Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 November 2014 - 02 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (1996)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test) (2000)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 1995)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): L 130
- Chemical name (IUPAC name): Hexastrontium boron pentaphosphate, europium doped
- Appearance: Faint light-green, powdery solid

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 10 weeks (males) or 11 weeks (females).
- Weight at study initiation: 294-330 g (males) and 186-223 g (females).
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
Temporary deviations from the daily mean relative humidity occurred in the room in which selected animals were kept temporarily for motor activity measurents (daily mean values were not lower than 37%). As laboratory historical data do not indicate an effect of the deviations, the study integrity was not affected.
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

- Experimental starting date: 11 November 2014 (randomization dose range finding study)
- Start treatment: 8 December 2014
- Experimental completion date: 2 February 2015 (end of in-life phase)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous carboxymethyl cellulose
Details on exposure:
- Method of formulation: Formulations (w/v) were prepared daily prior to dosing and homogenized to a visually acceptable level. No correction was made for the purity/composition of the test substance. Initially (day 1-3), formulations were prepared within 6 hours prior to dosing. From day 4 onwards, the time between preparation of the test substance formulations and dosing was kept as brief as possible. The actual time between preparation and dosing of the test substance formulations was maximally about 4 hours on days 1-3 and maximally about 2 hours thereafter.
- Storage conditions of formulations: At room temperature
- Justification for use and choice of vehicle: The vehicle was choosen based on trial formulations performed at WIL Research Europe.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analyses were conducted on a single occasion during the treatment phase (10 December 2014), according to a validated method (WIL Research Project 506993, see IUCLID section 8).
- Samples of formulations 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 85-115% of the target concentration.
- Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
- Stability of the test substance in the formulations was not determined because this was not possible with the analytical method used (inductively coupled plasma – mass spectrometry (ICPS-MS) based on strontium in the test substance). The test substance is assumed to be stable according to its structure.
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
Duration of treatment / exposure:
Males were exposed for 32 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily, 7 days/week
Duration of test:
Males: 32 days
Females: 42-56 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 10-day dose range finding study (WIL Research Project 507000). In this DRF study, 3 females were dosed with either 500 or 1000 mg/kg bw/day. No signs of toxicity were noted in this study.
- Selection of animals for selected measurements: 5 animals/sex/group were selected for functional observations, locomotor activity, clininal pathology, macroscopic examination (full list), organ weights (full list) and histopathology. Only females with live pups were selected.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals during the treatment period, this was also performed outside the home cage in a standard arena.

BODY WEIGHT:
Yes
-Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on days 1 and 4.

FOOD CONSUMPTION:
Yes
- Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on days 1 and 4 of lactation.

FOOD EFFICIENCY:
Yes. (average food consumption [per animal per day]/average body weight per cage)x1000

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: The selected males were tested during week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: Hearing ability, pupillary reflex and static righting reflex; Fore- and hind-limb grip strength; Locomotor activity.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
The following parameters were examined in F1 offspring:
- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS: Yes
If possible, defects or cause of death were evaluated.
Statistics:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on histopathology findings.
Indices:
Reproductive indices; For each group, the following calculations were performed:
- Mating index: (Number of females mated/Number of females paired) x 100
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Conception index: (Number of pregnant females/Number of females mated) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY
No mortality was observed, except for one male dosed at 1000 mg/ kg bw/ day, which died at blood sampling (death not substance-related).

CLINICAL SIGNS
No clinical signs of toxicity were noted in any of the rats.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were not affected by the treatment. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.

BODY WEIGHTS
No toxicologically relevant changes in body weights and body weight gain were noted. In males at 1000 mg/kg bw/ day lower cumulative body weight gain was noted during the mating period. The differences from control were statistically significant at days 1 and 15 of this period (-4 g and - 5 g resp.). As mean body weights of males at 1000 mg/kg bw/ day did not differ significantly from those of controls, the differences in body weight gain were considered not to be toxicologically relevant. Mean terminal body weight of females at 1000 mg/kg bw/ day was lower than that of controls (relative difference: 10%). At day 4 of the lactation period, females at 1000 mg/kg bw/ day and controls had similar mean body weights based on 7 and 10 females, respectively. The terminal body weights were determined at days 5, 6 or 7 of lactation from a selection of these females (5/group) after overnight fasting. The difference in mean terminal body weights was likely to be the result, at least in part, of the selection and fasting of the females. Therefore, this difference was considered not to reflect treatment-related toxicity.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight showed no toxicologically or statistically significant differences between treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes were observed in males. Females at 1000 mg/kg bw/ day had higher values for activated partial thromboplastin time (+ 3.8s). As this value falls within historical range, this increase was not found to be toxicologically relevant. No other toxicologically relevant changes were observed.

