reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are currently no experimental data available to assess effects on fertility for the test substance. A data waiver is set in place to justify that no further testing on reproductive toxicity is required.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Effects on developmental toxicity

Description of key information

No data are available for calcium hydrogenorthophosphate.

Reliable data is available for calcium dihydrogenorthophosphate monohydrate (CAS 10031-30-8):

Prenatal developmental toxicity (similar to OECD 414), mice: NOAELmaternal ≥ 465 mg/kg bw/day; NOAELdevelopmental ≥ 465 mg/kg bw/day

Prenatal developmental toxicity (similar to OECD 414), rat: NOAELmaternal ≥ 410 mg/kg bw/day; NOAELdevelopmental ≥ 410 mg/kg bw/day

Prenatal developmental toxicity (similar to OECD 414), rabbit: NOAELmaternal ≥ 217 mg/kg bw/day; NOAELdevelopmental ≥ 217 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

(1) All salts are inorganic phosphates, composed of a phosphate anion and a calcium cation.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Ca2+ cation and the PO43-anion.
(3) A number of studies are provided to show that monovalent calcium and/or magnesium inorganic orthophosphates exhibit low acute oral toxicity. These data are provided in Section 7.2.1 of this dossier. The information provided in these records is considered to be of suitable relevance and reliability to underpin the read across for the acute dermal and inhalation endpoints. All substances are ionic and will readily dissociate to their ionic forms in aqueous environments. The orthophosphate ion can undergo ionisation with loss of H+ from each of the three –OH groups and therefore can occur in the -1, -2 or -3. The degree of ionisation is dependent upon the associated cation and the ambient pH (if in solution). Calcium and phosphate are key elements in various cellular processes their import and export over cell membranes is regulated via pore systems and usually tightly regulated. Orthophosphate salts of these types are not considered to differ in their systemic toxicity profiles. Differences arise in their local effects profile due differences in pH and buffering capacities. This does not have an effect on systemic toxicity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: no data

Deviations:

not specified

Principles of method if other than guideline:

Adult female albino CD-1 mice were mated with young adult males. Observation of a vaginal sperm plug was considered as Day 0 of gestation. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose or aspirin at 150 mg/kg) or test article in a water suspension (10 mL/kg bw) at 4.65, 21.6, 100.0 and 465.0 mg/kg was carried out daily on Days 6 to 15 of gestation. Observations of body weight, appearence, behaviour, and food consumption were performed. On Day 17 of gestation all dams underwent Caesarean section. Implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Urogenital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. One third foetuses of each litter underwent detailed visceral examination and the remaining two thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects.

GLP compliance:

no

Remarks:

Study predates GLP

Limit test:

no

Species:

mouse

Strain:

other: albino CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Outbred
- Age at study initiation: No data
- Weight at study initiation: 29.2 - 30.6 g
- Fasting period before study: No data
- Housing: Gang housing in disposable plastic cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: No data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data

IN-LIFE DATES: No data

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE: Water
- Amount of vehicle (if gavage): 10 mL/kg bodyweight

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

10 days (Day 6 to Day 15 of gestation)

Frequency of treatment:

Daily

Duration of test:

17 days

Remarks:

Doses / Concentrations:
4.65 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
21.6 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
100.0 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
465.0 mg/kg
Basis:
nominal in water

No. of animals per sex per dose:

Table 1 Number of animals dosed
Material Dose (mg/kg) Total
Mated Pregnant
Sham 0.0 25 22
Aspirin 150.0 25 20
FDA 71-81 4.65 25 24
21.6 26 19
100.0 23 22
465.0 25 23

Control animals:

yes, sham-exposed

other: positive control: 150 mg/kg aspirin

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Appearence, behaviour, food consumption and weight observed daily.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights recorded on days 0, 6, 11, 15 and 17.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 17
- Organs examined: uterus and urogenital tract

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of resorptions: Yes

Fetal examinations:

- External examinations: Yes: one third per litter
- Soft tissue examinations: Yes: one third per litter
- Skeletal examinations: Yes: two thirds per litter
- Head examinations: Yes: two thirds per litter

Statistics:

No data

Indices:

No data

Historical control data:

No

Details on maternal toxic effects:

Maternal toxic effects:no effects

Key result

Dose descriptor:

NOAEL

Effect level:

> 465 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

mortality

Key result

Dose descriptor:

NOAEL

Effect level:

> 465 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

dead fetuses

necropsy findings

number of abortions

pre and post implantation loss

Fetal body weight changes:

not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

not examined

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

increase of incomplete ossification of sternebrae and extremities was observed in the highest dose group (see table 3 below for details)

