Germanium dioxide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 June 2017- 26 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Germanium dioxide

EC Number:

215-180-8

EC Name:

Germanium dioxide

Cas Number:

1310-53-8

Molecular formula:

GeO2

IUPAC Name:

Germanium dioxide

Test material form:

solid: particulate/powder

Details on test material:

Batch number: 968
Description: White Solid (powder)
Purity: 99.9%
Storage conditions: Controlled room temperature (15-25°C, below 70% RH%)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses and face mask) for unknown materials were applied to assure personnel health and safety."
Grain size: d50: 14.3 µm
Bulk density: 0.9-1.2 kg/l
Moisture: ≤ 0.5%
Chlorine (Cl): 118 ppm
Aluminium (Al): <0.2 ppm
Calcium (Ca) <0.2 ppm
Iron (Fe) <0.2 ppm
Silicon (Si) <1ppm
solubility >4.5g/l

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes/no]: yes
- Age at study initiation: (P) males: 9-10 wks; females: 13-14 wks
- Weight at study initiation: (P) Males: 271-304 g; Females: 206-244 g
- Fasting period before study: No info
- Housing:
On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam.
The cages containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The room in which the animals were kept was documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study
- Water: Municipal tap water was freely available to each animal via water bottles
- Acclimation period: acclimate to the Test Facility toxicology accommodation for at least 5 days prior to start of the pretest period (females) or at least 5 days before the commencement of dosing (males).
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 40-58
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
First administration: 29 June 2017
End of in-life part (males): 28 Juli 2017
End of in-life part (females): 17 August 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared daily as a solution (Groups 1: vehicle and 2: 10mg/kg/day) or suspension (Groups 3: 30 mg/kg/day and 4: 100 mg/kg/day) and dosed within 6 hours adding the vehicle to the test item. Adjustment was made for density of the test item. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item. 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.

VEHICLE: 1% carboxymethyl cellulose
- Justification for use and choice of vehicle (if other than water): carefully tested in a preceeding study 'Development and Validation of an Analytical Method for the Analysis of Germanium dioxide in Vehicle including Formulation Analysis'

Details on mating procedure:

After 14 days 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.
For one couple of Group 2 (male no. 11, female no. 51), detection of mating was not confirmed in first instance. Therefore, it remained housed together until the end of the mating period of 14 days. Retrospectively, 16 July 2017 (i.e. mating Day 4) was taken as the date of mating, based on the results from the estrous cycle determination indicating sperm in the vaginal lavage on Days 4 and 5 of the mating period,.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration Analysis:
Duplicate sets of samples for all groups (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of target concentration.
Homogeneity Analysis:
Duplicate sets of samples for Groups 2 and 4 (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days.
Males were treated for 29 days, up to and including the day before scheduled necropsy, i.e. 14 days prior to mating and during the mating period.
Females were treated for 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy (females with offspring: 50-56 days (most females) or 61/64 days (two females); females without offspring: 43 days). The first day of dosing was designated as Day 1.

The spare male that replaced the original male no. 32 (Group 4) on Day 2 received its first dose on Day 2. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.

Female nos. 41, 47 (Group 1), 64 (Group 3), 73 and 79 (Group 4), were not dosed on one occasion as these females were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.

One control male (no. 2) inadvertently received two doses of the vehicle on Day 11 of the pre-mating period. This was considered not to have affected the outcome of the study

Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube.

Frequency of treatment:

once daily oral gavage 7 days a week for a minimum of 28 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately
- Rationale for animal assignment (if not random):
The dose levels were selected based on the results of a 28-day oral (gavage) dose range finding study with Germanium Dioxide in the rat (30, 100, 300 mg/kgbw/day) (Test Facility Study No. 516776- Results not included here) and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
During the dosing period, these observations were performed at least at 1 hour (± 30 min) after dosing (based on the peak effect of occurrence of clinical signs observed in the dose range finder (Test Facility Study No. 516776, see Appendix 6).
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; Food consumption was quantitatively measured weekly, except for males and females which were housed together for 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 AND COMPOUND INTAKE (if drinking water study): No data: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
:

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females

Sperm parameters (parental animals):

Parameters examined in male parental P generation (control group and highest dose group (100 mg/kg/day):
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplusn pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable..

