Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July, 2016
Deviations:
no
Remarks:
No deviations occurred that impacted the validity of the study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company, Batch 38015
- Expiration date of the lot/batch: 26 April, 2021
- Purity test date: 20 April, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature
- Stability under test conditions: The stability of the test substance in gavage liquid was determined during the study in the batch prepared on 17 November and and additionally on 8 December 2017 because of the decrease in dosing volume (decreased from 10 ml/kg to 5 ml/kg, see section 4.3). For this purpose, samples of each concentration were analyzed in duplicate on the day of preparation (t=0) and after storage for at least 10 days at room temperature (t=10).


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was mixed with propylene glycol prior to dosing.
- Final dilution of a dissolved solid, stock liquid or gel: The test article was diluted appropriately to deliver dose levels of 30, 100 or 300 mg/kg at a dose volume of 10 ml/kg body weight/day until 24 November 2017 at which point a dose volume of 5 mL/kg body weight/day was utilized.

FORM AS APPLIED IN THE TEST: Diluted in propylene glycol.

Test animals

Species:
rat
Strain:
other: Wistar Han
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (P) 9 weeks for males, 10 weeks for females
- Weight at study initiation: Body weight variation did not exceed 20% of the mean weight for each sex.
- Fasting period before study: None
- Housing: Animals were housed in Makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental
enrichment. During the pre-treatment period the animals were housed four rats to a cage (separate by sex) and there was one cage with two males and two cages with five females (this included the spare animals, see also section 3.2). For mating, one male and one female were housed together. Mated females were housed individually in cages, which were placed in another cage rack. The location of the mated females in the new cage racks was determined by the date of mating (females found sperm-positive on the same date were considered a ‘lot’) and by animal number (within each lot the mated females were housed in numerical order). After delivery, the cage containing the dam with a litter was transferred to another cage rack. The location of the dam with the litter in this cage rack was determined by the delivery date and the animal number.
- Diet (e.g. ad libitum): Ad libitum (Rodent Diet VRF1 (FG) - SDS Special Diets Services, Witham, England).
- Water (e.g. ad libitum): Ad libitum (Tap water)
- Acclimation period: At least a week.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data
IN-LIFE DATES: From: 25 October, 2017 To: 16 January, 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Stability and solubility of the test article.
- Concentration in vehicle: The test article was diluted appropriately to deliver dose levels of 30, 100 or 300 mg/kg at a dose volume of 10 ml/kg body weight/day until 24 November 2017 at which point a dose volume of 5 mL/kg body weight/day was utilized.
- Amount of vehicle (if gavage): 10 ml/kg body weight/day until 24 November 2017 at which point a dose volume of 5 mL/kg body weight/day was utilized.
- Lot/batch no. (if required): K48209424
- Purity: 99.9%
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 5 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: No data
- After successful mating each pregnant female was caged (how): Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the homogeneity and content of the test substance in the dilutions were conducted using a LC MS/MS method.
Duration of treatment / exposure:
The test substance was administered daily by oral gavage at dose levels of 30, 100 or 300 mg/kg body weight during a premating period of 2 weeks and during mating (1 week), gestation and lactation until 14 days after delivery. Male animals were sacrificed after 29 days of treatment. Female animals and pups were sacrificed at or shortly after day 13 of lactation.
Frequency of treatment:
Daily
Details on study schedule:
The test substance was administered daily by oral gavage at dose levels of 30, 100 or 300 mg/kg body weight during a premating period of 2 weeks and during mating (1 week), gestation and lactation until 14 days after delivery. Male animals were sacrificed after 29 days of treatment. Female animals and pups were sacrificed at or shortly after day 13 of lactation.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a range finding study.
- Rationale for animal assignment (if not random): Random
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each adult animal was observed daily in the morning hours by cage-side observations. All cages were checked again in the afternoon for dead or moribund animals to prevent loss of tissues by cannibalism or autolytic degeneration. All abnormalities, signs of ill health or reactions to
treatment were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed clinical examinations were conducted in an area outside the home cage in all (surviving) rats prior to the first exposure and then once weekly throughout the study, except for the last days of pregnancy and during the initial phase of the lactation period. In the last week of the study the detailed clinical examinations were part of the Functional Observational Battery tests (FOB) in the animals concerned. Signs noted included but were not limited to changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic movements, stereotypies and bizarre behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of male and female animals were recorded just before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the pre-mating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on days 0, 4, 7 and 13 of lactation. Non-mated females were weighed once per week after the mating period. The adult animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.
Oestrous cyclicity (parental animals):
During the pre-treatment period, vaginal smears were made of every female rat (including surplus animals) for monitoring estrous cycle in order to exclude any possible female animal with an irregular estrous cycle.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations: Epididymides (paired), prostate (dorsolateral and ventral), seminal vesicles and coagulation glands (paired), tested, levator ani plus bulbocavernosus muscle complex, Cowper's glands, glans penis
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups, runts (pups that are smaller than control pups) and grossly malformed pups was evaluated on days 0, 4, 7 and 13 of lactation. The alive pups were individually weighed on days 0, 4, 7 and 13 of lactation. Mean pup weight was calculated per sex and for both sexes combined per dose group. At day lactation day 4 the anogenital distance (AGD) was measured of each pup before culling of the litter. The AGD is reported as such and corrected for body weight and using the cube root of body weight. On postnatal day 13 all surviving male pups were examined for the presence of nipples or areolas. Any abnormal behavior of pups was recorded on day 0, 4, 7 and 13 of lactation. Grossly malformed pups were sacrificed and examined. A necropsy was performed on stillborn pups and pups that died during the study and macroscopic abnormalities of these pups were recorded. At necropsy of the surviving dams and their litter, at or shortly after day 13 of lactation, pups were examined externally for gross abnormalities and killed by decapitation (except for the two pups per litter of which blood was collected for hormone analysis

