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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
July 11, 2005 - December 30, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study is conducted to an appropriate guideline and under the conditions of GLP. The reliability has been amended (from reliability 1 to 2) to reflect the use of this study for read across. Read across justification: sodium and potassium orthophosphates. The following substances are considered to be similar enough to facilitate read across for systemic toxicity endpoints: Sodium dihydrogenorthophosphate, CAS: 7558-80-7 Disodium hydrogenorthophosphate, CAS: 7558-79-4 Trisodium orthophosphate, CAS: 7601-54-9 Potassium dihydrogenorthophosphate, CAS: 7778-77-0 Dipotassium hydrogenorthophosphate, CAS: 7758-11-4 Tripotassium orthophosphate, CAS: 7778-53-2 Potassium pentahydrogen bis(phosphate), CAS: 14887-42-4 All members of the group are structurally similar ionic inorganic compounds with the anion only changing by the number of hydrogen atoms to account for changes in charge due to increase in cation numbers. Progression through the group sees an increase in cation number from one to three followed by a change in cation from sodium to potassium and again an increase in number from one to three. Both cations are group 1 alkali metals with the same ionic charge, similar chemical behaviour and both sodium and calcium are essential biological elements. Both the Na+ and the K+ cation have a similar biological function and therefore orthophosphate salts of these types are not considered to differ in their systemic toxicity profile. No members of the group are classified for acute toxicity and generally exhibited no mortalities at the classification limit. Two members of the group are classified for local effects only (i.e. skin/eye irritation) which will not have an impact on the systemic toxicity of the compounds and all are highly water soluble. Read-across between these substances has also been justified for repeated dose toxicity. .
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Potassium phosphate, dibasic (CAS No. 7758-11-4)
- Physical state: solid (messed colourless powder)
- Analytical purity: 1158906: 99.5%, 1118496:99.6%
- Impurities (identity and concentrations): See Appendix 21 for Certificate of Analysis
- Composition of test material, percentage of components: See Appendix 21 for Certificate of Analysis
- Purity test date: No data
- Lot/batch No.: 1158906, 1118496
- Storage condition of test material: Normal temperature, dehumidify
- Other: pH: 9.2

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Orient Co., Ltd
- Age at study initiation: 7 weeks
- Housing: Stainless steel wire cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C):21.5 - 25°C
- Humidity (%): 41.8 ± 68.7%
- Air changes (per hr): 10- 15 times per hr
- Photoperiod (hrs dark / hrs light): lighting: 12 hrs per day



Administration / exposure

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

PREPARATION OF DOSING SOLUTIONS: Solution of test substance was prepared daily by suspending in water for injection after measurement of expected test substances expected consumption. Stability and homogeneity analysis of prepared test substance were not conducted after agreement with sponsor.
Details on mating procedure:
Mating period was a 2 week period. Males and females were mated at a 1:1 ratio in each cage. Coitus was confirmed by dropping of vaginal plug at every morning and afternoon. Gestation day 0 was determined to be the coitus confirmation day.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
2 weeks before copulation to 4 days after parturition in female rats.
2 weeks before copulation to 2 weeks after copulation in male rats.
Frequency of treatment:
Daily (number of applications is not clearly defined in study report)
Details on study schedule:
No data
Doses / concentrations
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
22 Males and 22 Females in the control and test groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In the preliminary test (R05078-P1, R05078-P2, Biotoxtech Co., Ltd) for dose range findings it was considered that dosing of 1000 mg/kg for 7 days did not have any influence. Therefore, 1,000 mg/kg bw was selected as the dose for the main test.

