, on days 11-14 post-coitum and days 1-4 of lactation statistically significant lower mean absolute and/or relative food consumption were noted at 150 and/or 500 mg/kg bw/ day. As these changes were only minor and no effects on body weight gain was noted, these changes were not considered toxicologically relevant.
Food efficiency:
no effects observed
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):
Males at 500 mg/kg bw/ day had a dose-related, statistically significant increase of approximately 8% in mean Prothrombin Time (PT). White blood cell count was increased in females at 150 and 500 mg/kg bw/ day (statistically significant increase), increase was not dose-related. In absence of alterations in other haematological parameters, the effects were found to be not toxicologically relevant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Total bilirubin was increased for high dose group males and females (+32% and 48% in males and females, respectively). Inorganic phosphate levels were increased statistically significantly for the high dose group males (+27%). Other effects were judged as not toxicologically relevant: Aspartate aminotransferase (ASAT) was statistically significantly decreased in males of the high dose group, for females ASAT levels were also decreased (statistically significant for low and high dose group). Based on the absence of dose-relationship and based on the fact that this was a decrease, these observations are not regarded to be toxicologically relevant. Alkaline phosphatase (ALP) was decreased in the low dose group (males), in absence of a dose-relationship this was found not toxicologically relevant. Cholesterol levels were decreased at 150 and 500 mg/ kg bw/ day in males only with -18% and -24%. Furthermore, bile acids were increased for males at 150 and 500 mg/ kg bw/ day. Changes in cholesterol and bile acid levels were however not statistically significant. These effects are considered to have arisen as a result of slightly high control values and/or remained within the range considered normal for rats of this age and strain and are thus regarded not to be of toxicological relevance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals and no toxicologically relevant effects on grip strength were observed. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative organ weights in males were statistically significantly affected for liver and kidney in high dose rats (both +13%). In females, absolute and relative kidney weights were statistically significantly increased at 500 mg/kg bw/ day (+16% and +13% resp.). Furthermore, males dosed at 500 mg/ kg bw/ day had a decreased thymus weight (-21%) and increased spleens (+14%) (not statistically significant). Seminal vesicles weights of all exposed males were decreased, however not in a dose-dependent way (-23%, -23% and -3.5% for resp. low, mid and high dose groups), therefore this was not regarded as toxicologically relevant. Furthermore, statistically significant decrease of prostate weight at 150 mg/ kg bw/ day (both absolute and relative) was measured. In absence of dose-relationship of the effect, this was not found to be toxicologically relevant. In females, absolute spleen weights were increased at 150 and 500 mg/ kg bw/ day (+20% and +22%; also relative values were increased), however this was not statistically significant. Uterus weights were increased for all dosed females, but not related to the dose (+30%, +27% and 16% for resp low, mid and high dose groups; also increased for relative values), therefore this was not regarded as a toxicologically relevant effect.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy did not reveal any toxicologically relevant alterations. Incidental findings among control and treated animals were within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. Incidental findings included pelvic dilation of the kidneys (1 male at 50 mg/kg bw/ day, 2 males at 500 mg/ kg bw/ day) and reduced thyroid glands for 2 females dosed at 1000 mg/ kg bw/ day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related morphologic alterations were present in the thyroid gland of 3 high dose females and consisted of minimal hypertrophy of follicular cells. Furthermore, in females dosed at 500 mg/ kg bw/ day hypertrophy of the urothelium was seen (minimal for two rats and slight for 2 rats). No effects on thyroids and urinary bladder were seen in any other female. There were no correlating morphologic alterations for the test item-related higher kidney weights noted in females treated at 500 mg/kg/day. Tubular basophilia was present in 1/5 females of the control group and in 4/5 females of the 500 mg/kg/day group, at minimal degrees and in all rats unilateral (only in one of the two kidneys present). Furthermore, there was no other evidence of regeneration/ degeneration, or signs of nephropathy (like for example thickening of basement membranes). A few basophilic cortical tubules can be a normal feature in young rats (within background levels of rats of this age and strain). Therefore this somehow higher incidence in females of the 500 mg/kg/day group was considered not to be toxicologic relevant. No test item-related morphologic alterations in males were noted.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
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:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Analysis of dose preparations: In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 150 and 500 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies 98% and 100% respectively). For the formulation of the group exposed to 50 mg/ kg bw/ day, the mean accuracy was negligibly below the target concentration (i.e. 88% of target) and therefore the results were accepted. The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation ≤ 7.1%). Recovery after 6 hours was found to be 78.7% and 92.9% for the low and mid dose respectively. Based on this, the formulations of the high dose group were found to be stable when stored at room temperature under normal laboratory light conditions for at least 6 hours whereas the formulations of the low dose group were found not to be stable when stored at room temperature under normal laboratory light conditions for 6 hours. Formulations of the low dose group were found to be stable when stored at room temperature under normal laboratory light conditions for at least one hour (relative difference of 4.2%).

