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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP and guideline
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and guideline
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 weeks
- Weight at study initiation: 254.2 - 297.8 g (males) and 182.7 - 208.2 g (females)
Route of administration:
oral: gavage
Duration of treatment / exposure:
35 days for male animals and 41-45 days for female animals
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control

Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
60
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
- Clinical observations performed and frequency: Clinical symptoms were observed once a day but were observed once a week in detail; a death rate was observed twice a day; and body weight was observed once a week and just before the necropsy, but in case of pregnant females, it was measured on the day 0, 7, 14, 20 of gestation period, date of delivery, and 4 days after the delivery; consumption rate of fodder was observed once a week except mating period.

Tests for sensory organ and reflex action: 5 animals were randomly selected from each test group. Both preyer reflex test and corneal reflex test were performed before necropsy and during lactation for males and females, respectively.

Behaviour test: 5 animals were randomly selected from each test group to do grip strength test in terms of behaviour test. This test was performed before necropsy and during lactation for males and females, respectively.

- Haematological and biochemical test of blood: randomly selected 5 male and female animals from each test group were fasted a day before necropsy for both tests. Animals were anesthetized using ether and cut the abdomen open to collect blood. In case of the haematological test, blood coagulation preventative chemicals for the test of blood coagulation and the calculation of blood-corpuscles were 3.2 % sodium citrate and EDTA- 2K, respectively. On the other hand, blood coagulation preventative chemical was not used for the biochemical test, but gathered blood was left itself in the room temperature then the sera were separated using a centrifuge. For haematological test, 6 following items were measured; Haematocrit, hemoglobin concentration, erythrocyte count, total and different leucocyte count, platelet count, prothrombin time, and active partial thromboplastin time. For biochemical test of blood, eleven following items were measured; sodium, potassium, glucose, total cholesterol, blood urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, aspatate aminotransferase, and total bilirubin.


Organs examined at necropsy: Organ weight: testes, epididymider (all males) liver, kidney, adrenals, thymus, spleen, brain and heart (5 male and female animals from each test group).

Fixation: 22 kinds of tissues were fixed to do histopathologic tests such as testes, epididymides, ovaries, accessory sex organs for all animals, brain (including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including peyer’s patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea, lungs, uterus, urinary bladder, lymph nodes (cervical mesenteric), peripheral nerve (sciatic or tibial), and bone marrow.




























Statistics:
Statistical decision tree, but in case of recovery group, either two-side Students t-test or two-side Apsin Welch t-test was used. In case of categorical data, two-sided Fishers exact test was used.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality: There was one death at day 8 for male, and each death on the day 7 and 14 for female in the treatment group of 300 mg/kg/day. These were occurred during the administration process, so it did not have relationship with test substance.

Body weight: In male groups, temporarily, a case of diminishment in the amount of body weight change was observed at week 2 within the
control group in which some clinical signs were observed such as damage to esophagus. Body weight loss for female animals was observed several times during lactation period in every treatment group including the control group. However, these were occurred temporarily because of the lactation.

- Clinical signs: In male control group, a case of salivation and bloodylike secretion was observed on the day 11 and 12. In the 1,000 mg/kg/day treatment group, a case of depilation, dcab and pus was observed on the left cheek between the day 25 and the closing day. However, the frequency of occurrence was low and no dose-response correlation. Thus these symptoms were not influenced by test substance. In female control group, a case of genitalia bloody-like secretion was observed at day 29. In the 100 mg/kg/day treatment group, each case of hypoactivity and depilation was observed on the day 8 and 9, and between day 44 and the closing day, respectively. However, these symptoms were disappeared in short, thus these did not have relationship with test substance.

Amount of fodder consumption: No crucial difference between the treatment group and the control group was observed for both male and female animals during test period. For recovery group, no significant change was observed within themselves.

Test of reflex action: Five male and female animals were randomly selected from each test group, in which no specific reaction was observed.

Grip strength test: For male animals, 6 animals were left out from the treatment group. For female animals, 5 animals were left out from the control group, and the treatment group. All things being considered, there was no dose-response correlation and was no illness at the related organs such as the cerebellum and muscle.