- Post-exposure recovery period in satellite groups: 2 weeks after termination of administration

Positive control:
Not applicable.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day, 30 minutes after mating
- Cage side observations checked in tables 2.1-2.2 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day, 30 minutes after mating

BODY WEIGHT: Yes
- Time schedule for examinations: Female: Once a week during first administration, day 1 of dosing, gestational days 0, 7, 14, 20 and potst-partum days 0, 4 and just before necropsy.
Males: Once a week during first administration and just before necropsy

Bodyweights were not measure during mating period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Female: Food consumption, was measured once a week after first administration involved the day before administration, food and water were supplied in gestational day 0, 6, 13, 19 and postpartum day 0, 3 and residue was measured in the next day.
Male: Food consumption was measured once a week after first administration involved the day. Before administration, food and water were supplied in coitus confirm day and the day before necropsy, residue was measured in the next day.


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: before necropsy
- Anaesthetic used for blood collection: Yes (ether anesthesia)
- Animals fasted: Yes (18 hours)
- How many animals: 6 females and 6 males
- Parameters stated in table 10 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: As for haematology
- Animals fasted: Yes (18 hours)
- How many animals: 6 females and 6 males
- Parameters stated in table 11 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: collected during 3-4 hr time period in the day before necropsy.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters stated in table 9 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: start of dosing, just before mating and the day before necropsy
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity (SOP/RDT/240), grip strength and motor activity (SOP/RDT/24)

ANALYISIS OF MATING RESULT:
Mating result was calculated from observations in coitus and gestation period, parturition day.
- Mating rate (%) = (No. females confirmed / no. males used in mating) x 100
- Mating period = coitus confirm day – coitus starting day
- Gestation period = postpartum 0 day – gestational 0 day
- male fertility (%) = number of pregnant females/no. males used in mating) x 100
- female fertility (%) = (No. pregnant females/no. females confirmed after coitus) x 100
- parturition rate (%) = (no . females delivering live neonates / no. females confirmed after coitus) x 100
Postmortem examinations (parental animals):

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. (see tables 12-15)

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 16 were prepared for microscopic examination and weighed, respectively. The number of corpus lutea verum and implantations were also recorded.
Statistics:
The Levene’s test was conducted to compare the homogeneity of the variance for the data collected from the examinations of the body weight, food and water consumption, haematology, blood chemistry, organ weight, and body weight and crown lump length of neonates. If the variance is homogenous, one-way ANOVA test was performed and if the results showed significance, Dunnets T-test was conducted to find out significance. If the variance was homogeneity after repeat Levene’s test about transformed data, one-way ANOVA test was carried out and if the results showed significance, Dunnets T-test was conducted to find out significance.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
(See table 1 and Appendix 1-1, 1-2)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
(See tables 2-1, 2-2 and Appendix 1-1, 1-2, (See figures 3,4,5,6 and Tables 4, 5 and Appendix 3-1, 3-2, 4-1, 4-2)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
(See tables 2-1, 2-2 and Appendix 1-1, 1-2, (See figures 3,4,5,6 and Tables 4, 5 and Appendix 3-1, 3-2, 4-1, 4-2)
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
(See tables 12, 13, 14 and Appendix 11-1, 11-2, 12-1, 12-2, 13-1, 13-2, 14-1, 14-2 )

Reproductive function / performance (P0)

Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
Mortality was not observed in the control and treatment group.
One case of fur loss at the bilateral forelimb in a male rate in the main experiment group and one case of fur loss at the region under the left ear in a female control group rat were observed.
In addition, one case of fur loss at the bilateral forelimb and abdomen region were observed in 1 female in the main test group. There were no other clinical signs.

BODY WEIGHT AND WEIGHT GAIN
There was no difference between the control and treatment groups, apart from a significant increase (p<0.01) of the females in the main experiment group as compared to the controls on postpartum day 0.

WATER CONSUMPTION AND FOOD INTAKE (if drinking water study)
Food consumption was significantly increase (p<0.05) in the male main experiment group compared to the control group on the 28th day of administration and significantly increased (p<0.01) on the 7th day of administration and gestation day 20 in the females in the main experiment. Significant increase (p<0.05) was on the 28th day of administration in the female recovery group.
Water consumption was significantly decreased (p<0.05) in the female main treatment on the gestational days 0 and 6.

