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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From October 2013 to January 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study is conducted on a read across test material. The complete read across justification is attached in section 13. The reliability of the original study is 2.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD TG 421
Deviations:
yes
Remarks:
many more endpoints were measured
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
A total of 90 Wistar Hannover (Crl:WI(Glx/BRL/Han)IGSBR) rats (45 males and 45 virgin females), at least 7 to 8 weeks old and weighing 226-250 g for males and 201-225 g for females, were ordered from Charles River Italia S.p.A., Calco (Lecco), Italy and were supplied by Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L’Arbresle Cedex, France.
After arrival, on 24 October 2013, the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was performed by a veterinarian.
An acclimatisation period of at least 2 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.
The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22 °C±2 °C and 55 % ±15 % respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring approximately 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During the mating period, animals were housed on the basis of one male to one female in polysulphone cages measuring 43x27x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese).
Each cage tray held absorbent material which was inspected and changed daily.
The males were re-caged after mating as they were before mating. After mating, the females were transferred to individual polysulphone solid bottomed cages measuring approximately 43x27x18 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Suitable nesting material was provided and changed at least 2 times a week.
Drinking water was supplied ad libitum to each cage via water bottles.
A commercially available powdered laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study. There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at RTC.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: powdered laboratory rodent diet
Details on exposure:
The test item was formulated, using powdered diet, by initial preparation of a pre-mix (using mortar and pestle) followed by dilution with further quantities of diet and mixing. The formulation was prepared at fixed concentrations of 100, 300 and 1000 ppm. Fresh diets were prepared at intervals for up to 5 days, according to the stability of the test item in the diet. Concentrations were calculated and expressed in terms of test item as supplied.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated before starting the in vivo protocol. It was based on IPC analysis of Al.
Details on mating procedure:
Matings were monogamous (one male to one female). Vaginal smears were taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plug found on the cage tray). The female was paired with the same male until positive identification of copulation occurred or 14 days had elapsed.
Duration of treatment / exposure:
Males:
Animals had access to their appropriate diets (treated or control diet, depending on treatment group) seven days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter for at least other two consecutive weeks until the day of necropsy, except for animals subjected to bleeding procedure.
Females:
Animals had access to their appropriate diets (treated or control diet, depending on treatment group) seven days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 4 post partum, (the day of sacrifice), except for animals subjected to bleeding procedure.
Frequency of treatment:
Daily in diet.
Duration of test:
Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 29 days.
Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum.
Pups: until Day 4 post partum.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
100 ppm
Dose / conc.:
300 ppm
Dose / conc.:
1 000 ppm
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, plain diet
Details on study design:
Doses was selected according to an existing study on repeated dose toxicity. Oral gavage was not feasible due to the low pH of a Phoslite IP-A solution. Full precipitation of Aluminium occurs when the pH is stabilised at the physiological pH (around 7). Higher doses in feed would have been problematic for palatability and analysis

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, approximately at the same time interval.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Ovaries and uterine content:
Ovaries: weight + microscopic examination
Fetal examinations:
After delivery:
- External examinations: Yes
- Skeletal examinations: Yes
- Sex ratio: Yes
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov.
Indices:
Females: pre-implantation loss (%) Pre-birth loss (%)
Offspring viability indices: Cumulative pup loss on Day 4 post partum: CumLossD4 (%)
Historical control data:
Yes, but not reported

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Hair loss and/or scab(s) in females at 0, 300 and 1000 ppm; piloerection and hunched posture in a single high dose female.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female from the control group was found dead on Day 23, during gestation phase.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Comparable between control and treated groups both in males and female.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Slightly higher than the expected ones for the treated males and moderately higher for the treated females.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some females dosed with 300 and 1000 ppm showed decreases of neutrophils (approximately 23 %) and monocytes (57 %). Due to the low severity, these changes were not considered adverse. In addition, a male showed moderate neutrophilia and lymphocytosis. Due to the incidence and the absence of dose-relation, this finding was considered incidental.
The statistically significant increase of prothrombin time, recorded in females receiving 100 ppm (7 %), was considered incidental.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some statistically significant fluctuations of biochemical parameters were observed in females dosed with 100 and/or 300 ppm, such as: decrease of bilirubin (64 %) and increase of glucose (63 %) in animals dosed with 300 ppm, decrease of protein (approximately 8 %) and globulin (approximately 10 %) in those receiving 100 and 300 ppm.
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, non-treatment-related
Description (incidence and severity):
Increased incidence of single or multiple depressed and red area/s in the glandular region of the stomach in some males dosed at 300 and 1000 ppm.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The histopathological changes reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under our experimental conditions.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The histopathological changes reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under our experimental conditions.
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, non-treatment-related
Total litter losses by resorption:
effects observed, non-treatment-related
Early or late resorptions:
effects observed, non-treatment-related
Dead fetuses:
effects observed, non-treatment-related
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Gestation periods were similar in treated groups and controls. All dams, with the exception of 1 control and 1 low dose dam which gave birth on Day 24 post coitum, gave birth between Days 22 and 23 post coitum.
Changes in number of pregnant:
effects observed, non-treatment-related

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Remarks:
correspondent to 95 mg/kg bw
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
Mean pup loss on Day 4 post partum was slightly higher in the high dose groups as all dams with 100 % litter loss in Groups 2 and 3 were excluded from the statistical analysis.
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Implantation, pre-birth loss data and gestation length of females: no treatment-related effects were observed for these parameters.
Litter data and sex ratios were unaffected by treatment.
Clinical signs of pups: there were no treatment-related effects.
Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.
Executive summary:

The toxic effects on rats of both sexes after repeated dosing with the test item, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptus, parturition and early lactation of the offspring were investigated in this study.

Three groups, each of 10 male and 10 female Wistar rats, received the test item in the diet at fixed concentrations of 100, 300 and 1000 ppm, corresponding to theoretical dosages of 7, 20 and 65 mg/kg/day. A fourth similarly constituted group received the untreated diet and acted as a control.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 29 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum.

To evaluate the developmental toxicity only clinical data regarding female rats different are reported in this summary:

- Implantation, pre-birth loss data and gestation length of females: no treatment-related effects were observed for these parameters.

- Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups: litter data and sex ratios were unaffected by treatment.

- Clinical signs of pups: there were no treatment-related effects.

- Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum: necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.

Conclusion

No treatment-related effects indicating systemic toxicity were observed in female animals at any of the dose levels investigated (fixed concentrations of 100, 300 and 1000 ppm, corresponding to mean achieved dose levels of 10-11, 25-31 and 95 mg/kg/day for females). No adverse effects on sexual function and fertility or in developmental parameters and lactation were observed at any of the dose levels investigated.

On the basis of the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for developmental toxicity is more than 1000 ppm (equivalent to 95 mg/kg/day).