Organ weight: Both absolute weight of the liver and the left kidney were increased at the recovery group with administration of 1,000 mg/kg/day as compared with that of the control group within the recovery group. There was no histopathological illness at the organs, so increased organ weight did not have relationship with test substance.

Necropsy opinions: For male animals, in the control group within the recovery group, a case of left and right caput epididymis cyst was observed and the 1,000 mg/kg/day recovery group had symptom of right caput epididymis cyst. However, its frequency of occurrence was low and it was even observed at the control group within the recovery group, so it did not have relationship with test substance. For female animals, in the 300 mg/kg/day treatment group, each animal was dead on the day 7 and 14 and; each case of lung dark-red discolouration was observed, but white particle in a lobe of the lung was observed just from one of carcasses. A case of spleen white nodule was observed for an animal in the 300 mg/kg/day treatment group. There was a case of right adrenal gland white spots at the 1,000 mg/kg/day treatment group. In the control group within the recovery group, each case of right adrenal gland hemorrhagia and atrophy and liver adhesion with diaphragm was observed.

Analysis of haematological test of blood: In the 1,000 mg/kg/day male recovery group, segments were increased (p < 0.05) in contrast with that of the control group within the recovery group. Prothrombin time (PT) was decreased in the 1,000 mg/kg/day female recovery group in comparison with that of the control group within the recovery group. However, these symptoms were not observed in the definitive test groups, so these symptoms were not influenced by test substance. In addition, level of WBC (white blood cell) was increased (p < 0.001) in the 100 mg/kg/day female treatment group in contrast with that of the control group. However, its increased value was in the normal range and no dose-response correlation, so it did not have relationship with test substance.

- Analysis of biochemical test of blood: In the 100 mg/kg/day treatment group for male animals, BUN (Blood urea nitrogen) was decreased as compared with that of the control group. The male treatment groups with both administration of 300 mg/kg/day and the 1,000 mg/kg/day decreased in TP (Total protein), ALB (Albumin), AST (Aspatate aminotransferase), ALT (Alanine aminotransferase), BUN (Blood urea nitrogen), CREA (Creatinine), Na (Sodium), TCHO (Total cholesterol), and Cl (Chloride) as compared with those of the control group. In case of the 1,000 mg/kg/day male recovery group, the value of AST was decreased significantly in contrast with that of the control group within the recovery group.
No significant difference was found at every test item between female control and treatment group. The female recovery group with administration of 1,000 mg/kg/day decreased in AST in contrast with that of the control group within the recovery group, but TCHO and GLU (Glucose) were increased. In fact, decreased values of AST and ALT could be no toxicological effects. In addition, the changed values of AST, TCHO, and GLU in this
test were in the normal range and no histopathological opinion in terms of related organs, so these changed values were not influenced by test substance. However, decreased values of TP, ALB, BUN, and CREA were possibly influenced by excretion process or metabolism of test substance in relation to the kidney, and these symptoms were possibly recovered in 2 weeks from reversible effects.

- Histopathology: For male animals, in the control group, each case of heart focal inflammatory cell infiltration, submadibular lymph node blood absorption, liver mononuclear cell foci, and adrenal gland cortical vacuolation was observed. In the 300 mg/kg/day treatment group, two cases of pancreas vacuolation, and a case of liver mononuclear cell foci were observed. In the treatment group with administration of 1,000 mg/kg/day, there were three cases of liver mononuclear cell foci; and a case of heart focal inflammatory cell infiltration was found.

