Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: combined 28-day rep. dose reproduction
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to guidelines.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550( Reproduction/Developmental Toxicity Screening Test July 200)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Molecular formula :Mg(OH)2
- Molecular weight : 58.32
-Stability in water: Stable
-Description: White Powder
-Test substance storage: At room temperature in the dark
-Stability under storage conditions: Stable
-Hygroscopic: yes store in a closed vessel
-pH: ±10 ( 10 %/H2O)
-Solubility in water: 0.00014 mol/L

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: Approximately 11 weeks
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18cm)
Mating: Females were caged together with males on a one-to-one basis in Macrolon cages(MIII type, height 18cm)
Post-mating: Males were housed in their home cage ( Macrolon cages, MIV type, height 18cm) with a maximum of 5 animals per cage. Females were individually housed in Macrolon cages ( MIII type, height 18cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages ( MIII type, height 18cm)
General: Sterilised sawdust as bedding material and paper as cage enrichment. During activity monitoring animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during activity monitoring.
- Diet: Free access to pelleted rodent diet.
- Water: Free access to tap water.
- Acclimation period: At least 5 days prior to the start of treatment.

ENVIRONMENTAL CONDITIONS
Animals were housed in a controlled environment.
- Temperature (°C): 21 ±3°C (actual range: 19.7-21.7°C)
- Humidity (%): A relative humidity of 40-70% (actual range: 34-73%)
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle ( with a maximum of 1 hour) occured due to performance of pupillary tests and/or opthalmoscopic examinations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Route=oral

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at Notox and on information provided my the sponsor.

PREPARATION OF DOSING SOLUTIONS:
- Formulations were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level.
-Storage conditions: At ambient temperature.
-Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
CHEMICAL ANALYSIS OF DOSE PREPARATIONS:
- Analyses were conducted during the treatment phase, according to a validated method. Samples of formulations were analysed for homogeneity and accuracy of preparation. 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%..
Details on mating procedure:
- M/F ratio per cage: 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.
- Length of cohabitation: Once mating had occurred, the males and females were separated.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged: Females were individually housed in Macrolon cages (MIII type, height 18cm). 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy and homogeneity were determined for formulations prepared for use during treatment.
-Duplicate samples( approx 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasksof 50ml. For determination of accuracy, samples were taken at 50% height or at 90%, 50% and 10% height. The latter set of samples was also used for the determination of the homogeneity of the samples.
The volumetric flasks were filled up to the mark with 4% aqueous HNO3. The solutions were further diluted with 4% aqueous HNO3 to obtain concentrations within the calibration range.
Duration of treatment / exposure:
EXPOSURE PERIOD
-Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Females 41, 46, 48, 49 (group 1, table 1), 53,59 (group 2, table 1), 61, 62, 68 (group3, table 1) and 76 ( group 4, table 1) were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Details on study schedule:
Male number paired with, mating date, confirmation of pregnancy, and delivery date were all recorded. Pregnant females were all examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. 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.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
other: control
Remarks:
Doses / Concentrations:
110 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
330 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
Four groups of ten male and ten female Wistar (Hans) rats were exposed by oral gavage to the test substance.
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on the results of a 10-day dose range finding study.
- Rationale for animal selection: This species and strain of rat has been recognised as appropriate for general and reproduction toxicity studies.

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily detailed clinical observations were made in all animals immediately after dosing. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating , or housed individually.

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

FOOD CONSUMPTION: Yes
- Food consumption: Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

FUNCTIONAL OBSERVATIONS:
The following tests were performed on the selected 5 animals/sex/group:
-hearing ability, pupillary reflex, static righting reflex and grip strength.
-motor activity test.
During the motor activity test, males were caged individually and females were caged with their pups. The selected males were tested during week 4 of treatment and the selected females were tested during lactation. In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 minutes.
Sperm parameters (parental animals):
Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
Litter observations:
STANDARDISATION OF LITTERS
- Each litter was examined.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Mortality/ Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined.
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.

GROSS EXAMINATION OF DEAD PUPS:
Yes, if possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: The day of necropsy for all survivng males was following completion of the mating period ( 29 days of dose administration)
- Maternal animals: The day of necropsy for females which delivered was lactation days 5-6.
GROSS NECROPSY
- All animals were subjected to macroscopic examination (including examination of the body surface, orifices and cranial, thoracic and abdominal tissues and organs and their contents), with special attentionbeing paid to reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [3] were prepared for microscopic examination and weighed, respectively.
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4( table 2)
-The additional slides of the testes of the selcted 5 males of Groups 1 and 4 to examine staging of spermatogenesis ( table 2)
-All gross lesions of all animals
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed by decapitation on lactation Day 5 or 6.
All pups were sexed and descriptions of all external abnormalities were recorded. The sromach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

Statistics:
For each group the following calculations were performed:

Mating (%) - (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.

