Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Qualifier:
according to
Guideline:
other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: white to off white powder or flakes
Storage conditions: at room temperature protected from light

Test animals

Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Details on species / strain selection:
Untreated, nulliparous, non-pregnant females and untreated males were used at the initiation of the study.
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
- At initiation of dosing males and females were 6 weeks old, males weighed between 154 and 187 g and females weighed between 120 and 145 g.
- Fasting period before study: no
- Housing: Pretest and pre-mating: animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm); Mating: Males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm); Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm); Lactation: Females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) (During motor activity measurements, animals did not have access to food for a maximum of 2 hours)
- Water: Free access to tap-water (During motor activity measurements, animals did not have access to water for a maximum of 2 hours)
- Acclimation period: for 7 days prior to start of treatment;

DETAILS OF FOOD AND WATER QUALITY:
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
(set to maintain)
- Temperature (°C): 20 to 22
- Humidity (%): 43 to 66
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 April 2017 To: 02 August 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily as solution (low and mid dose) or suspension (high dose) within 5 hours prior to dosing and were homogenized to a visually acceptable level. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.
Details on mating procedure:
After 8 weeks of treatment, animals were cohabitated on a 1:1 basis within 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 had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which the females who had not shown evidence of mating were separated from her male.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of samples of formulations taken in week 1, 5 and 13 during the treatment phase were analysed according to a validated method. The samples were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were in agreement with the target concentrations (i.e. 90-110% for the low and mid dose and 85-115% for high dose). Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Stability of formulations over 5 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study.
Duration of treatment / exposure:
Males were treated for 92 days, i.e. 8 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were treated for 93-98 days, i.e. during 8 weeks prior to mating, the variable time to conception, the duration of the pregnancy and 14-15 days after delivery up to and including the day before scheduled necropsy.
Females which failed to deliver healthy offspring were treated for 85-96 days.
Routinely, females that are littering are left undisturbed. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.
Frequency of treatment:
Once daily for 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 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: Based on the results of a 14-day dose range finding study the dose levels for this combined 90-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg bw/day. In this DRF no mortality or no abnormalities were noted at 500 and 1000 mg/kg bw/day.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, 19 and 20 postcoitum and during lactation on PND 1, 4, 7 and 13.

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 post-coitum and during lactation on PND 1, 4, 7 and 13

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

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes were examined using an ophthalmoscope after application of a mydriatic agent prior to initiation of treatment in all animals, and towards the end of the treatment period in Week 13 in all males and during lactation in all females.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Blood of F1 was collected on PND 4 and PND 13-15 if possible.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes for F0, o/n (with a maximum of 24h)
- How many animals: all F0 animals; 2 pups per litter of F1
- Parameters checked according to Guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Blood of F1 was collected on PND 4 and PND 13-15 if possible.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes for F0,, o/n (with a maximum of 24h)
- How many animals: all F0 animals; 2 pups per litter of F1
- Parameters checked according to Guidelines. Included thyroid hormone analyzed (T4 and TSH).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations:
The selected males were tested during Week 13 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 8-12). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined:
5 animals/sex/group
- Battery of functions tested: hearing ability, pupillar reflex and static righting reflex, fore- and hind-limb grip strength, locomotor activity.

IMMUNOLOGY: No

OTHER: reproduction and developmental parameters, plasma thyroid hormone analysis
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 11 days prior to the mating period and during mating until evidence of copulation was observed. Vaginal lavage continued for those
females with no evidence of copulation until termination of the mating period.
On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, staging of spermatogenesis
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:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups.
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation. Clinical pathology included measurement of plasma thyroid hormone level.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes, according to Guidelines
HISTOPATHOLOGY: Yes, according to Guoidelines
Organ weights according to Guidelines

The numbers of former implantation sites were recorded for all paired females.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Postmortem examinations (offspring):
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/ determined for pups born or found dead.