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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of administration).
- Maternal animals which delivered: All surviving animals on PND 14-16
- Maternal animals which failed to deliver (no 74 and 78): with evidence of mating: post-coitum day 26

GROSS NECROPSY
- Gross necropsy consisted of external and internal macroscopic examination for any abnormalities of pathological changes with special attention to reproductive organs
- The number of implantation sites were recorded. In case no macroscopically visible implantation sites are present, nongravid uteri will be stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea will be recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS
tissues prepared for microscopic examination and weighed:
All animals: Epididymis, thyroid gland, gross lesions/masses, ovaries, and testes.
Males that failed to sire and females that failed to delivery pups: Cervix, coagulation gland, prostate gland, seminal vesicles, uterus and vagina


organ weights: epididymis, gland, coagulation, gland, parathyroid gland, prostate gland, seminal vesicle gland, thyroid testes

Postmortem examinations (offspring):

SACRIFICE
- Unscheduled Deaths - F1-Generation
Pups that died before scheduled termination were examined externally and sexed (both externally and internally).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.
- Scheduled Euthanasia - F1-Generation
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 13-15. Sex was determined both externally and
internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter and the
thyroid from two pups per litter (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.

GROSS NECROPSY
- Gross necropsy consisted of external macroscopic examination for any abnormalities of pathological changes with special attention to reproductive genitals to examine signs of altered development

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 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 matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Reproductive indices:

Mating (%): (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 with living pups on Day 1/Number of pregnant females) x100
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 generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All females treated at 100 mg/kg showed pale feces, starting in the fifth treatment week and lasting for nearly two weeks. In addition, two 100 mg/kg females showed piloerection on a few days in the fifth treatment week. Based on its incidental occurrence, the latter finding was considered not to be toxicologically relevant.
No clinical signs were noted in males up to 100 mg/kg.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.

One male of Group 4 (no. 32) was sacrificed for humane reasons on Day 2 of treatment and subsequently replaced by a spare male. Findings in this animal included an emaciated appearance (consistent with its low body weight), swollen abdomen, enlarged kidneys with irregular surface (bilateral), and gross lesions of the adrenal gland and renal lymph node (unilaterally). Considering the time of occurrence (body weight was already low prior to initiation of treatment on Day 1), this condition was regarded unrelated to treatment with the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight (gain) at 100 mg/kg in both sexes (about 10% lower compared to controls). No treatment related changes in body weights or body weigth gain were noted in rats treated up to 30 mg/kg. The statistically significant differences noted in 10 mg/kg males (lower weight gain on Days 1 and 8 of the mating period) were considered unrelated to treatment due to the lack of similar changes at the next higher dose level; moreover the difference from controls was slight.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced food consumption at 100 mg/kg in both sexes: during the pre-mating period (about 10% in both sexes), post-coitum (about 20%) and lactation (most markedly, about 25%, between lactation Days 1-4)

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):

Thyroid hormone analyses: serum levels of T4 in F0-males were considered not to be affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A test item-related microscopic finding was noted in the thyroid gland of females treated at 100 mg/kg
Vacuolation of the colloid in the follicles of the thyroid gland was present in 5/10 females at 100 mg/kg at a minimal or slight degree. This thyroid gland finding was not a clear correlate for the increased thyroid weight noted in 100 mg/kg females because the amount of colloid in the follicles appeared normal and vacuolation in itself will not automatically increase the thyroid gland weight.

