Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
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
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD, 422 (2016) and also based on EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (2000)
Deviations:
yes
Remarks:
However, none of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dilithium tetraborate
EC Number:
234-514-3
EC Name:
Dilithium tetraborate
Cas Number:
12007-60-2
Molecular formula:
B4Li2O7
IUPAC Name:
dilithium(1+) bicyclo[3.3.1]tetraboroxane-3,7-bis(olate)
Test material form:
solid: bulk

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The test laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
At initiation of dosing, males were 10 weeks old and weighed between 273 and 309 g and females were 13 weeks old and weighed between 198 and 233 g.The animals were allowed to acclimate to the Test Facility toxicology accommodation for 8 days prior to start of the pre-test period (females) or 8 days before the commencement of dosing (males).
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20 to 22°C with an actual daily mean relative humidity of 42 to 73%. A 12 hour light/12 hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Pelleted rodent diet was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours. It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: MOL WO M 46 medicinal white oil
Details on exposure:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item.Test item dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle.

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. The dosing formulations were stirred continuously during dose administration. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
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
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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure.
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 29 days. Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating and up to and including the day before scheduled necropsy. Females that delivered were treated for 50-56 days, i.e. 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 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 38-46 days.
Frequency of treatment:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage.
Details on study schedule:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 29 days. Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating and up to and including the day before scheduled necropsy. Females that delivered were treated for 50-56 days, i.e. 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 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 38-46 days.
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
Dose / conc.:
0 mg/kg bw/day
Remarks:
(Vehicle) MOL WO M 46 medicinal white oil
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels in this study were selected to be 0, 15, 50 and 150 mg/kg/day, based on the results of the dose range finder. 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.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- (F0-Generation ). Time schedule: On treatment Days 1-15, these clinical observations were at least conducted 1 hour ± 15 minutes after dosing. From Day 16 of treatment onwards, these clinical observations were conducted during the intervals 0-15 minutes and 1 hour ± 15 minutes after dosing

BODY WEIGHT: Yes
- (F0-Generation ). 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 terminal weight was recorded on the day of scheduled necropsy (fasted for males, non-fasted for females).

FOOD CONSUMPTION: Yes
-Food consumption was quantitatively measured 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: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottle.

FUNCTIONAL TESTS:
-Functional tests were performed on the selected 5 males during Week 4 of treatment and on 5 selected females during the last week of lactation (i.e. PND 6-13 for Groups 1-3 and PND 2-6 for Group 4) or post-coitum Day 23-26 (Group 4). These tests were started at least 1 hour ± 15 minutes after dosing, after completion of clinical observations.
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 (pre-test period), the first 14 days of treatment and during mating until evidence of copulation was observed.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for three females.
Sperm parameters (parental animals):
Parameters examined in selected males (P) and all males that failed to sire [testis weight, epididymis weight], a detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:

- Clinical observations at least once daily for all pups
-Body weights - live pups were weighed individually on PND 1, 4, 7 and 13.
- Sex was externally determined for all pups on PND 1 and 4.
-Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
-All male pups in each litter were examined for the number of areola/nipples on PND 13.
-On PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus 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 prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Which sired and failed to sire. Following completion of the mating period (a minimum of 28 days of administration). All males surviving to scheduled necropsy were fasted overnight with a maximum of approximately 24 hours before necropsy. Water was available.
- Maternal animals: Females which delivered (PND 14-16) and females which failed to deliver (with evidence of mating, post-coitum day 25-27. Dams with no surviving pups were euthanized within 24 hours after the last pup is found dead or missing

GROSS NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.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 and the number of corpora lutea was recorded in addition.

-ORGAN WEIGHTS/TISSUE COLLECTION AND PRESERVATION
See 'any other information on materials and methods incl. tables'.

-HISTOPATHOLOGY– F0-Generation
For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. All tissues as outlined in table





Postmortem examinations (offspring):
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 7-16), except for the two pups per litter selected for blood collection, were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).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.
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

Pups that died or were euthanized 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.

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.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 was 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.
Reproductive indices:
Mating index %, precoital time, fertility index %, gestation index %, duration of gestation, post implantation survival index %,
Offspring viability indices:
Live birth index%, % live males at first litter check, % live females at first litter check, viability index%, lactation index%