GROSS NECROPSY
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered
formalin. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or % CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Precoital time = Number of days between initiation of cohabitation and confirmation of mating
Fertility 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 viability indices:
Post-implantation survival index (%) = (Total number of offspring born/Total number of uterine implantation sites) x 100
Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
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
Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Lactation index (%) = (Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Salivation seen among animals treated with the test item was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
Any other clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Mortality occurred in two incidences only; the premature death of male no. 26 (300 mg/kg) and female no. 80 (1000 mg/kg) was considered unrelated to treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Compared to controls, body weight gain of male rats was reduced, to about the same extent, at 300 and 1000 mg/kg from start treatment onwards, resulting in about 10% lower mean body weights from treatment Day 36 onwards. Statistical significance was achieved in most weeks of the pre-mating period at 1000 mg/kg and occasionally at 300 mg/kg. To a lesser extent, a similar trend was noted in males treated at 100 mg/kg.
Body weights and body weight gain of females were considered not to be affected by treatment. The slightly higher mean body weights noted in 1000 mg/kg females at most time points (up to 6%, not statistically significant) were considered to represent normal biological variation and, therefore, regarded as unrelated to treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption (before and after correction for body weight) was considered not to be affected by treatment.
For females at 1000 mg/kg a statistically significant decreased relative food consumption was noted on Days 4-7 of lactation. This was caused by the lower food consumption of female no. 75, which was related to her small litter size (i.e. one pup).
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to treatment.
The nature and incidence of ophthalmology findings noted during pretest and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significantly lower mean number of platelets was noted at 1000 mg/kg in males (relative difference from controls 17%). Individual values in 1000 mg/kg males generally remained within the historical control range .
There were no treatment-related changes in red and white blood cell parameters. The few statistically significant variations noted in females (higher mean values for total white blood cells, lymphocytes and red blood cell distribution width at 100 or 300 mg/kg) were considered unrelated to treatment due to the lack of a dose-related response.

A statistically significantly higher mean prothrombin time (PT) was noted at 1000 mg/kg in males (relative difference from controls 5%). Values in 1000 mg/kg males remained within the historical control range (and mostly also in the concurrent control range). There were no treatment-related changes in activated partial thromboplastin time (APTT).

Historical control data for the number of platelets (10E9/L) in male Wistar Han rats
Mean: 741, P5- 95: 566–900 (n=91; period 2014 - July 2016)
Historical control data for prothrombin time (s) in male Wistar Han rats
Mean: 17.1, P5-P95: 15.6-18.9 (n=91, period 2014 - July 2016)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes, all at 1000 mg/kg in males, distinguished treated animals from control animals. Relative changes in mean values compared to the control group are indicated between parentheses.
• Higher (26%) plasma activity of aspartate aminotransferase (ASAT)
• Higher (55%) plasma activity of alkaline phosphatase (ALP).
• Higher albumin (7%)
• Higher total bilirubin (50%)
• Lower cholesterol (31%).
Mean values for ASAT, ALP and bilirubin in treated males remained within the historical control ranges, those for albumin and cholesterol were slightly out of these ranges .
The other (mostly statistically significant) variations noted in clinical biochemistry values were considered unrelated to treatment due to the lack of a dose-related response (albumin, creatinine and sodium in 100 and/or 300 mg/kg treated males) or an abnormal value in a single animal (high bile acids in male no. 40 at 1000 mg/kg; values for other liver-related clinical chemistry parameters were also relatively high in this male). Lower ASAT levels in treated females (all dose groups) was related to the relatively high mean control value, due to an abnormal high values in two control females (no. 45 and 49).

Historical control data for male Wistar Han rats (n=94, period 2014 – July 2016)
ASAT (U/L) mean: 75.2, P5-P95: 63.3-92.5
ALP (U/L) mean: 137, P5-P95: 82-246
Albumin (g/L) mean: 32.1, P5-P95: 30.8-33.9
Total bilirubin (umol/L) mean: 2.2, P5-P95: 1.6-3.0
Cholesterol (mmol/L) mean: 1.98, P5-P95: 1.57-2.41