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals. All males were sacrificed after the mating period on 19 December 2017 (after 29 days of dosing).

- Maternal animals: All surviving animals. Dams were sacrificed, after overnight fasting, on day 14 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table were prepared for microscopic examination and/or weighed.

Weighed and Microscopically examined:
Epididymides
Prostate
Seminal vesicles and coagulation glands
Testes
Ovaries
Uterus
Adrenal glands
Brain
Heart
Kidneys
Liver
Spleen
Thymus
Thyroid

Microscopically Examined:
Vagina
All gross lesions
Bone marrow
Eye
Lungs
Mesenterial and axillary lymph nodes
Nasopharyngeal tissue
Peripheral nerve
Pituitary
Skeletal muscle
Small and large intestines
Spinal cord
Stomach
Urinary bladder

Postmortem examinations (offspring):
SACRIFICE and GROSS NECROPSY
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Any abnormal behavior of pups was recorded on day 0, 4, 7 and 13 of lactation. Grossly malformed pups were sacrificed and examined. A necropsy was performed on stillborn pups and pups that died during the study and macroscopic abnormalities of these pups were recorded. At necropsy of the surviving dams and their litter, at or shortly after day 13 of lactation, pups were examined externally for gross abnormalities and killed by decapitation (except for the two pups
per litter of which blood was collected for hormone analysis.

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid glands were weighed and microscopically examined.
Reproductive indices:
- Pre-coital time
- Duration of Gestation
- Male mating index
- Female mating index
- Male fertility index
- Female fertility index
- Female fecundity index
- Gestation index
- number of adult females with normal or abnormal estrous cycle and cycle duration
- number of females placed with males (and vice versa)
- number of females (not) mated and number of males (not) mated
- number of females (not) pregnant
- number of males that became sire
- number of females killed moribund or found dead
- number of females completing delivery
- number of females with liveborn pups
- number of females with (all) stillborn pups
- total and mean number of pups delivered (liveborn + stillborn)
- total and mean number of implantation sites
Offspring viability indices:
- total number of liveborn pups
- total number of stillborn pups
- total and mean number of live pups(/litter) at day n
- total number of culled pups
- total number of pups lost (dead, missing, cannibalized) (period)
- number and incidence of litters lost entirely (period)
- total and mean number of live male pups at day n

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs noted in the high-dose group (300 mg/kg) were related to the poor health status of these animals and included dyspnoea, thin, muscle weakness, lethargic, hunched posture, piloerection, soiled fur, salivation, sniffing, encrustation of nose, grunting and blepharospasm of the eyes. Weekly detailed clinical examinations confirmed the above daily clinical observations.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Four males of the high-dose group were sacrificed in moribund condition on test days and their moribund condition was considered test substance-related. In the female animals no treatment-related moribundity was observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights and body weight change were decreased in the males dosed with 300 mg/kg. In all other dose groups mean body weights and body weight change were comparable to their respective control groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In conjunction with the reduced body weights, male rats dosed with 300 mg/kg test article also demonstrated reduced mean food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Hematology was conducted in plasma of 5 rats/sex/group at necropsy. There were no toxicologically relevant changes noted.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Male rats dosed with 300 mg/kg demonstrated test substance related and adverse increases in serum BUN and creatinine. Female rats dosed with 300 mg/kg test article demonstrated a test substance-related increase in serum calcium levels that were outside the testing facilities historical control range.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No abnormalities were observed in the FOB.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
males dosed with 300 mg/kg demonstrated increased relative kidney, liver and testes weights and the cowpers glands were decreased. The mean absolute weights of the seminal vesicles and cowpers glands were also decreased males dosed with 300 mg/kg.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gross organ observations at necropsy revealed enlarged and/or pale kidneys, in one animal the urinary bladder was firm to touch, and in two animals the thymus was small in males dosed with 300 mg/kg. In the gross macroscopy of the four sacrificed moribund males, several findings were noticed, including small spleen, cryptorchism, stomach with gaseous content, red discoloration of the glandular stomach, small thymus, small white discolored heart, pale kidneys, pale glandular stomach with ulcers, small prostate gland and small seminal vesicles and red discoloration of
the glandular stomach.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No abnormal neurobehavioral observations were indicated in the FOB.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic evaluation of these moribund animals revealed decreased cellularity of the spleen and thymus (4/4 and 3/4, respectively), hyperkeratosis of the stomach (2/4) and renal tubular dilation, inflammation, and crystalline material in the renal pelvis, and mild hyperplasia of the transitional epithelium .