CLINICAL BIOCHEMISTRY
Males at 1000 mg/kg bw/ day had higher blood values for alkaline phosphatase (ALP) and inorganic phosphate (+49% and + 23% resp.), but the values of these parameters at 1000 mg/kg bw/ day remained in the normal range for male rats of this age and strain. As this effect was not accompanied by other relevant changes in other parameters or histopaythological changes, therefore these differences in ALP and inorganic phosphate were considered not to be adverse. Furthermore, at all dose levels total protein levels and albumin levels were decreased. In absence of dose-relationship and given the fact that changes were less than 10% compared to control values, the changes were not found to be adverse. In females, no toxicological relevant changes in clinical biochemistry were noted.

MACROSCOPIC EXAMINATION
There were no test item-related gross observations. Incidental findings in males consisted of several reddish foci in thymus of two rats dosed at 300 mg/ kg bw/ day and one rat with its right medial lobe grown together with its diaphragm. At 1000 mg/kg bw/ day, one animal was found to have an enlarged right side auricle, several reddish foci in the lung and several observations in liver (diaphragmatic hernia, enlarged and discoloured right medial lobe (dark red)). Several occasional observations were seen (irregular surface of glandular mucosa of the stomach and enlarged mandibular lymph nodes in one female each dosed at 300 mg/kg bw/ day, fluid in uterus (2 rats at 300 mg/kg bw/ day, 1 rat at 1000 mg/kg bw), stomach irregularities (one with irregular surface of the glandular mucosa and one with dark red foci, at 1000 mg/ kg bw/ day), reddish focus on clitoral gland and an enlarged spleen (resp 2 and 1 rats at 1000 mg/ kg bw/ day), but as there was no dose-related increase in incidence and/or severity, they were found not to be related to exposure to L 130.

ORGAN WEIGHTS
Males dosed at 1000 mg/kg bw/ day had statistically significantly lower thymus weights (absolute: -17%, -13.5% and - 29% and relative: -13.6%, -11.6% and -26%, for groups dosed at 100, 300 and 1000 mg/kg bw/ day resp.). The lower thymus weight was not associated with histological changes and within the historical control range of the laboratory. Therefore, this finding was considered not to be toxicologically relevant. Absolute and relative spleen weight was decreased related to control in all male groups (absolute: -10%, -3.8% and - 10% and relative: -6.5%, -1% and -15%, for groups dosed at 100, 300 and 1000 mg/kg bw/ day resp.). In absence of histopathological effects this effect was found to be not adverse.
Females at 100 and 1000 mg/kg had higher heart to body weight ratios than controls. These differences showed no dose-related response and were considered to be due to incidental intergroup differences in terminal body weights of the selected females. Absolute and relative mean liver weights of females were decreased compared to the control (absolute: -17.6%, -22% and -15%, relative: -10%, -15% and -4% for females dosed at 100, 300 and 1000 mg/ kg bw/ day resp.) This effect can be accounted for by one control female with a very high liver weight (12.2 g versus average of other control animals of 7.9 g) and was thus not found to be toxicologically relevant. Absolute and relative thymus weight was decreased for females dosed at 100 and 1000 mg/kg bw/ day (absolute: -20% and -22%, relative: -14% and -13% resp.) In absence of a dose-relationship and given the fact that one female was found with a relatively high thymus weight in the control group influencing the results, this effect was found to be not toxicologically relevant.
Furthermore, uterus weight (absolute and relative) was increased in all exposed groups (absolute: +11%, +2.7% and +14%, relative: +19%, +10% and +27% for females dosed at 100, 300 and 1000 mg/ kg bw/ day resp.). In both the groups exposed to 100 and to 1000 mg/ kg bw/ day the high average is related to presence of one female with a relatively higher uterine weight. Therefore, this observation has not found to be adverse. The other organ weights and organ to body weight of treated animals were similar to those of control animals.