Serum T4 levels in F0-males and F0-females were statistically significantly reduced (by 57% and 19%, respectively) at 1000 mg/kg. The statistically significantly lower (about 20%) mean T4 value noted in males at 100 mg/kg was considered unrelated to treatment due to the lack of a dose response (mean T4 at 300 mg/kg was similar to the control mean). Moreover, individual T4 values at 100 mg/kg generally remained within the concurrent and historical control range .
There were no treatment-related changes in serum TSH levels of both F0-males and F0-females. Overall, mean values of males and females at 100 and 300 mg/kg and males at 1000 mg/kg appeared higher than concurrent control mean values. Due to the relatively high standard deviations of these groups and a lack of a dose response, the higher mean values of the treated animals were considered as not toxicologically relevant. The statistically significantly higher (about four times higher than the control value) mean TSH value noted at 300 mg/kg was considered unrelated to treatment as the increase was caused by abnormal values in two single males (nos. 22 and 29).

Historical control data for T4 values in Wistar Han rats from OECD 422 studies
Males mean: 4.65, P5-P95: 2.970-6.440 (n=448; period 2015-2017)
Females mean: 3.57, P5-P95: 2.183-5.250 (n=40; period 2015-2017)
Note: males within historical control data were 14 weeks, whereas males within the current study were about 19 weeks old at time of blood sampling.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Grip strength was considered not to be affected by treatment. The statistically significant differences noted in hind limb grip strength (lower mean values in males at 100 and 1000 mg/kg and in females at 300 mg/kg) showed no clear dose-related response and were not accompanied by corroborative changes in other measures in the neuromuscular domain (including fore limb grip strength, gait, static righting reflex and motor activity). Moreover, the difference from controls was slight and all values remained within normal limits. Therefore, these findings were regarded as unrelated to treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity 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):
Organ weights were considered not to be directly affected by treatment.
The statistically significant organ weight differences noted in 1000 mg/kg males (13% higher relative testes weight and 15% lower absolute prostate weight) were considered secondary to the (test item-related) lower body weights of these males (10% lower mean terminal body weight). Body weights of 300 mg/kg males were decreased to a similar degree but the changes in their testes and prostate weights (which were slightly smaller than those at 1000 mg/kg) were not statistically significant.
The other statistically significant variations noted in organ weight values for males (brain, thymus and adrenals in males at 100 and/or 300 mg/kg) were considered unrelated to treatment due to the lack of a dose-related response.
No treatment-related effects were observed in organ weights of females up to 1000 mg/kg.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related gross observations.
A slightly higher incidence of irregular surface of the stomach was noted in 3/10, 2/10, 4/10 and 1/9 females in the control, 100, 300 and 1000 mg/kg groups, respectively. This finding was considered unrelated to treatment due to the lack of a dose response.
All of the remaining recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the thyroid gland, an increased incidence of follicular cell hypertrophy was present in 1000 mg/kg females (up to slight).
In the urinary bladder, an increased incidence of hyperplasia/hypertrophy of the urothelium was present in 1000 mg/kg females (minimal). The urothelial change was characterized by a mixture of hyperplasia of the basal cell layer and/or hypertrophy of the dome-shaped surface cells (umbrella cells). The cells of the urothelium formed a uniform thickness (diffuse) without prominent focal outward or inward growth and without cellular atypia. There was no inflammation or cell death involved in this process.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A finding of note was observed in an ovary of 1000 mg/kg/day treated female (no.78) consisting of a tubular adenoma (also known as tubulostromal adenoma). Since there were no other test item-related proliferative findings observed in the ovaries, this incidental finding was considered as unrelated to the test item.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment.
Most females had regular cycles of four days. Irregular cycles occurred in two females at 100 mg/kg (no. 56 which was not pregnant and no. 57 with a normal litter), and one female at 300 mg/kg was acyclic with extended di-estrus (no. 67 which was not mated; abnormal cyclicity was also indicated by the morphology of her ovaries and vagina). These findings were not attributed to treatment due to their incidental occurrence and lack of a dose-related trend.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
One female at 300 mg/kg showed no evidence of mating (no. 67, mated with male no. 27) and two females were not pregnant despite evidence of mating (no. 46 of the control group and no. 56 of the 100 mg/kg group, mated with male nos. 06 and 16, respectively.
Female no. 67 showed slight vacuolation and hypertrophy of the corpora lutea of the ovaries and a marked increase in mucification of the vagina epithelium indicative of an abnormal cycle. This was consistent with the abnormal estrous cyclicity shown by the vaginal lavage data (female no. 67 was acyclic with persistent di-estrus). This was considered as an incidental finding and unrelated to treatment with the test item. For the two other couples, no abnormalities were seen in the reproductive organs which could account for their non-pregnancy.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