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

No effects

Key result

Dose descriptor:

NOAEL

Remarks:

fetal toxicity

Effect level:

>= 465 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 465 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 2 Reproduction data

Dose (mg/kg)	Sham	Aspirin	4.65	21.6	100.0	465.0
Pregnancies
Total No.	22	20	24	19	22	23
Died or aborted (before Day 17)	0	1	0	0	0	0
To term (on Day 17)	22	19	24	19	22	23
Live litters
Total No.*	22	19	24	19	22	22
Implant Sites
Total No.	261	224	283	225	244	265
Average/dam*	11.9	11.8	11.8	11.8	11.1	11.5
Resorptions
Total No*	8	20	19	4	12	28
Dams with 1 or more sites resorbed	7	9	13	3	10	12
Dams with all sites resorbed	--	--	--	--	--	1
Per cent partial resorptions	31.8	47.4	54.2	15.8	45.5	52.2
Per cent complete resorptions	--	--	--	--	--	4.35
Live foetuses
Total No	252	201	261	221	230	233
Average/dam*	11.5	10.6	10.9	11.6	10.5	10.1
Sex ratio (M/F)	1.02	0.88	0.78	0.92	1.13	0.93
Dead Foetuses
Total No.*	1	3	--	--	2	4
Dams with 1 or more dead	1	3	--	--	2	3
Dams with all dead	--	--	--	--	--	--
Per cent partial dead	4.55	15.8	--	--	9.09	13.0
Per cent all dead	--	--	--	--	--	--
Average foetus weight (g)	0.84	0.81	0.88	0.87	0.82	0.85

* Includes only those dams examined at term

** Positive control: 150 mg/kg

 

Table 3 Summary of skeletal findings

Findings	Dose (mg/kg)
	Sham	Aspirin	4.65	21.6	100.0	465.0
Live foetuses examined (at term)	179/22	141/19	186/24	155/19	162/22	164/22
Sternebrae
Incomplete oss.	10/8	31/10	18/9	27/15	22/10	28/15
Scrambled
Bipartite	8/7	3/3	11/8	9/7	10/8	9/6
Fused
Extra		6/3
Missing	23/10	28/11	13/9	13/6	32/14	23/7
Other
Ribs
Incomplete oss.						9/4
Fused/split		2/2
Wavy		1/1	2/2
Less than 12	1/1		1/1			1/1
More than 13	48/18	30/12	42/18	18/11	32/15	34/17
Other
Vertebrae
Incomplete oss.	9/6	9/3	2/2	1/1	11/4	13/4
Scrambled
Fused
Extra ctrs. oss.
Scoliosis
Tail defects
Other
Skull
Incomplete closure		2/2
Missing			1/1
Craniostosis
Other; facial bones, inc						2/1
Extremities
Incomplete oss.	7/5	5/3	2/2		10/5	14/4
Missing
Extra
Miscellaneous
Hyoid; missing	37/16	33/11	31/15	22/12	52/15	33/13
Hyoid; reduced	17/11	25/12	17/11	21/14	15/11	27/14

* Numerator = Number of foetuses affected; Denominator = Number of litters affected

** Positive control: 150 mg/kg

 

Table 4 Summary of soft tissue abnormalities

Material	Dose level (mg/kg)	Dam	Number of pups	Description
Aspirin	150.0	A 6068	1	Cleft palate; gastroschisis

Conclusions:

Under the conditions of the study, the test material administered to pregnant mice for 10 days up to a dose level of 465 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and developmental toxicity is > 465 mg/kg bw.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

(1) All salts are inorganic phosphates, composed of a phosphate anion and a calcium cation.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Ca2+ cation and the PO43-anion.
(3) A number of studies are provided to show that monovalent calcium and/or magnesium inorganic orthophosphates exhibit low acute oral toxicity. These data are provided in Section 7.2.1 of this dossier. The information provided in these records is considered to be of suitable relevance and reliability to underpin the read across for the acute dermal and inhalation endpoints. All substances are ionic and will readily dissociate to their ionic forms in aqueous environments. The orthophosphate ion can undergo ionisation with loss of H+ from each of the three –OH groups and therefore can occur in the -1, -2 or -3. The degree of ionisation is dependent upon the associated cation and the ambient pH (if in solution). Calcium and phosphate are key elements in various cellular processes their import and export over cell membranes is regulated via pore systems and usually tightly regulated. Orthophosphate salts of these types are not considered to differ in their systemic toxicity profiles. Differences arise in their local effects profile due differences in pH and buffering capacities. This does not have an effect on systemic toxicity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: no data