For female animals, in the control group, two cases of liver mononuclear cell foci, a case of kidney cortical scaring, and a case of pancreas vacuolation were observed. In the treatment group with administration of 100 mg/kg/day, one case of esophagus submucosal gland proliferation was observed. In the 300mg/kg/day treatment group, a case of trachea submucosal gland proliferation was observed. In the 1,000 mg/kg/day treatment group, each case of pancreas vacuolation and liver mononuclear cell foci was observed. However, these symptoms for both sexes were just subtle level and were occurred spontaneously, so there was no significant difference between the treatment group and the control group.















































Dose descriptor:
NOAEL
Effect level:
79 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
calcium sulfate anhydrous
Sex:
male
Basis for effect level:
other: see remarks
Dose descriptor:
LOAEL
Effect level:
237 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
calcium sulfate anhydrous
Sex:
male
Basis for effect level:
other: The LOAEL for calcium sulfate dihydrate was determined to be 300 mg/kg/day for males. The value has been corrected for calcium sulfate anhydrous
Dose descriptor:
NOAEL
Effect level:
790 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
calcium sulfate anhydrous
Sex:
female
Basis for effect level:
other: No effects were observed in females.
Remarks on result:
other: The NOAEL for calcium sulfate dihydrate was 1000 mg/kg/day and has been corrected for calcium sulfate anhydrous
Critical effects observed:
no
Conclusions:
The study was performed with calcium sulfate dihydrate and the results calculated for calcium sulfate anhydrous.

The NOAEL for calcium sulfate anhydrous was 79 mg/kg/day for males based on a significant decrease in TP (Total Protein), ALB (Albumin), BUN (Blood Urea Nitrogen), and CREA (Creatinine) at the 300 mg/kg b.w./day and 1,000 mg/kg b.w./day groups. No effects were observed in females.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Calcium sulfate, dihydrate
- Analytical purity: 99.9%
- Lot/batch No.: Sigma Aldrich Corporation, LOT No. - 109H0166

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 weeks
- Weight at study initiation: 254.2 - 297.8 g (males) and 182.7 - 208.2 g (females)

Administration / exposure

Route of administration:
oral: gavage
Details on mating procedure:
- M/F ratio per cage: 1:1 (from same test group)
- Length of cohabitation: 4 days
- Proof of pregnancy:sperm in vaginal smear
Duration of treatment / exposure:
Premating exposure period of 2 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
60
Control animals:
yes, concurrent no treatment
Details on study design:
A pre-treatment test was conducted with 0, 125, 250, 500 and 1,000 mg/kg/day of test substance for 7 days to determine the appropriate starting dose level.

Examinations

Parental animals: Observations and examinations:
Clinical observations performed and frequency: Clinical symptoms were observed once a day but were observed once a week in detail; a death rate was observed twice a day; and body weight was observed once a week and just before the necropsy, but in case of pregnant females, it was measured on the day 0, 7, 14, 20 of gestation period, date of delivery, and 4 days after the delivery; consumption rate of fodder was observed once a week except mating period.







Litter observations:
Observation of F1:
The number of survivors and deaths during delivery
Body weight and Survival rate: measured on the day 0 and 4 after the delivery
Postmortem examinations (parental animals):
A number of implantation and corpus luteum: while female animals were necropsied, the number of corpus leteum and implantation were
counted; and the former was measured in the ovary and the latter was measured in the uterus.

Organ weight: testes, epididymider (all males) liver, kidney, adrenals, thymus, spleen, brain, and heart (5 male and female animals from each test group).

- Fixation: 22 kinds of tissues were fixed to do histopathologic tests such as testes, epididymides, ovaries, accessory sex organs for all animals, brain (including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including peyer’s patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea, lungs, uterus, urinary bladder, lymph nodes (cervical mesenteric), peripheral nerve (sciatic or tibial), bone marrow.






Statistics:
Statistical decision tree, but in case of recovery group, either two-side Students t-test or two-side Apsin Welch t-test was used. In case of categorical data, two-sided Fishers exact test was used.

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

Pregnancy and delivery: There was no significant difference between the treatment groups and the control group in terms of delivery and; the number of corpus luteum and implantation. Some animals in the test groups including the control group had quite higher loss rate of the embryo prior to the implantation and the fetus after the implantation but significant difference was not observed between the treatment group and the control group. These higher loss rate were occurred spontaneously and no dose-response correlation, thus no influence under test substance.


Index of copulation, fertility and gestation: Every test group including control group was succeeded in the mating, but each animal from every group was not succeeded in the gestation. However, all pregnant female animals were succeeded in the delivery. Therefore, no significant difference between the treatment group and the control group was found.