HAEMATOLOGY AND CLINICAL CHEMISTY
In haematological test results, there was no difference between the male control and male main experiment group. Haematoloigcal test result of female main experiment group showed that the total erythrocyte count (p<0.01), haemoglobin concentration (P<0.001) and haematocrit (p<0.05) showed significant decrease and mean cell haemoglobin showed significant increase of female treatment group compared to control. There was no difference between recovery control and treatment group except significant decrease (p<0.05) of mean cell haemoglobin of male treatment group.
In the results of blood biochemical tests, alanine aminotransferase showed a significant increase (p<0.05), potassium showed significant decrease (p<0.05) in the males in the main treatment group. Creatinine, total bilirubin (p<0.05), albumin (p<0.05), phosphorus (p<0.01) and calcium showed significant decreases, cholinesterase showed significant increase (p<0.05) in female main experiment group. There was no difference between the control and treatment groups in each sex recovery group

URINALYSIS
There was no change between control and treatment group.

NEUROBEHAVIOUR
There was in the observation of sensory functions through the auricle, acoustic, corneal and pupilary reflex among the control and experimental groups of both the main experiment and recovery experiment groups.
In traction, rota rod and open field test, Significant increase (p<0.05) in the female main experiment group and significant decrease (P<0.01) in the female recovery group were about the number of squares that animal moved in spontaneous locomotor activity box.

ORGAN WEIGHTS
In the absolute organ weight there was no difference between the control and treatment group except significant increase (p<0.05) of thymus compared to control in male recovery group. In the relative organ weight there was no difference between the control and treatment groups except significant decrease of the heart and liver compared to the control in the female main experimental group.
There was no significant change in the male sex absolute and relative organ weights.

GROSS PATHOLOGY
In the scheduled necropsy on the main experiment group, there was no specific finding in the control and treatment group. In the case of recovery group, there were no specific findings in the control and treatment group except unilateral yellow cyst of caudal epididymis.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no specific findings in the histopathological tests of six males and six females in the main experiment group and male and female recovery group. In the result of the histopathological tests on the case of unilateral cyst of the caudal epididymis in the recovery group, it was confirmed that the spermatic granuloma observed were cause by accidiential shock causing the epididymal duct to move to the institial tissue.

It was considered that spontaneous reference findings were observed randomly and there was no dose response relation on the cellular infiltration and microgranuloma of liver and cellular infiltration and inorganic matter permeation of the liver in control and treatment groups.
Therefore no findings relating to the test substance were observed in the recovery and treatment groups.

PRE AND POST IMPLANTATION LOSS (Table 17 and Appendix 17)
In the control and treatment group, group pre-implantation loss rates by calculation of numbers of corpus lutea verum and implantation sites were 43.59 and 42.54%. Individual pre-implantation loss rates were 39.96 and 39.99%. Group post-implantation loss rates were 6.82 and 4.76%. Individual pre-implantation loss rates were 7.77 and 4.60%.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Fertility
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Results: F1 generation

Details on results (F1)

OBSERVATIONS OF NEONATES (Tables, 18, 19, 20 and Appendix 18, 19-1, 19-2, 19-3, 19-4, 20 and Figures 7, 8, 9 and 10)
Mean litter sizes (No.) were 12.8 and 13.8, birth rates were 98.5 and 97.3%, postpartum 0 days survival rates were 100%, postpartum 4 days survival rates were 99.5% and 99.1%, sex ratio in postpartum 0 and 4 days body weight and crown lump length. There was no abnormalities about external examination of neonates in control and treatment groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
It is considered that the NOAEL for reproductive parameters of the parent animals (fertility) and the development of neonates is 1000 mg/kg bw because all animals in the control and treatment groups were successful in mating, pregnancy and parturition. No effects on neonatal development were noted.