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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 2016 - Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA OPPTS 870.3550
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
422-150-1
EC Name:
-
Molecular formula:
Hill formula: Mg4.3 Al2(OH)12.6 (CO3)0-0.75 (ClO4)0.5-2.0 . (0-5)H2O CAS formula: Mg4.3 Al2(OH)12.6 (CO3)0-0.75 (ClO4)0.5-2.0 . (0-5)H2O
IUPAC Name:
Aluminium-magnesium-carbonate-hydroxide-perchlorate-hydrate
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: white powder
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Details on test animals or test system and environmental conditions:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies.
Charles River Den Bosch 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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing (actual maximum time: 4 hours and 46 minutes) and were homogenized to a visually acceptable level. Adjustment was made for specific gravity/density of the test item and vehicle. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch
- Dose volume: 5 mL/kg bw
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 (09 January 2017) according to a validated method (Test Facility Study No. 511114).
Samples of formulations were analysed for homogeneity (lowest and highest 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 item in the formulations was not determined because this was not possible with the analytical method used (inductively coupled plasma – mass spectrometry (ICP-MS) based on aluminium in the test item). Due to the inorganic nature of the substance, stability measurements, based on an element in the test item, are not meaningful.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 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 was confirmed, the males and females were separated.
Duration of treatment / exposure:
Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were treated for 42-53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 4-5 days of lactation (i.e. up to the day prior to scheduled necropsy. Females which failed to deliver healthy offspring were treated for 42 or 53 days.
Frequency of treatment:
daily
Duration of test:
53 days
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

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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 and 20 post-coitum and during lactation on PND 1 and 4.

FOOD CONSUMPTION:
- Food consumption for each animal determined: 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 and 4.

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

CLINICAL BIOCHEMISTRY: yes
Blood samples were collected from all F0-animals at the end of treatment period on the day of scheduled necropsy.
Parameters determined: ALAT, ASAT, ALP, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium and inorg phosphate.

GENERAL REPRODUCTION DATA: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Cage debris of pregnant females were examined for evidence of premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
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:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
Mortality/viability:
The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective tables.
Body weights: Live pups were weighed on PND 1 and 4.
Sex: Sex was determined for all pups on PND 1 and 4.

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


Statistics:
The following statistical methods were used to analyse the data:
• 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. Test statistics were calculated on the basis of exact values for means and pooled variances.
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
Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Percentage of postnatal loss = (number of dead pups before planned necropsy/number of live pups at first litter check) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation seen after dosing among animals of the 100, 300 and 1000 mg/kg dose group from the second week of the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Incidental findings that were noted included scabbing, scales, wounds, alopecia and rales. These findings 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. At the incidence observed, these were not considered to be signs of toxicological relevance.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In females at 1000 mg/kg a trend towards slightly lower food intake (absolute and relative) was noted during lactation. As changes compared to the control group were relatively small (approximately 15%), reaching no statistically significance, and values were within normal limits, no toxicological relevance was attached to this finding.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males and females of the 1000 mg/kg dose group, alkaline phosphatase (ALP) was significantly increased. When compared to the concurrent control group, mean values were 51% (males) and 62% (females) higher.
The statistically significant higher concentrations of sodium in males at 300 mg/kg (141.7 mmol/L) and of potassium in females at 1000 mg/kg (4.29 mmol/L) were not considered to be toxicologically relevant as they occurred in the absence of a treatmentrelated distribution (sodium) and/or remained within the historical range for rats of this age and strain.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher thyroid gland weights (absolute and relative to body weights) were noted in the 100, 300 and 1000 mg/kg treated males and females as shown in the table below.
Mean Percent Thyroid Gland Weight Increases from Control Groups
Males Females
Dose level (mg/kg): 100 300 1000 100 300 1000
Thyroid Gland
Absolute 55** 68** 77** 5 21* 42**
Relative to bw 67** 83** 83** 0 25* 38**
*: P<0.05, **: P<0.01
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related enlarged thyroid glands were observed in the 10/10 males treated at 100, 300 and 1000 mg/kg and in 6/10 females treated at 1000 mg/kg. In addition, discoloration, dark red in the thyroid glands was observed in 10/10 males treated at 100, 300 and 1000 mg/kg and in 2/10 females treated at 1000 mg/kg.
The remainder of the macroscopic findings recorded in control and treated animals were within the range of background gross observations encountered in rats of this age and strain, and did not show a dose-related incidence trend.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with ALCAMIZER5 were noted in the thyroid gland of the 100, 300 and 1000 mg/kg treated males and females and are summarized in the table below.