Microscopic histopathological evaluation of male rats dosed with 300 mg/kg revealed a nephropathy and mitotic epithelium noted in the urinary bladder. No other microscopic changes were noted.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no test article-related effects on the estrous cycle.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were test article-related effects on male reproductive parameters.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were test article-related effects on reproductive performance.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No effects were observed on pup observations.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No effects were observed on pup survival.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects were observed on pup weight.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No effects were observed on gross pup observations.
Histopathological findings:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were observed on pup observations, pup sex, pup survival and pups weight.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, the no observed adverse effect level (NOAEL) for parental toxicity is 100 mg/kg/day and the reproductive and developmental NOAEL is 300 mg/kg/day.
Executive summary:

The reproductive and developmental toxicity potential of the test article was evaluated in Wistar rats. The study was conducted according to OECD Guideline 422 (2016) and was conducted in compliance with OECD GLP (1997). The test substance was administered daily by oral gavage at dose levels of 30, 100 or 300 mg/kg body weight during a premating period of 2 weeks and during mating (1 week), gestation and lactation until 14 days after delivery. Male animals were sacrificed after 29 days of treatment. Female animals and pups were sacrificed at or shortly after day 13 of lactation. The results of test substance analysis in the carrier showed that, the test substance was homogenously distributed in the carrier and that the concentrations of the test substances in the carrier were within acceptable variation from target concentration.

Four males of the high-dose group were sacrificed in moribund condition on test days and their moribund condition was considered test substance-related. In the female animals no treatment-related moribundity was observed. Clinical signs noted in the high-dose group (300 mg/kg) were related to the poor health status of these animals and included dyspnoea, thin, muscle weakness, lethargic, hunched posture, piloerection, soiled fur, salivation, sniffing, encrustation of nose, grunting and blepharospasm of the eyes. Weekly detailed clinical examinations confirmed the above daily clinical observations. Neurobehavioral observations and FOB did not indicate any neurotoxic potential of test article in rats. Mean body weights and body weight change were decreased in the males dosed with 300 mg/kg. In all other dose groups mean body weights and body weight change were comparable to their respective control groups. In conjunction with the reduced body weights, male rats dosed with 300 mg/kg test article also demonstrated reduced mean food consumption.  Hematology was conducted in plasma of 5 rats/sex/group at necropsy. There were no toxicologically relevant changes noted. Male rats dosed with 300 mg/kg demonstrated test substance related and adverse increases in serum BUN and creatinine. Female rats dosed with 300 mg/kg test article demonstrated a test substance-related increase in serum calcium levels that were outside the testing facilities historical control range. Terminal body weights of male animals of the high-dose group (300 mg/kg) were statistically significantly decreased (8%) when compared to the control group. In addition, males dose with 300 mg/kg demonstrated increased relative kidney, liver and testes weights and the cowpers glands were decreased. The mean absolute weights of the seminal vesicles and cowpers glands were also decreased males dosed with 300 mg/kg. Gross organ observations at necropsy revealed enlarged and/or pale kidneys, in one animal the urinary bladder was firm to touch, and in two animals the thymus was small in males dosed with 300 mg/kg. In the gross macroscopy of the four sacrificed moribund males, several findings were noticed, including small spleen, cryptorchism, stomach with gaseous content, red discoloration of the glandular stomach, small thymus, small white discolored heart, pale kidneys, pale glandular stomach with ulcers, small prostate gland and small seminal vesicles and red discoloration of the glandular stomach. Microscopic evaluation of these moribund animals revealed decreased cellularity of the spleen and thymus (4/4 and 3/4, respectively), hyperkeratosis of the stomach (2/4) and renal tubular dilation, inflammation, and crystalline material in the renal pelvis, and mild hyperplasia of the transitionalepithelium. Microscopic histopathological evaluation of male rats dosed with 300 mg/kg revealed a nephropathy and mitotic epithelium noted in the urinary bladder. No other microscopic changes were noted. There were no effects of the test substance on estrous cycle and on fertility and reproductive performance. Results of T4 hormone analysis in male parental animals and in male and female pups of postnatal day 13 did not show any significant effects between the groups. In female pups of the high-dose group the absolute weight of the thyroid was decreased; however, histopathological evaluation of the pup thyroid revealed no adverse findings and this weight change is considered non-adverse. No effects were observed on pup observations, pup sex, pup survival and pups weight. Based on the unexpected moribundity of four male animals in the high-dose group, the increased serum BUN and creatinine that correlated with nephropathy observed in the microscopic evaluation of the kidneys, and the elevated serum calcium in females, the NOAEL for parental toxicity was placed at 100 mg/kg body weight per day. Based on the absence of effects on fertility, the reproductive NOAEL is 300 mg/kg.  Based upon the absence of adverse effects on litters and pups, the developmental NOAEL is 300 mg/kg.