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Repeated dose toxicity: oral

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Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 April, 1995 - 01 May, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
screening for reproductive / 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA Health Effects Test Guidelines OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
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.
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
- Age at study initiation: approx. 10 weeks for males and 11 weeks for females
- Weight at study initiation: (P) Males: 266-294 g; Females: 191-219 g
- Fasting period before study: no
- Housing: in Macrolon plastic cages; pre-mating animals were housed in groups of 5 animals/sex/cage; during mating females were caged together with males on a one-to-one-basis; post-mating males were housed in their home cage with a max. of 5 animals/cage and females were housed individually. Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material.
- 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
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 2 January 2017 To: 24 February 2017
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
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.
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.
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
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of an acute oral toxicity study in the rat (LD50 was > 2000 mg/kg) and a 28-day repeated-dose oral toxicity study in the rat (NOTOX Study No. 137745). In the latter study, dose levels of 50, 200 and 1000 mg/kg/day were tested. A No Observed Effect Level of 50 mg/kg/day was established based on microscopic findings in the kidneys at 200 and 1000 mg/kg/day. The kidneys of some male and female rats of the mid- and high dose groups showed degenerative changes which were characterized by cystic dilated tubules containing proteinaceous casts. Regenerative changes, characterized as basophilic tubules were seen in the kidneys of almost all male and female animals of the mid- and high-dose groups. Based on the very slight to slight severity of both kidney findings and in the absence of any other clear signs of toxicity, a NOAEL of 1000 mg/kg/day was concluded.
Parental animals: Observations and 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.
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
yes, spermatogenic profiling
Litter observations:
STANDARDISATION OF LITTERS : no

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.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All animals following completion of the mating period (min. 28 days administered)
- Maternal animals: PND 5-6 for females which delivered and post-coitum day 27 for females which failed to deliver, but with evidence of mating.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
According to OECD 421 (1995).
Postmortem examinations (offspring):
SACRIFICE
- day 5-6 of lactation

GROSS NECROPSY
- Gross necropsy consisted of external examinations. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

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.
Reproductive indices:
For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Precoital time = Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%) = (Number of pregnant females/Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
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
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One male at 1000 mg/kg was observed with hunched posture (3 days) or piloerection (2 days) during the mating period. In the absence of any further signs of toxicity in this male and due to the isolated occurrence of this finding, it was not considered to be adverse.
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
Description (incidence):
There were no premature decedents in the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
See under observations.
Ophthalmological findings:
not examined
Haematological findings:
not examined
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.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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):
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
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There were 1/10 couples treated at 100 mg/kg/day and 2/10 couples treated at 1000 mg/kg that were not pregnant. Histopathology did not reveal any changes in the reproductive organs that could explain this observation.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males
examined. See further under details on results.
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.
Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: increased thyroid weight and higher incidence of follicular hypertrophy of thyroid
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
See below.
Body weight and 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.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.
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.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
no

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%).

Conclusions:
In an oral screening reproduction/developmental toxicity study in rats performed according to OECD 421 and GLP, the parental LOAEL was determined to be 100 mg/kg bw/d based on thyroid effects, while the reproduction and developmental NOAEL was determined to be at least 1000 mg/kg bw/d based on the absence of effects. 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.

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.

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, no parental NOAEL could be established and the level of 100 mg/kg bw/d was considered to be a LOAEL. A parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived in this study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
(1981)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
(1992)
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 in the dark

Test animals

Species:
rat
Strain:
other: Wistar Crl:(WI)BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: males: 182 - 209 g; females: 153 - 170 g
- Fasting period before study: Overnight prior to dosing.
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors.
- Diet: Free access to standard pelleted laboratory animal diet (from Carfil Quality BVBA, Oud-Turnhout, Belgium)
- Water: Free access to tap-water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Air changes (per hr): Approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 April, 1995 To: 30 April, 1995

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily immediately prior to dosing. Adjustment was made for specific gravity of vehicle (1.036).

DOSE VOLUME: 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations, prepared after completion of the in-life phase, were analysed to check homogeneity (highest and middle concentration) and accuracy of preparations (all concentrations).
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 d/w.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 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:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of a 5-day dose range finding study. No mortality was observed in the 50, 200 and 1000 mg/kg bw. No clinical signs were noted except for 3 animals at the 200 mg/kg bw dose level which showed alopecia and scabs. Based on the results, dose levels for the main study were selected to be 50, 200 and 1000 mg/kg bw.
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from day 1 onwards. The time of onset, degree and duration were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly and on the day preceding termination, prior to overnight fasting.

FOOD CONSUMPTION
- Weekly

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m.
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes, overnight
- How many animals: all rats/sex/group
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m.
- Animals fasted: Yes, overnight
- How many animals: all rats/sex/group
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
NECROPSY:
All animals surv1v1ng to the end of the observation period (day 29) were deeply anaesthetised using ether vapour and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.

ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, Heart, Kidneys, Liver, Spleen and Testes.

HISTOPATHOLOGY: Yes
HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

HISTOPATHOLOGY
Slides of adrenals, heart, kidneys, liver, spleen, stomach and testes, collected at the scheduled sacrifice from all animals of the control and the highest dose group, and all gross lesions of all animals were examined by a pathologist.
Based on the treatment related morphologic changes, kidneys were also examined from all rats of the intermediate dose groups.
All abnormalities were described and included in the report.
Statistics:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnetttest (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-one rank test) was applied when the data could not be assumed to follow a normal distribution.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period .
There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.

FOOD CONSUMPTION
There were no differences in food consumption before or after allowance for body weight between treated and control animals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment.

CLINICAL CHEMISTRY
There were no differences noted between control and treated rats that were considered to be related to treatment with ALCAMIZER 5.

ORGAN WEIGHTS
Spleen weights and spleen:body weight ratios were increased in males receiving 1000 mg/kg when compared to control weights. The spleen weights of the control animals, however, were considered to be slightly low when compared to values in other 28-day toxicity studies with similar rats (where control group mean spleen weights varied from 0.585 to 0.689 gram). The group mean value of 1000 mg/kg treated males remained within this range of historical data. In addition, there were no findings noted in the spleen microscopically and no corroborative findings were noted in the opposite sex. Therefore, it is unlikely that this change represents a toxic effect of the test substance.
Other organ weights and organ:body weight ratios of treated animals were considered to be similar to those of control animals.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
The kidneys of some male and female rats receiving 200 and 1000 mg/kg/day showed degenerative changes which were characterised by cystic dilated tubules containing proteinaceous casts. Regenerative changes, characterised as basophilic tubules, were seen in the kidneys of almost all male and female animals receiving 200 and 1000 mg/kg/day. The severity of both findings was very slight to slight.
One male receiving 1000 mg/kg/day showed a slight inflammatory change diagnosed as pyelonephritis. This finding was considered not to be a clear sign of toxicity as it can incidentally be noted in untreated rats of this age and strain. The small number of other findings recorded are within the normal range of background alterations.

Analysis of dose preparations:
Test substance formulations in propylene glycol formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 90% to 106% of nominal. One analysis resulted in 122% when compared to the nominal concentration based on Mg.
However, this sample revealed 95% based on the analysis of Al. Therefore, the results were considered to represent an acceptable level of accuracy for formulations of this type.
The stability of the test substance in the vehicle was not determined.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on kidneys were observed in the OECD421 study, up to and including 1000 mg/kg bw/day, the highest dose tested.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a 28-day repeated dose oral toxicity study in rats performed according to OECD 407 guideline and GLP principles, the NOAEL was determined to be 1000 mg/kg bw/day.
Executive summary:

A 28-day repeated dose oral toxicity study in rats was performed with Alcamizer5 according to OECD 407 guideline and GLP principles. Based on the results of a 5-day dose range finding study, dose levels for the main study were selected to be 50, 200 and 1000 mg/kg bw/day. No mortality occurred during the study period. There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment. Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.

Spleen weights and spleen:body weight ratios were increased in males receiving 1000 mg/kg bw/day when compared to control weights. The spleen weights of the control animals, however, were considered to be slightly low when compared to values in other 28-day toxicity studies with similar rats (where control group mean spleen weights varied from 0.585 to 0.689 gram). The group mean value of 1000 mg/kg bw/day treated males remained within this range of historical data. In addition, there were no findings noted in the spleen microscopically and no corroborative findings were noted in the opposite sex. Therefore, it is unlikely that this change represents a toxic effect of the test substance. Other organ weights and organ:body weight ratios of treated animals were considered to be similar to those of control animals.

The kidneys of some male and female rats receiving 200 and 1000 mg/kg bw/day showed degenerative changes which were characterised by cystic dilated tubules containing proteinaceous casts. Regenerative changes, characterised as basophilic tubules, were seen in the kidneys of almost all male and female animals receiving 200 and 1000 mg/kg bw/day. The severity of both findings was very slight to slight.

One male receiving 1000 mg/kg bw/day showed a slight inflammatory change diagnosed as pyelonephritis. This finding was considered not to be a clear sign of toxicity as it can incidentally be noted in untreated rats of this age and strain. The small number of other findings recorded are within the normal range of background alterations.

In a recently performed OECD 421 study in the same rat strain (Wistar), additional parental parameters were included in order to study the reported minimal effects on the kidney observed in the 28-day repeated dose toxicity study. No effects on kidneys were observed in the study, up to and including 1000 mg/kg bw/day, the highest dose tested. Based on the absence of any effects on the kidneys in the same rat strain in the recently performed study, it is concluded that the previously observed minimal effects on the kidneys should not be considered adverse.

Based on the results of the study, the NOAEL was determined to be 1000 mg/kg bw/day.