Conclusions:
In an oral OECD422 screening study with rats, the NOAEL was determined to be 150 mg/kg bw/day, based on adverse effects on kidney, liver and thyroid glands at 500 mg/kg bw/day.
Executive summary:

A combined 28d repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. PEMP product was administered by daily oral gavage to male and female rats at dose levels of 50, 150 and 500 mg/kg bw/ day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 40-46 days). No mortality occurred. No clinical signs were noted apart from salivation related to substance administration. Neurobehavioural examination did not show effects of PEMP product exposure. Furthermore, no effects were seen on body weight gain and food consumption. Several blood parameters were affected by the treatment in the highest dose group: males had a dose-related, statistically significant increase in inorganic phosphate levels (+27%); total bilirubin was increased for males and females (+32% and 48%, respectively). In males, relative liver and kidney weights were increased (both at +13%). In females, absolute and relative kidney weights were statistically significantly increased (+16% and +13% resp.). Follicular cell hypertrophy of the thyroid gland was observed with a slightly higher incidence in 3 high dose females. Furthermore, in females dosed at 500 mg/ kg bw/ day hypertrophy of the urothelium was seen (minimal for two rats and slight for 2 rats). No test item-related morphologic alterations in males were noted. Based on the adverse effects on thyroid glands (females only), liver and kidney (both sexes) at 500 mg/ kg bw/ day, a No Observed Adverse Effect Level (NOAEL) for PEMP product of 150 mg/kg bw/day was established.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was performed according to OECD/ EC guidelines and GLP prinicples (Klimisch 1).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A combined 28d repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. PEMP product was administered by daily oral gavage to male and female rats at dose levels of 50, 150 and 500 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 40-46 days). No mortality occurred. No clinical signs were noted apart from salivation related to substance administration. Neurobehavioural examination did not show effects of PEMP product exposure. Furthermore, no effects were seen on body weight gain and food consumption. Several blood parameters were affected by the treatment in the highest dose group: males had a dose-related, statistically significant increase in inorganic phosphate levels (+27%); total bilirubin was increased for males and females (+32% and 48%, respectively). In males, relative liver and kidney weights were increased (both at +13%). In females, absolute and relative kidney weights were statistically significantly increased (+16% and +13% resp.). Follicular cell hypertrophy of the thyroid gland was observed with a slightly higher incidence in 3 high dose females. Furthermore, in females dosed at 500 mg/ kg bw/day hypertrophy of the urothelium was seen (minimal for two rats and slight for 2 rats). No test item-related morphologic alterations in males were noted. Based on the adverse effects on thyroid glands (females only), liver and kidney (both sexes) at 500 mg/ kg bw/day, a No Observed Adverse Effect Level (NOAEL) for PEMP product of 150 mg/kg bw/day was established.

Justification for classification or non-classification

Based on the available data, PEMP is not classified for repeated dose exposure according to CLP Regulation (EC) No. 1272/2008.