- Clinical signs: In male control group, a case of salivation and bloodylike secretion was observed on the day 11 and 12. In the 1,000 mg/kg/day treatment group, a case of depilation, dcab and pus was observed on the left cheek between the day 25 and the closing day. However, the frequency of occurrence was low and no dose-response correlation. Thus these symptoms were not influenced by test substance. In female control group, a case of genitalia bloody-like secretion was observed at day 29. In the 100 mg/kg/day treatment group, each case of hypoactivity and depilation was observed on the day 8 and 9, and between day 44 and the closing day, respectively. However, these symptoms were disappeared in short, thus these symptoms did not have relationship with test substance.


Necropsy opinions: For male animals, in the control group within the recovery group, a case of left and right caput epididymis cyst was observed and the 1,000 mg/kg/day recovery group had symptom of right caput epididymis cyst. However, its frequency of occurrence was low and it was even observed at the control group within the recovery group, so it did not have relationship with test substance. For female animals, in the 300 mg/kg/day treatment group, each animal was dead on the day 7 and 14 and; each case of lung dark-red discolouration was observed, but white particles in a lobe of the lung was observed just from one of carcasses. A case of spleen white nodule was observed for an animal in the 300 mg/kg/day treatment group. There was a case of right adrenal gland white spots at the 1,000 mg/kg/day treatment group. In the control group within the recovery group, each case of right adrenal gland hemorrhagia and atrophy and liver adhesion with diaphragm was observed. However, their frequencies of occurrence were low and no dose-response correlation, so these did not have relationship with test substance.













Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
790 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The result has been corrected to calcium sulfate anhydrous

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

Details on results (F1)

Number of pups born, viability and sex ratio: No significant difference was observed between the treatment group and the control group at the time of the delivery and on the day 4 after the delivery. There were reconfirmed of sex ratio at the day 7 after the delivery since total 8 cases of sex were decided again. For instance, each 2 cases of misconfirmation of sex was found at the following three treatment groups such as 100, 300, 1,000 mg/kg/day as their sexes were replaced from male to female; and each case of mis-confirmation of sex was found at the 300 mg/kg/day and 1,000 mg/kg/day, for their sexes were replaced to male.(refer to table 1 for results)


Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
790 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The result has been corrected for calcium sulfate anhydrous

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Table 1:

DOSE: (mg/kg)

0

100

300

1000

No. of mated males

Copulation index (%)

Fertility index (%)

No. of mated females

Copulation index (%)

Fertility index (%)

Gestation index (%)

No. of corpora lutea

Mean ± S.D

No. of implantations

Mean ± S.D

Mean % preimplantation loss

No. of embryo/fetal death

No. of live pups born

Mean ± S.D

Mean pregnancy period (day)

Viability index on day at birth(%)

Viability index on day 4 pp (%)

Body weights of pups (g)

Male (at birth)

4 DAY

Female (at birth)

4 DAY

12

100.0

91.7

12

100.0

91.7

100.0

17.2

2.6

15.1

2.5

11.6

1.5

13.5

2.2

21.8

99.0

98.0

 

6.49

9.78

6.19

9.44

12

100.0

91.7

12

100.0

91.7

100.0

17.0

3.4

13.6

4.1

20.2

1.0

12.6

4.0

21.7

99.4

98.3

 

6.42

9.86

6.06

9.13

10

100.0

90.0

10

100.0

90.0

100.0

17.4

3.5

15.7

1.9

9.0

0.7

15.0

1.8

22.0

98.0

97.8

 

6.56

9.70

6.23

9.37

12

100.0

91.7

12

100.0

91.7

100.0

17.2

1.9

15.4

4.3

10.3

0.7

14.6

4.5

22.0

100.0

97.6

 

6.55

9.52

6.26

8.74

Applicant's summary and conclusion

Conclusions:
According to the results of the reproductive toxicity test the NOAEL was the highest dose tests (1000 mg/kg) which was corrected to 790 mg/kg for calcium sulfate anhydrous. Calcium sulfate was not considered to be toxic to reproduction or development