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

The following statistical methods were used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-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 was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test was applied to frequency data.
All test were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY: No mortality occurred during the study period. No clinical signs of toxicity were noted during the observation period. Incidental findings that were noted in single females included alopecia or piloerection. 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 considered signs of no toxicological relevance.

BODY WEIGHTS: Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

REPRODUCTIVE PERFORMANCE: No toxicologically relevant effects on gestation index and duration, parturation, maternal care and early postnatal pup development ( mortality, clinical signs, body weight and macroscopy) were observed.
The gestation index was 100% for all groups and the duration of gestation was similar between control and treated groups.
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.

ORGAN WEIGHTS: No toxicologically relevant changes were noted in organ weights and organ to body weight ratios. THe significantly lower absolute thymus weight observed for females at 1000 mg/kg was considered to be due to a low weight for female no.72. Individual weights of other females of this dose group remained within the range observed among other treated females.

GROSS PATHOLOGY: Animals survivng to the scheduled necropsy were deeply anaesthetised using iso-flurane vapour and subsquently exsanguinated. All animals were subjected to a macroscopic examination with special attention being paid to the reproductive organs. Description of all macroscopic abnormalities were recorded.

HISTOPATHOLOGY : The following slides were examined by a pathologist:
-The preserved organs and tissues of the selected 5 animals/sex groups 1 and 4( see table 2)
-The additional slides of the testes of the selected 5 males of groups 1 and 4 to examine staging of spermatogenesis (see table 2)
-All gross lesions of all animals.

FUNCTIONAL OBSERVATIONS: Hearing ability,pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment.

FOOD CONSUMPTION: Food consumption before or after allowance for body weight was similiar between treated and control animals.

HAEMOTOLOGY: No toxicologically relevant changes occurred in haematological parameters of treated rats. Any statistically significant changes were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

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

CLINICAL BIOCHEMISTRY: The following statistically significant changes in clinical biochemistry parameters distinguished treated animals from control animals:
-lower total protein levels in males at 330 and 1000 mg/kg,
-lower albumin levels in males at 1000 mg/kg,
-lower calcium levels in males at 330 and 1000mg/kg.
Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain.

URINALYSIS:
The following statistically significant changes in urinary parameters distinguished treated males from control males:
-Lower sodiumexcretion (mmol/TPV) at 330 and 1000 mg/kg,
-Lower potassium excretion (mmol/TPV) at 1000 mg/kg,
-Higher calcium concentration (mmol/L) at 1000 mg/kg.

Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain. the significant higher specific gravity seen at 330 mg/kg was not considered to be toxicologically relevant as it occurred in the absence of a treatment-related trend.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
No observed adverse effect level
Effect level:
>= 1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No reproduction/developmental toxicity was observed at any dose level
Remarks on result:
other: Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

MORTALITY: Two pups of the control group, three pups at 110 mg/kg, and one pup at 330 mg/kg were found dead or missing during lactation. the missing pups were most likely cannibalised. No pups were found dead or missing at 1000 mg/kg. 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.

CLINICAL SIGNS: Incidental clinical symptoms of pups consisted of blue spot on the back and scabbing of the snout or back. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT:Body weights of pups were considered to be unaffected by treatment.

MACROSCOPY: Incidental macroscopic findings for pups that were found dead included autolysis and absence of milk in the stomach. Scabbing on the snout was noted for one surviving pup. The nature and incidence of these findings remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 4: Clinical signs Males parental animals:

 

 

 

Pre-Mating

Reproduction period

Sign (Max Grade)

 

Week

 

1

 

2

 

1

 

2

 

3

 

4

Location

day

1,2,3,4,5,6,7

1,2,3,4,5,6,7

1,2,3,4,5,6,7

1,2,3,4,5,6,7

1,2,3,4,5,6,7

1,2,3,4,5,6,7,1,2,3

 

 

 

 

 

 

 

 

Group 1 (control)

No clinical signs noted

 

-

-

-

-

-

-

Group 2 (110 mg/kg)

No clinical signs noted

 

-

-

-

-

-

-

Group 3 (330 mg/kg)

No clinical signs noted

 

-

-

-

-

-

-

Group 4 (1000 mg/kg)

No clinical signs noted

 

-

-

-

-

-

-

 

Table 5: Clinical signs Females parental animals:

 

 

 

Pre-Mating

Reproduction Period

Sign (Max Grade)

Week

1

2

1

2

3

4

Location

Day

1,2,3,4,5,6,7

1,2,3,4,5,6,7

1,2,3,4,5,6,7

1,2,3,4,5,6,7

1,2,3,4,5,6,7

1,2,3,4,5,6,7,1,2,3

 

 

 

 

 

 

 

 

Group 1 (control)

No clinical signs noted

 

 

-

 

-

 

-

 

-

 

-

 

-

Group 2 (110 mg/kg)

Skin/ fur

 Alopecia

G:

 

%:

-

 

-

-

 

-

 

-

 

-

1,1,1,1,1,1,1

 

1,1,1,1,1,1,1

1,1,1,1,1,1,1

 