There were no other test item-related histologic changes. 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:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to be affected by treatment. All females had regular cycles, mostly of four days.
Extended di-estrus during pairing occurred in one female at 30 mg/kg (no. 67) and one female at 100 mg/kg (no. 71). Both females had normal litters. These findings were considered to be unrelated to treatment due to their incidental occurrence and lack of a dose-related trend.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

There were 2/10 couples at 100 mg/kg that failed to deliver healthy pups (female/male nos. 74/34 and 78/38). Female no. 74 showed an implantation site in the uterus at necropsy, and the uterus of female no. 78 contained one dead fetus. Histopathology did not reveal any changes in the reproductive organs that could explain the lack of healthy offspring for these two couples.
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.

Mating index: was not affected by treatment. All females showed evidence of mating.
Precoital time: was not affected by treatment. Most females showed evidence of mating within four days.
Number of implantation sittes: The mean number of implantation sites was statistically significantly decreased at 100 mg/kg (8.5 versus 12.2 in controls). This was at least in part due to two 100 mg/kg females that had only one implantation site each. The mean number of implantation sites of the remaining 8/10 females was 10.4, which is still at the lower end of the available historical control range. (Historical control range implantation sites: mean ± SD: 12 ± 3; n= 387 (period: 2015-2017))
Fertility index: was not affected by treatment. All mated females were pregnant (fertility index 100% for all groups).

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

reproductive performance

Remarks on result:

other: reproduction NOAEL is based on a reduced number of implantation sites at 100mg/kg bw/d

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

uterus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were noted among pups of females treated with the test item.
The clinical signs observed among pups of the control group (mostly alopecia) remained within the range considered normal for pups of this age.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No death pups at first litter check or thereafter.
Except the two pregnant 100 mg/kg females which delivered no live offspring had only one implantation (the uterus of one of these females contained a dead fetus). These unsuccessful pregancies were considered to be related to the low number of implantations rather than a direct effect of the test item on the female's ability to maintain pregancy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

When compared to the concurrent control group, mean body weights of male and female pups in the 100 mg/kg group were statistically significantly lower on PND 13 (by 10 and 11%, respectively). Body weights of these pups were also lower on PND 4 and PND 7 (on average by 8 and 10%, respectively; statistical significance was achieved only for male pups and combined male and female pups on PND 7). At birth (PND 1), body weights of 100 mg/kg pups were close to control values (mean weights were 4% and 3% lower in male and female pups, respectively).

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 not to be affected by treatment.

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 macroscopic findings were noted among pups of females administered the test item that were considered to be related to treatment.
Findings in pups of treated females were limited to situs inversus in a surviving male pup of the 30 mg/kg group (pup 1/litter 61), selected for collection of the thyroid gland. Situs inversus is an incidental background finding in this strain of rats. As situs inversus was present in only one pup at 30 mg/kg and was not observed at the next higher dose level, it was considered to be unrelated to treatment.
The macroscopic findings among pups of the control group (mostly alopecia) remained within the range considered normal for pups of this age.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this reproduction/developmental toxicity screening test, the following No-Observed-Adverse-Effect levels (NOAELs) of Germanium Dioxide were established:
Parental NOAEL: 30 mg/kg, based on reduced body weight (gain) and food consumption at 100 mg/kg in both sexes.
Reproduction NOAEL: 30 mg/kg, based on a reduced number of implantation sites at 100 mg/kg.
Developmental NOAEL: 30 mg/kg, based on reduced pup body weight gain at 100 mg/kg.

Reduced number of implantation sites at 100mg/kg- 2 on 10 pregnant females have only 1 implantation site while average is 12 +/-3. this effect effect is seen as very exceptional and considered treatment related. However, histopathology did not reveal any changes in the reproductive organs that could explain the lack of healthy offspring for these two couples.