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.

Details on results (P0)

In females treated at 1000 mg/kg microscopic examination revealed an increased incidence of follicular cell hypertrophy (minimal or slight) in the thyroid. This was accompanied by a decrease of thyroid hormone T4 (on average 19%). In males treated at 1000 mg/kg serum levels of thyroid hormone T4 were also decreased (on average by 57%), unaccompanied by treatment related changes in thyroid weight or morphology. For both males and females no corroborative findings were observed in TSH levels. Based on the minimal or slight increase in follicular cell hypertrophy in combination with a decrease in T4 levels in 1000 mg/kg treated females and the magnitude of decreased T4 levels in 1000 mg/kg treated males, adversity could not be excluded.
Exposure to the test item up to 1000 mg/kg was not associated with obvious toxicity in the remaining parental parameters examined in this study (i.e. mortality, clinical appearance, functional tests, ophthalmoscopy, body weight, food consumption, clinical pathology, macroscopic examination and organ weights). Other changes noted in treated rats, mostly at 1000 mg/kg, were considered non-adverse as discussed below.
Slight salivation observed after dosing among treated animals (all dose levels) was considered a physiological response rather than a sign of systemic toxicity.
Male rats administered the test item showed reduced body weight gain from start treatment onwards, which was not accompanied by reduced food consumption. This occurred to a similar degree at 300 and 1000 mg/kg, and to a lesser degree at 100 mg/kg. The resulting reduction in mean body weights was modest (about 10% from Day 36 onwards at 300 and 1000 mg/kg) and the growth rate of treated males had returned to control levels after five weeks of treatment. Therefore, the decreased body weights of treated male rats were regarded as non-adverse within the context of this study.
A further treatment-related morphological change observed in females treated at 1000 mg/kg consisted of an increased incidence of minimal hyperplasia/hypertrophy of the urothelium of the urinary bladder. There was no inflammation or cell death involved in this process. Therefore, this minimal change was considered as non-adverse (Frazier et al., 2012).
Clinical pathology showed several changes in males treated at 1000 mg/kg: higher values for aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total bilirubin and albumin; and lower values for cholesterol. Additionally, the increased prothrombin time (PT) for males at 1000 mg/kg could be related to the decrease in the number of platelets. These changes were not accompanied by adverse anatomic pathology alterations and the mean values for these parameters in 1000 mg/kg males generally remained within the range considered normal for rats of this strain and age (see results section). As such, these clinical pathology changes were regarded as non-adverse (Palazzi et al., 2016).

Frazier, KS, Seely, JC, Hard, GC, Betton, G, Burnett, R, Nakatsuji, S, Nishikawa, A, Durchfeld-Meyer, B, Bube A (2012). Proliferative and Non-proliferative Lesions of the Rat and Mouse Urinary System. Toxicological Pathology 40 (4) suppl., 54S-55S.

Palazzi X, Burkhardt JE, Caplain H, Dellarco V, Fant P, Foster JR, Francke S, Germann P, Gröters S, Harada T, Harleman J, Inui K, Kaufmann W, Lenz B, Nagai H, Pohlmeyer-Esch G, Schulte A, Skydsgaard M, Tomlinson L, Wood CE, Yoshida M (2016). Characterizing "Adversity" of Pathology Findings in Nonclinical Toxicity Studies: Results from the 4th ESTP International Expert Workshop. Toxicological Pathology 44(6), 810-24.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) at 1000 mg/kg appeared lower than that at the other dose levels (9.8 versus 11.4, 12.2 and 11.2 at 0 (control), 100 and 300 mg/kg, respectively). The difference was not statistically significant and could largely be explained by the low number of live pups in a single litter (at first litter check, 1000 mg/kg female no. 75 had 2 live pups and 7 dead pups). Such a small live litter size occasionally occurs in untreated controls. All other litters at 1000 mg/kg had normal live litter sizes . Therefore, the apparently lower mean live litter size at 1000 mg/kg was considered not to reflect an adverse effect of the test item.