1,1,1,1,1,1,1

1,1,1,1,1,1,1

 

1,1,1,1,1,1,1

Group 3 (330 mg/kg)

Skin/fur

 Piloerection

G:

 

%:

-

 

-

-

 

-

1

 

1

-

 

-

-

 

-

-

 

-

Group 4 (1000 mg/kg)

No clinical signs noted

 

 

-

 

-

 

-

 

-

 

-

 

-

Key:

G: Median value of the highest individual daily grades

%: Percent of affected animals (0= less than 5%, 1= between 5% and 15%, A= more than 95%)

- observation performed, no sign detected

Table 6: Reproduction Data

 

 

Group 1

(Control)

Group 2

(110 mg/kg)

Group 3

(330 mg/kg)

Group 4

(1000 mg/kg)

Females paired

10

10

10

10

Females Mated

10

10

10

10

Non-pregnant

0

0

0

0

Pregnant females

10

10

10

10

Females with living pups on Day 1

10

10

10

10

Mating Index (%)

(females mated/females paired) * 100

100.0

100.0

100.0

100.0

Fertility Index (%)

(Pregnant females/females paired) * 100

100.0

100.0

100.0

100.0

Conception index (%)

(Pregnant females/females mated) * 100

100.0

100.0

100.0

100.0

Gestation index (%)

(Females with living pups on Day 1/pregnant females) * 100

100.0

100.0

100.0

100.0

 

Table 7: Body weights (gram) Summary

Parental Males/Females:

 

 

 

Group 1

Control

Group 2

110 mg/kg

Group 3

330 mg/kg

Group 4

1000 mg/kg

 

 

Male

Female

Male

Female

Male

Female

Male

Female

Pre-mating

 

 

 

 

 

 

 

 

 

Day 1

Mean

314

180

312

175

319

177

317

180

Week 1

St.Dev

19.8

7.9

17.3

4.9

22.6

4.5

29.2

11.2

 

N

10

10

10

10

10

10

10

10

Day 8

Mean

337

190

334

186

342

186

337

188

Week 2

St.Dev

23.1

12.0

17.7

6.6

22.7

7.2

28.1

8.1

 

N

10

10

10

10

10

10

10

10

Mating period

 

 

 

 

 

 

 

 

 

Day 1

Mean

353

195

346

193

356

192

351

195

Week 1

St.Dev

29.0

12.2

19.1

7.5

24.9

6.5

30.1

7.6

 

N

10

10

10

10

10

10

10

10

Day 8

Mean

365

 

354

 

365

 

356

 

Week 2

St.Dev

33.1

 

21.7

 

25.1

 

32.5

 

 

N

10

 

10

 

10

 

10

 

Day 15

Mean

385

 

368

 

381

 

376

 

Week 3

St.Dev

34.2

 

22.1

 

26.9

 

34.7

 

 

N

10

 

10

 

10

 

10

 

 

Table 8: Food consumption (G/Animal/Day) Males/Females Parental:

 

 

 

Group 1

Control

Group 2

110 mg/kg

Group 3

330 mg/kg

Group 4

1000 mg/kg

 

 

Males

Females

Males

Females

Males

Females

Males

Females

Pre mating

 

 

 

 

 

 

 

 

 

Days 1-8

Mean

23

14

23

14

23

14

22

14

Weeks 1-2

St.Dev

0.0

0.4

1.9

0.2

0.2

0.3

0.5

0.3

 

N (cage)

2

2

2

2

2

2

2

2

Days 8-15

Mean

24

15

23

16

24

15

24

15

Weeks 2-3

St.Dev

0.0

0.4

1.2

0.6

0.6

0.1

0.0

1.3

 

N (cage)

2

2

2

2

2

2

2

2

Mean f Means over pre mating: mean

 

24

15

23

15

24

14

23

15

Mating Period

 

 

 

 

 

 

 

 

 

Days 1-8

Mean

26

-

26

 

26

 

27

 

Weeks 1-2

St.Dev

0.8

-

3.2

 

0.9

 

0.3

 

 

N (cage)

2

0

2

 

2

 

2

 

Days 8-15

Mean

24

 

23

 

24

 

25

 

Weeks 2-3

St.Dev

0.3

 

1.3

 

1.2

 

0.4

 

 

N (cage)

2

 

2

 

2

 

2

 

Mean of means over mating period: mean

 

25

 

25

 

25

 

26

 

Applicant's summary and conclusion

Conclusions:
No reproduction/developmental toxicity were observed at any dose level. based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was determined.
Executive summary:

A number of clinical biochemistry and urinary changes were noted at 330 and 1000 mg/kg in males which included lower total protein, albumin and calcium levels in blood, and lower sodium and potassium excretion and higher calcium concentration in urine. Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain. Moreover, there were no histopathological correlates in eg. liver or kidneys that would support these changes. Therefore, these changes were considered not to be of toxicological relevance.

Overall, no toxicologically relevant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).