Registration Dossier

Administrative data

Endpoint:
developmental toxicity
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
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (March 1996)
Deviations:
no
Qualifier:
according to
Guideline:
other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)
Deviations:
no
Qualifier:
according to
Guideline:
other: other guidance as listed under Principles of method if other than guideline
Deviations:
no
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): PEMP product
- Substance type: organic
- Physical state: Colourless transparent liquid
- Storage condition of test material: In refrigerator (2-8°C)

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on 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%.
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
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. Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.
Frequency of treatment:
Once daily for 7 d/w.
Duration of test:
Males: 29 days
Females: 40-46 days
Doses / concentrationsopen allclose all
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.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Examinations

Maternal examinations:
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)

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
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation days 5-7.

HISTOPATHOLOGY / ORGAN WEIGTHS
No.
Statistics:
For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Indices:
Reproductive indices; For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.
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 (and including) 500 mg/kg bw/ day.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
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 females (+48%). For females, aspartate aminotransferase (ASAT) was statistically significantly decreased 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.
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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In females, absolute and relative kidney weights were statistically significantly increased at 500 mg/kg bw/ day (+16% and +13% resp.). 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 reduced thyroid glands for 2 females dosed at 500 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. No abnormalities were seen in the reproductive organs, which could account for their infertility.
Histopathological findings: neoplastic:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
maternal
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
organ weights and organ / body weight ratios

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: kidney, liver and thyroid glands at 500 mg/ kg bw/ day

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered to have been unaffected by treatment up to 500 mg/kg bw. The slightly lower body weights at 150 mg/kg bw were not statistically significant different from control values, and did not show a dose-response.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Body weights of pups were considered to have been unaffected by treatment up to 500 mg/kg bw.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Five pups of the control group, 2 pups at 50 mg/kg bw, 9 pups at 150 mg/kg bw and 6 pups at 500 mg/kg bw were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was unaffected by the treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.
External malformations:
no effects observed
Description (incidence and severity):
Incidental macroscopic findings of pups included scabbing, beginning autolysis, cannibalism, and bent tail. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Details on embryotoxic / teratogenic effects:
Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
development
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects seen at 500 mg/kg bw/day

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

REPRODUCTIVE DATA

No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. There were 1/10 couples of the control group, 3/10 couples of the 50 mg/kg bw/ day group, 1/10 couples of the 150 mg/kg bw/ day group and 1/10 of the 500 mg/kg bw/ day group which failed to deliver health pups. One female treated with 50 mg/ kg bw/ day had implantation sites only, one female in the highest dose group did not mate; all others were not pregnant.

GESTATION

No toxicologically relevant effects on gestation index and duration were noted.

PARTURITION/MATERNAL CARE

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

PARTURITION/MATERNAL CARE

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

EARLY POSTNATAL PUP DEVELOPMENT

Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

MORTALITY PUPS

Five pups of the control group, 2 pups at 50 mg/kg, 9 pups at 150 mg/kg and 6 pups at 500 mg/kg were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS PUPS

Incidental clinical symptoms of pups consisted of swelling of the flank, pale appearance, blue spots, absence of milk in the stomach, wound, and small size. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT PUPS

Body weights of pups were considered to have been unaffected by treatment up to 500 mg/kg. The slightly lower body weights at 150 mg/kg were not statistically significant different from control values, and did not show a dose-response.

MACROSCOPY PUPS

Incidental macroscopic findings of pups included scabbing, beginning autolysis, cannibalism, and bent tail. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Applicant's summary and conclusion

Conclusions:
In an oral OECD 422 screening study, the NOAEL parental was derived to be 150 mg/kg bw/day, while the NOAEL for development was determined to be 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). Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces. At the highest dose, adverse effects were seen in males and females on kidney, liver and thyroid glands (females only) at 1000 mg/ kg bw/ day, which was seen in females as minimal hypertrophy of follicular cells of the thyroid glands and hypertrophy of the urothelium, increased absolute and relative kidney weights, increase bilirubin levels. In males increased (relative) liver and kidney weights were measured, furthermore changes in blood parameters were found (increase prothrombin time, bilirubin levels, inorganic phosphate levels and aspartate aminotransferase). No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed at any dose level.

Based on these data, the parental NOAEL for PEMP product was set at 150 mg/kg bw/day, whereas the NOAEL for development was set at ≥5 00 mg/kg bw/day.