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) at 1000 mg/kg was relatively low compared to the concurrent control level (84% versus 89%). Also compared to historical control data, the calculated post-implantation survival index at 1000 mg/kg was at the lower limit of the normal range .
In the nine pregnant females treated at 1000 mg/kg, the individual difference between number of implantation sites and number of pups born (the so-called unaccounted for (implantation) sites) varied between 0 and 4, were 4/9 females had 3, 1/9 had 4 and 4/9 had 0-2 unaccounted for sites, respectively. For comparison, in the nine pregnant control group females 1/9 females had 3 and 8/9 females had 0-2 unaccounted for sites. These results indicated a trend of increased number of females with a higher number of unaccounted for sites, outside the range of that observed in concurrent controls, at 1000 mg/kg.
For female no. 70 (300 mg/kg) the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation.

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) at 1000 mg/kg (91%) was lower than that at the other dose levels (100, 100 and 99% at 0 (control), 100 and 300 mg/kg, respectively). At first litter check, one pup at 300 mg/kg (no. 66) and 9 pups at 1000 mg/kg (one of litter nos. 71 and 79, seven of litter no. 75) were found dead.

Viability indices (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment up to 300 mg/kg. The viability indices were 100, 98, 100 and 95% at 0 (control), 100, 300 and 1000 mg/kg, respectively.
One pup at 100 mg/kg (litter no. 52) and four pups at 1000 mg/kg (litter nos. 75, 78 and 79) went missing (presumably cannibalized) between PND 2-4. One pup at 300 mg/kg (litter no. 66) and nine pups at 1000 mg/kg (litter nos. 71, 75 and 79) were found dead on PND 1. In addition, one pup at 300 mg/kg was sacrificed early on PND 4, based on its poor physical condition (i.e. lean appearance). At 1000 mg/kg, the postnatal loss differed statistically significantly from that in the control group, in which no pups died. Also compared to historical control data the postnatal loss at 1000 mg/kg was higher .

Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100% for all groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, mean pup weights on PND 1 were 9% (male pups) or 8% (female pups) lower compared to controls. Combined mean pup weights at 1000 mg/kg were statistically significantly lower, however remained within the normal range . Mean pup weights at 1000 mg/kg remained slightly lower at PND 4 and 7, but were close to control values at PND 13. A finding of note was the abnormally low birth weight and severely reduced postnatal growth of the single surviving female pup of 1000 mg/kg female no. 75. Based on the incidental occurrence, the reduced growth of this pup was regarded not toxicologically significant.
Pup weights at 100 and 300 mg/kg showed no remarkable differences from control weights.
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:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were considered unaffected by treatment.
Urinalysis findings:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the findings noted (i.e. alopecia) remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Description (incidence and severity):
The sex ratio of living pups at first litter check in the 1000 mg/kg group differed statistically significantly from that in the control group (% males/females: 34/66 at 1000 mg/kg versus 57/43 in the control group). On an individual litter basis, at 1000 mg/kg the percentage of female pups was about 70-80% in 5/8 litters of normal sizes and 100% in the litter that included only two live pups. For comparison, in the control group the percentage of female pups was about 80% in 1/9 litters and 55% or lower in the remaining litters. The sex ratios at 100 and 300 mg/kg were unremarkable (55/45 and 45/55, respectively).