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical findings were noted only in females at 150 mg/kg and consisted of piloerection, mostly on one or a few days at the beginning of the lactation period, and, less frequently, hunched posture on a few days during gestation or lactation. The piloerection noted incidentally in a few females at 50 mg/kg and piloerection and hunched posture at 150 mg/kg was considered not to reflect an adverse effect on general health.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain was observed in three 150 mg/kg females between Days 17-20 of gestation and during lactation. For two females the lower weight gain at the end of gestation was related to a low number of growing fetuses (one or none). The reduced weight gain during lactation was accompanied by a marked decrease in food consumption (two of the three lactating females at 150 mg/kg had total litter loss at PND 4 or 6).
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related, non-adverse hematological changes were observed at 50 mg/kg (females only) and 150 mg/kg (both sexes). The changes at 150 mg/kg consisted of higher numbers of reticulocytes and lower hemoglobin concentration in both sexes, and increased number of platelets, higher red blood cell distribution width (RDW) and lower hematocrit and mean corpuscular hemoglobin (MCH) in females. Altered red blood cell values in 150 mg/kg females mostly occurred in the females that had a different physiological status (i.e. no healthy offspring) and shorter study duration than controls. Females at 50 mg/kg had lower values for hemoglobin, hematocrit and numbers of white blood cells (total, lymphocytes and neutrophils).
The red blood cell changes at 150 mg/kg were associated with extramedullary hematopoieses in the spleen in both sexes and increased spleen weights and macroscopic enlargement/thickening of the spleen in females. A slight increase in splenic extramedullary hematopoiesis noted in 50 mg/kg males occurred in the absence of changes in red blood cell parameters. The extramedullary hematopoiesis in the spleen was not accompanied by any degenerative changes and, therefore, regarded as non-adverse.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Non-adverse clinical chemistry changes were observed at 150 mg/kg. Cholesterol was decreased in both sexes, other changes generally occurred in only one sex (lower sodium and higher total bilirubin and bile acids in males; lower urea and higher albumin and calcium in females) or the direction of the changes was different in the two sexes (alanine aminotransferase (ALAT), total protein, potassium). The changes in 150 mg/kg females mostly occurred in the females that had a different physiological status and study duration than controls (exceptions: lower cholesterol and ALAT). The changes were regarded as non-adverse as they were not associated with adverse anatomic pathology findings and remained within physiological ranges.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Increased extramedullary hematopoiesis was present in males starting at 50 mg/kg and in females at 150 mg/kg (See above - not considered to be adverse) .Inflammatory cell infiltrate and necrosis of the heart was present in a single female (no. 75) at 150 mg/kg, which as only ocurring in one test animal is unlikely to be a treatment related effect.
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:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
No reproduction toxicity was observed up to the highest dose level tested (150 mg/kg).
No treatment-related changes were noted the reproductive parameters examined 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).

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 50 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg, most of the 150 mg/kg pups that died postnatally were cold and several of them had less milk in the stomach. Abnormal position of the limbs, lean appearance and/or scabs on hind legs were noted in the two 150 mg/kg pups that were sacrificed for humane reasons. The finding of an abnormal position of the limbs occasionally occurs and is considered a change finding. The few 150 mg/kg pups that survived until scheduled necropsy (5 of litter no. 76) were cold and had less milk in the stomach at PND 1 and/or 2.
No treatment-related clinical signs were noted among pups of females treated at 15 or 50 mg/kg. The clinical signs observed incidentally at these dose levels remained within the range considered normal for pups of this age.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The decreased postnatal survival resulted in a high number of total litter losses which occurred mostly at PND 1 and 2. Pups found dead at first litter check showed autolysis, cannibalism or absence of milk in the stomach. Many pups that died postnatally were cold and several of them had less milk in the stomach.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced pup birth weight and postnatal growth at 150 mg/kg (bw). Body weights of pups at 15 and 50 mg/kg were not affected by treatment.
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):
At 150 mg/kg, insufficient data were available for evaluation. Serum T4 results were obtained from only one male and one female PND 14-16 pup (both from litter no. 76). The T4 values in these pups were within normal limits.
Serum T4 levels in male and female PND 14-16 pups at 15 and 50 mg/kg were considered not to be affected by treatment. Note, the low number of living pups at 150 mg/kg precluded sound conclusions on possible test item-related changes in the other developmental parameters examined (anogenital distance, sex ratio, areola/nipple retention and serum concentration of thyroid hormone T4).
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 150 mg/kg, body weights of pups were reduced by about 20% at PND 1 (statistically significant; based on only 16 pups from five litters). Thereafter, body weight gain of the few surviving pups was markedly reduced. At PND 13, mean body weight of the five surviving 150 mg/kg pups (litter no. 76) was about 35% lower than that of control pups. Body weights of pups at 15 and 50 mg/kg were not affected by treatment.
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: based on reduced gestation index, marked reductions in post-implantation survival, live litter size, live birth, viability and lactation indices, a high number of total litter losses, and reduced pup birth weight and postnatal growth at 150 mg/kg.

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a combined 28 -day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422) of dilithium tetraborate by oral gavage in rats using doses 0, 15, 50 and 150 mg/kg/day, no treatment-related changes were noted the reproductive parameters examined in this study up to the highest dose level tested. The reproduction NOAEL is therefore at least 150 mg/kg.

The NOAEL (Developmental toxicity) = 50 mg/kg (bw). This is based on reduced gestation index, marked reductions in post-implantation survival, live litter size, live birth, viability and lactation indices, a high number of total litter losses, and reduced pup birth weight and postnatal growth at 150 mg/kg 
The parental NOAEL= 50 mg/kg (bw). This is based on reduced body weight gain at gestation and lactation.
Executive summary:

In a combined 28 -day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422) of dilithium tetraborate by oral gavage in rats using doses 0, 15, 50 and 150 mg/kg/day, no treatment-related changes were noted the reproductive parameters examined 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) up to the highest dose level tested ( 150 mg/kg)

Developmental toxicity was observed at 150 mg/kg.  There were no explanatory morphological findings in parental reproductive organs.  The developmental toxicity at 150 mg/kg was characterized by a reduction in the gestation index, marked reductions in post-implantation survival index, live litter size, live birth index, viability index and lactation index, and reduced birth weight and postnatal growth of pups.  

The parental NOAEL based on the outcome of this study is 50 mg/kg (bw). This is based on reduced body weight gain at gestation and lactation.