MICROSCOPIC EXAMINATION
There were no test item-related histologic changes.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Details on embryotoxic / teratogenic effects:
There was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

REPRODUCTIVE DATA

Detection of mating was not confirmed for one female dosed at 300 mg/ kg bw/ day which had implantation sites. The mating date of this animal could not be determined. For one female dosed at 100 and one female dosed at 300 mg/ kg bw/ day, which delivered live offspring, detection of mating was not confirmed but pregnancy was confirmed by palpation. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated day 0 postcoitum. There were 3/10 couples treated at 300 mg/kg and 3/10 couples treated at 1000 mg/kg without offspring. This was within historical control limits of the laboratory and thus not found to be adverse. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring. Spermatogenic staging profiles were normal for all males examined.

The mating index, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. The numbers of litters were 10, 10, 7 and 7 at 0, 100, 300 and 1000 mg/kg, respectively. Lower numbers of corpora lutea and implantations were noted at 300 mg/kg. The differences from the control group were not statistically significant and there was no dose-related response. Therefore, these findings were not attributed to treatment.

PARTURITION/MATERNAL CARE

No signs of difficult or prolonged parturition were noted among the pregnant females, except for one female at 1000 mg/kg bw/ day which had a parturition period between one and two days. This single occurrence of prolonged parturition was considered not to be related to treatment because it was seen for only one female which had a litter of normal size and healthy pups. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

EARLY POSTNATAL PUP DEVELOPMENT

The numbers of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings. At 300 mg/kg the mean number of living pups at first litter check was statistically significantly lower than in the control group. This difference was not attributed to treatment because there was no dose-related response and values were within normal limits.

MORTALITY PUPS

At the first litter check, two pups of the control group, two pups at 300 mg/kg bw/ day and four pups (all of one litter) at 1000 mg/kg bw/ day were found dead. During lactation, one pup of the control group and one pup at 300 mg/kg bw/ day went missing, and one pup of the control group died spontaneously. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the historical range.

CLINICAL SIGNS PUPS

Pups that went missing or died during lactation showed no clinical signs. Clinical signs in surviving pups were limited to scabs on the head in one pup of a litter of the control group. This was reported to be a normal background finding in pups of this age.

BODY WEIGHT PUPS

Body weights of pups were not adversely affected by treatment. At 1000 mg/kg bw/ day mean body weights of male and female pups were higher compared to controls at days 1 and 4 of lactation. The pups in one litter at 1000 mg/kg bw/ day were heavier than those in control litters because the pups of this litter were born on day 23 post-coitum. The pup weights in the other litters at 1000 mg/kg bw/ day were within normal limits, therefore the higher mean pup weights at 1000 mg/kg bw/ day were considered not to be related to treatment.

MACROSCOPY PUPS

Surviving pups showed no macroscopic abnormalities. Incidental macroscopic findings of pups that were found dead consisted of beginning autolysis. The nature and incidence of these finding remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Applicant's summary and conclusion

Conclusions:
In an oral OECD 422 study with rats, the maternal and developmental NOAELs were determined to be at least 1000 mg/kg bw/day, based on absence of adverse effects seen at the highest dose level.
Executive summary:

An oral OECD 422 study was performed with rats according to OECD/ EC guidelines and GLP principles. L 130 was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 32 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-56 days). Accuracy and homogeneity of formulations were confirmed by analyses. No parental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/ day) as evidenced by the absence of clinical signs of toxicity or adverse changes in functional observational results, body weight (gain), food consumption, haematology and clinical biochemistry parameters, organ weights, macroscopic findings, and microscopic findings.

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). In the 300 and 1000 mg/kg dose groups, there were 7 females with living pups. At 300 mg/kg, out of ten females one was not pregnant and two had implantation sites only. At 1000 mg/kg, out of ten females two were not pregnant and one had implantation sites only. This finding was considered a chance finding and not treatment related, as the offspring at 300 and 1000 mg/kg was healthy and litter size was normal. Furthermore, the numbers of litters at 300 and 1000 mg/kg showed no dose-related response. Therefore, it was concluded that the test substance did not affect the number of females with living pups and that enough offspring was produced to assure a meaningful evaluation of possible developmental (from implantation onwards) toxicity of the test substance. It was therefore concluded that no developmental toxicity was observed up to the highest dose level tested. In conclusion, the maternal and developmental NOAELs of L 130 were determined to be at least 1000 mg/kg bw/day , based on absence of adverse effects seen at the highest dose level.