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
A statistically significantly higher mean value for normalized anogenital distance in female pups at 1000 mg/kg was observed when compared to mean control value (0.59 vs. 0.53, respectively). As the mean values remained within historical control data , the difference between mean control and mean 1000 mg/kg values was minimal and due to the lack of a dose-related response, the increased mean value at 1000 mg/kg was not attributed to treatment.


Treatment up to 1000 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)


No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).
No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling and histopathological examination of reproductive organs).

A skewed sex ratio towards females was noted at 1000 mg/kg: 66% female pups versus 43% in the control group (the difference was statistically significant). With such a high proportion of female pups it could not be excluded that the skewed sex ratio was related to treatment. This was strengthened by the finding that the percentage of female pups was about 70-80% in 5/8 litters of normal size at 1000 mg/kg versus 1/9 in the control group. There were no treatment-related changes in nipple retention in male pups, anogenital distances in male and female pups, or reproductive organs of parental animals. Therefore, it was considered unlikely that the skewed sex ratio resulted from an endocrine mediated (antiandrogenic) effect. An alternative explanation for the skewed sex ratio could be selective loss of male embryos due to a sex difference in sensitivity to toxic effects of the test item. The slightly increased number of unaccounted for implantation sites noted at 1000 mg/kg (resulting in a slightly lower live litter size) might reflect higher sensitivity of male embryos but does not constitute conclusive evidence of selective loss of male embryos.
Live birth index at 1000 mg/kg was lower compared to controls (91% versus 100%). In total, 9 pups of the 1000 mg/kg group were found dead at first litter check: 7/9 of one litter (2 male and 5 female pups) and one male pup in two other litters. Additionally, a slight increase in postnatal loss was noted: 4 pups in three litters died between PND 1 and 4. Furthermore, decreased mean pup weights were noted at 1000 mg/kg on PND 1 (statistically significant for combined mean weights, relative to control 11%). Mean pup weights at 1000 mg/kg remained slightly lower at PND 4 and 7, but were close to control values at PND 13. Based on the combination of a decreased live birth index, a slight increase in postnatal loss and decrease mean pup weights at PND 1, a treatment-related effect on the early viability of the pups could not be excluded.
No treatment-related relevant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation index and duration, lactation indices, parturition, maternal care and the postnatal pup parameters clinical signs, anogenital distance (PND 1), areola/nipple retention (PND 13 males), serum concentration of thyroid hormone T4 (PND 13-15) and macroscopy).

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: sex ratio, live birth index, postnatal loss

Target system / organ toxicity (F1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
other: sex ratio, birth index, postnatal loss
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on decreased T4 levels (for females in combination with increased incidence of follicular cell hyperthrophy in the thyroid) at 1000 mg/kg bw/day, a NOAEL of 300 mg/kg bw/day is derived for parental toxicity.
No fertility effects were observed, therefore, the reproduction NOAEL is concluded to be at least 1000 mg/kg bw/day.
Based on a skewed sex ratio towards females, a reduced live birth index, increased postnatal loss and decreased body weights at 1000 mg/kg bw/day, a NOAEL of 300 mg/kg bw/day is derived for developmental toxicity.
Executive summary:

Wistar Han rats were treated with Ethylene glycol dibenzoate by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, polyethylene glycol 400, alone. Males were treated for 8 weeks prior to mating, during mating, and up to termination (for 92 days). Females that delivered offspring were treated for 8 weeks prior to mating, during mating, duringpost-coitum, and 14-15 days of lactation (for 93-98 days). Females without offspring were treated for 85 days (two non-pregnant females) or 96 days (one female without evidence of mating).

Formulation analysis showed that the formulations were prepared accurately and homogeneously.

Parental results

In females treated at 1000 mg/kgmicroscopic examination revealed an increased incidence of follicular cell hypertrophy (minimal or slight) in the thyroid. This was accompanied by a decrease of thyroid hormone T4 (on average 19%). In males treated at 1000 mg/kg serum levels of thyroid hormone T4 were also decreased (on average by 57%), unaccompanied by treatment related changes in thyroid weight or morphology. For both males and females no corroborative findings were observed in TSH levels. Based on the minimal or slight increase in follicular cell hypertrophy in combination with a decrease in T4 levels in 1000 mg/kg treated femalesand the magnitude of decreased T4 levels in 1000 mg/kg treated males, adversity could not be excluded.