Executive summary:

The objective of this study was to evaluate the potential of Germanium Dioxide, when given orally by gavage for a minimum of 28 days to Wistar Han rats, to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No-Observed-Adverse-Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 10, 30 and 100 mg/kg/day, based on the results of the dose range finder. The study design was as follows:

Group No°	Test item identification	dose level (mg/kg/day)	dose volume (mL/kg)	dose concentration (mg/mL)	number of animals		animal numbers
					Males	Females	Males	Females
1	-	0 (vehicle) ^a	5	0	10	10	01 -10	41 -50
2	Germanium dioxide	10	5	2	10	10	11 -20	51 -60
3	Germanium dioxide	30	5	6	10	10	21 -30	61 -70
4	Germanium dioxide	100	5	20	10^b	10	31 -40	71 -80

a The vehicle was 1% aqueous carboxymethyl cellulose.

b Male no. 32 was euthanized in extremis on treatment Day 2 due to non-test item-related health issues and was replaced by a spare male which became male no. 32.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters were evaluated in this study: mortality/moribundity, clinical signs,

body weight and body weight gain, food consumption, estrous cycle length and regularity, serum level of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights (thyroid and male reproductive organs) and histopathologic examination (thyroid and reproductive organs).

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 13-15 pups, and macroscopy).

Accuracy and homogeneity of dosing formulations were confirmed by analyses.

Parental results:

Females treated at 100 mg/kg showed pale feces during a period of about two weeks, starting in the fifth treatment week. Based on its transient occurrence, the pale feces was considered a non-adverse change within the context of this reproduction/ developmental toxicity screening test.

Reduced body weight gain was observed at 100 mg/kg in males throughout the treatment period and in females up to the first week of the lactation period. Several 100 mg/kg females showed weight loss, generally slight, during the pre-mating period and the first week of lactation. At the end of the treatment period, mean body weights for both sexes at 100 mg/kg were about 10% lower compared to controls. This effect on body weight (gain) was regarded as adverse based on the magnitude of the lower body weight (about 10%) in combination with (slight) weight loss in females.

The reduced body weight gain at 100 mg/kg was accompanied by reduced food consumption during the pre-mating period (about 10% in both sexes), post-coitum (about 20%) and lactation (most markedly, about 25%, between lactation Days 1-4).

Vacuolation of the colloid in the follicles of the thyroid at a minimal or slight degree was observed in females at 100 mg/kg. The low degree of this vacuolation in absence of any additional morphologic alteration was considered to be a non-adverse microscopic finding. Additionally, females at 100 mg/kg showed a modest, statistically significant increase in the weight of the thyroid (relative to body weight). A relation to the observed vacuolation of the colloid is unlikely. In the absence of a clear microscopic correlate, the increase in thyroid weight was regarded as non-adverse.

Reproductive results

The number of implantation sites was statistically significantly decreased in females treated at 100 mg/kg (mean 8.5 versus 12.2 in controls). This was at least in part due to two 100 mg/kg females that had only one implantation site each. The mean number of implantation sites of the remaining 8/10 females was 10.4, which is still at the lower end of the available historical control range (Historical control range implantation sites: mean ± SD: 12 ± 3; n= 387 (period: 2015-2017))

Developmental results

The gestation index for the 100 mg/kg group appeared lower than that for the other groups (80% versus 100%). The two unsuccessful pregnancies (i.e. failure of delivery) at 100 mg/kg were probably related to the low number of implantations (only one) for both females. Body weight gain of male and female pups was reduced at 100 mg/kg, resulting in about 10% lower mean body weights on PND 13. At birth, body weights of 100 mg/kg pups were not significantly affected (mean weights were 3% lower).

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No-Observed-Adverse-Effect levels (NOAELs) of Germanium Dioxide were

established:

Parental NOAEL: 30 mg/kg, based on reduced body weight (gain) and food consumption at 100 mg/kg in both sexes.

Reproduction NOAEL: 30 mg/kg, based on a reduced number of implantation sites at 100 mg/kg.

Developmental NOAEL: 30 mg/kg, based on reduced pup body weight gain at 100 mg/kg.