Summary Test Item-Related Microscopic Findings – Thyroid Glands
Males Females
Dose level (mg/kg): 0 100 300 1000 0 100 300 1000
THYROID GLANDS (a) 10 10 10 10 10 10 10 10
Hypertrophy follicular cell
Minimal 8 2 - - 2 4 - -
Slight 1 5 1 - - 6 5 5
Moderate - 3 9 8 - - 5 3
Marked - - - 2 - - - 2
(a) = Number of tissues examined from each group.
An increased incidence and severity of follicular cell hypertrophy was present in the thyroid glands of males and females starting at 100 mg/kg, reaching up to marked degree.
There were no other test item-related histologic changes, including in the kidneys. 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

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Mating index was not affected by treatment. All females showed evidence of mating.
Precoital time was not considered to be affected by treatment. The majority of females mated within the first 4 days, except for two females in the high dose group that had a precoital time of 12 days. One of these two females delivered a completely normal litter. The other female was not pregnant. As comparable longer mating periods are more often seen for individual rats of this age and strain and all remaining reproductive parameters (including histopathology of the reproductive organs) were unaffected by treatment, the longer precoital time for these two females in the 1000 mg/kg dose group was not considered to be related to treatment with ALCAMIZER5.
Fertility index was not considered to be affected by treatment. A single female at 300 mg/kg and two females at 1000 mg/kg were not pregnant. This was not considered to be related to treatment, since these cases of nonpregnancy showed no dose-related incidence across the dose groups, and no histopathological changes were observed in the reproductive organs of these females and their males they had been mated with. These incidences were within the normal range of biological variation.
Numbers of corpora lutea and implantation sites in pregnant females of the control and treated groups remained within the normal range of biological variation.

Effect levels (maternal animals)

Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: increased thyroid weight and higher incidence of follicular hypertrophy in thyroid

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A trend towards slightly lower mean body weights was noted for pups in the 1000 mg/kg group (both sexes) when compared to the concurrent control group on lactation Days 1 and 4 (10% M+F). This change could not be explained by larger litters in the high dose group. As changes compared to the control group were relatively slight (reaching no statistically significance) and in the absence of any other effects on developmental parameters determined in this study (i.e. viability/mortality, clinical signs and macroscopy), it was considered non-adverse.
Description (incidence and severity):
Gestation index and duration of gestation were not affected by treatment. All pregnant females delivered live pups after 21-23 days of gestation.
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.
The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. One female at 1000 mg/kg with 13 uterine implantation sites had only one live pup at first litter check. This relatively high post-implantation loss is occasionally seen in this type of study. As the remaining litters in the high dose group had normal sizes, it was not considered to be related to treatment. Also in the control group one female was noted that had only 5 pups (3 alive and 2 dead) at first litter check compared to 13 implantation sites.
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment.
Three pups of the control group, one pup at 100 mg/kg, one pup at 300 mg/kg and two pups at 1000 mg/kg were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The number of live pups before planned necropsy compared to the number of pups born alive was not affected by treatment. One pup of the control group, two pups at 100 mg/kg and two pups at 300 mg/kg were found dead or missing. All pups born in the high dose group survived until scheduled necropsy. 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 range considered normal for pups of this age.
Sex ratio was not affected by treatment.
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)

Any other information on results incl. tables

Accuracy of preparation

A very small response was measured in the Group 1 formulation prepared for use during treatment. In the first Group 1 sample the maximum contribution to the Group 2 samples based on analysed concentration was 0.18%. The response in the second sample of Group 1 was comparable in magnitude with the response obtained in the analytical blanks. Taken together, the very small response detected in the Group 1 formulation samples was not considered to derive from the formulation, but was most probably introduced during pretreatment of the samples.