Exposure to the test item up to 1000 mg/kg was not associated with obvious toxicity in the remaining parental parameters examined in this study (i.e. mortality, clinical appearance, functional tests, ophthalmoscopy, body weight, food consumption, clinical pathology, macroscopic examination and organ weights). Other changes noted in treated rats, mostly at 1000 mg/kg, were considered non-adverse as discussed below.

Slight salivation observed after dosing among treated animals (all dose levels) was considered a physiological response rather than a sign of systemic toxicity.

Male rats administered the test item showed reduced body weight gain from start treatment onwards, which was not accompanied by reduced food consumption. This occurred to a similar degree at 300 and 1000 mg/kg, and to a lesser degree at 100 mg/kg. The resulting reduction in mean body weights was modest (about 10% from Day 36 onwards at 300 and 1000 mg/kg) and the growth rate of treated males had returned to control levels after five weeks of treatment. Therefore, the decreased body weights of treated male rats were regarded as non-adverse within the context of this study.

A further treatment-related morphological change observed in females treated at 1000 mg/kg consisted of anincreased incidence of minimal hyperplasia/hypertrophy of the urothelium of the urinary bladder. There was no inflammation or cell death involved in this process. Therefore, this minimal change was considered as non-adverse (Frazieret al., 20121).

Clinical pathology showed several changes in males treated at 1000 mg/kg: higher values for aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total bilirubin and albumin; and lower values for cholesterol. Additionally, the increased prothrombin time (PT) for males at 1000 mg/kg could be related to the decrease in the number of platelets. These changes were not accompanied byadverse anatomic pathology alterations and the mean values for these parameters in 1000 mg/kg males generally remainedwithin the range considered normal for rats of this strain and age (see results section). As such, these clinical pathology changes were regarded as non-adverse (Palazziet al., 20162).

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling and histopathological examination of reproductive organs).

Developmental results

A skewed sex ratio towards females was noted at 1000 mg/kg: 66% female pups versus 43% in the control group (the difference was statistically significant). With such a high proportion of female pups it could not be excluded that the skewed sex ratio was related to treatment. This was strengthened by the finding that the percentage of female pups was about 70-80% in 5/8 litters of normal size at 1000 mg/kg versus 1/9 in the control group. There were no treatment-related changes in nipple retention in male pups, anogenital distances in male and female pups, or reproductive organs of parental animals. Therefore, it was considered unlikely that the skewed sex ratio resulted from an endocrine mediated (antiandrogenic) effect. An alternative explanation for the skewed sex ratio could be selective loss of male embryos due to a sex difference in sensitivity to toxic effects of the test item. The slightly increased number of unaccounted for implantation sites noted at 1000 mg/kg (resulting in a slightly lower live litter size) might reflect higher sensitivity of male embryos but does not constitute conclusive evidence of selective loss of male embryos. 

Live birth index at 1000 mg/kg was lower compared to controls (91% versus 100%). In total, 9 pups of the 1000 mg/kg group were found dead at first litter check: 7/9 of one litter (2 male and 5 female pups) and one male pup in two other litters. Additionally, a slight increase in postnatal loss was noted: 4 pups in three litters died between PND 1 and 4. Furthermore, decreased mean pup weights were noted at 1000 mg/kg on PND 1 (statistically significant for combined mean weights, relative to control 11%). Mean pup weights at 1000 mg/kg remained slightly lower at PND 4 and 7, but were close to control values at PND 13. Based on the combination of a decreased live birth index, a slight increase in postnatal loss and decrease mean pup weights at PND 1, a treatment-related effect on the early viability of the pups could not be excluded.

No treatment-related relevant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation index and duration, lactation indices, parturition, maternal care and the postnatal pup parameters clinical signs, anogenital distance (PND 1), areola/nipple retention (PND 13 males), serum concentration of thyroid hormone T4 (PND 13-15) and macroscopy).