The concentrations analysed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

Homogeneity

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Applicant's summary and conclusion

Conclusions:
In an oral screening reproduction/developmental toxicity study in rats performed according to OECD 421 and GLP, the parental LOAEL was 100 mg/kg bw/d based on effects on the thyroid and the reproduction and developmental NOAEL was determined to be at least 1000 mg/kg bw/d. No indication of any adverse effects on kidney was observed (included specifically to investigate the observed effects on kidney in the previously performed 28 day repeated dose toxicity study in the same rat strain.
Executive summary:

In an oral screening reproduction/developmental toxicity study performed according to OECD 421 and GLP, rats were administered 0, 100, 300 or 1000 mg/kg bw/d of ALCAMIZER5 daily by gavage. Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were treated for 42-53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 4-5 days of lactation (i.e. up to the day prior to scheduled necropsy. Females which failed to deliver healthy offspring were treated for 42 or 53 days. The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), body weight and food consumption (at least at weekly intervals), clinical biochemistry, macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, precoital time, fertility index, numbers of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex and macroscopy).

In males and females of the 1000 mg/kg dose group, alkaline phosphatase (ALP) was significantly increased. When compared to the concurrent control group, mean values were 51% (males) and 62% (females) higher. However, in the absence of any corroborative findings in the remaining clinical biochemistry parameters and macroscopic findings at necropsy, this observation was not considered to be adverse.

Microscopic examination revealed an increase in incidence and/or severity (up to marked degree) of follicular cell hypertrophy in males and females starting at 100 mg/kg. This

change was accompanied by a substantial increase in thyroid organ weight in males at 100, 300 and 1000 mg/kg (increase in relative weight of 67%, 83% and 83%, respectively), and in females at 300 and 1000 mg/kg (increase in relative weight of 25% and 28%, respectively). At the macroscopic level, enlarged thyroid glands were observed in 10/10 males treated at 100, 300 and 1000 mg/kg and in 6/10 females treated at 1000 mg/kg. These findings in the thyroid gland were remarkable.

There were no other test item-related histologic changes. No indication of any adverse effects on kidney was observed (included specifically to investigate the observed effects on kidney in the previously performed 28 day repeated dose toxicity study in the same rat strain. No treatment-related changes were noted in any of the remaining parental parameters investigated in this study (i.e. mortality, clinical appearance, body weight and food consumption).

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, numbers of corpora lutea and implantations, spermatogenic profiling, and histopathological examination of reproductive organs).

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg). A trend towards slightly lower mean body weights was noted for pups in the 1000 mg/kg

group (both sexes) when compared to the concurrent control group on lactation Days 1 and 4. This change could not be explained by larger litters in the high dose group. As changes compared to the control group were relatively slight (reaching no statistically significance) and in the absence of any other effects on developmental parameters determined in this study (i.e. viability/mortality, clinical signs and macroscopy), it was considered non-adverse.

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 and macroscopy).

Conclusion

Treatment with ALCAMIZER5 by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg resulted in follicular cell hypertrophy in the thyroid

glands, with correlating higher organ weights and enlargement observed at necropsy, in males and females starting at 100 mg/kg. These findings in the thyroid gland were remarkable.

The increase in alkaline phosphatase (ALP) noted in males and females at 1000 mg/kg was regarded treatment-related, but not adverse. No indication of any adverse effects on kidney was observed (included specifically to investigate the observed effects on kidney in the previously performed 28 day repeated dose toxicity study in the same rat strain.

No reproduction toxicity was observed for treatment up to 1000 mg/kg.

A trend towards slightly lower mean body weights was noted for pups in the 1000 mg/kg group (both sexes). Also this finding was regarded treatment-related, but not adverse.

Based on the histologic findings in the thyroid glands, a parental LOAEL of 100 mg/kg bw/d could be established. A reproduction and developmental NOAEL of at least 1000 mg/kg was derived in this study.

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