Deviations:

not specified

Principles of method if other than guideline:

Adult female Dutch-belted rabbits were dosed on Day 0 with a single injection of 0.4 mL human chorionic gonadotropin (400 IU) via the marginal ear vein. After three hours each doe was artificially inseminated with 0.3 mL diluted semen from a donor buck using 20 x 10E06 motile sperm. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose) or test article in a water suspension (10 mL/kg bw) at 2.17, 10.10, 46.7 and 217.0 mg/kg was carried out daily on Days 6 to 18 of gestation. The positive control 6-aminonicotinamide at 2.5 mg/kg was dosed on Day 9. Observations of body weight, appearence, behaviour, and food consumption were performed. On Day 29 all does underwent Caesarean section. Number of corpora lutea, implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Urogenital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. Live foetuses of each litter were then placed in an incubator for 24 hours for evaluation of neonatal survival. All pups underwent detailed visceral examination. All foetuses were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects.

GLP compliance:

no

Remarks:

Study predates GLP

Limit test:

no

Species:

rabbit

Strain:

Dutch

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Outbred
- Age at study initiation: No data
- Weight at study initiation: 1.84 - 2.21 kg
- Fasting period before study: No data
- Housing: Individually housed in mesh-bottomed cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: No data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data

IN-LIFE DATES: No data

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE: Water
- Amount of vehicle (if gavage): 1 mL/kg bw at doses equal to or below 250 mg/kg bw and 2 mL/kg at doses up to 500 mg/kg bw

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: artificial insemination
- Proof of pregnancy: No data

Duration of treatment / exposure:

13 days (Day 6 to Day 18 of gestation)

Frequency of treatment:

Daily

Duration of test:

29 days

Remarks:

Doses / Concentrations:
2.17 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
10.10 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
46.7 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
217.0 mg/kg
Basis:
nominal in water

No. of animals per sex per dose:

Table 1 Number of animals dosed
Material Dose (mg/kg) Total
Mated Pregnant
Sham 0.0 21 12
6-AN 2.5 18 9
FDA 71-81 2.17 21 12
10.10 27 17
46.7 15 10
217.0 27 10

Control animals:

yes, sham-exposed

other: positive control: 2.5 mg/kg 6-aminonicotinamide (6-AN)

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Appearence, behaviour, food consumption and weight observed daily.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights recorded on days 0, 6, 12, 18 and 29.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: uterus and urogenital tract

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes

Statistics:

No data

Indices:

No data

Historical control data:

No

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Total died/Total in group:
0 mg/kg bw 2/21
2.17 mg/kg bw 2/21
10.10 mg/kg bw 5/27
46.7 mg/kg bw 1/15
217 mg/kg bw 0/27
6-AN mg/kg bw 2/18

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Description (incidence and severity):

Urogenital tract was observed but no data regarding this point were described in the report.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

effects observed, non-treatment-related

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

decreased number of corpora lutea in highest dose group observed

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

> 217 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

mortality

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

> 217 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

dead fetuses

number of abortions

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

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:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No dose related response observed.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 217 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 2 Reproduction data

Dose (mg/kg)	Sham	6-AN	2.17	10.10	46.7	217.0
Pregnancies
Total No.	12	9	12	17	10	10
Died or aborted (before Day 29)	2	0	1	4	0	0
To term (on Day 29)	10	9	11	13	10	10
Corpora Lutea
Total no.	160	117	132	191	110	121
Average/dam mated	11.4	9.75	9.43	9.55	8.46	6.37
Live litters
Total No.*	10	8	10	13	9	8
Implant Sites
Total No.	58	57	62	79	50	49
Average/dam*	5.80	6.33	5.64	6.08	5.00	4.90
Resorptions
Total No*	7	6	9	3	7	4
Dams with 1 or more sites resorbed	4	4	2	2	4	2
Dams with all sites resorbed	--	1	1	--	1	2
Per cent partial resorptions	40.0	44.4	18.2	15.4	40.0	20.0
Per cent complete resorptions	--	11.1	9.09	--	10.0	20.0
Live foetuses
Total No	51	49	49	76	43	45
Average/dam*	5.10	5.44	4.45	5.85	4.30	4.50
Sex ratio (M/F)	0.96	1.13	1.29	1.30	0.87	1.05
Dead Foetuses
Total No.*	--	2	4	--	--	--
Dams with 1 or more dead	--	2	1	--	--	--
Dams with all dead	--	--	--	--	--	--
Per cent partial dead	--	22.2	9.09	--	--	--
Per cent all dead	--	--	--	--	--	--
Average foetus weight (g)	39.6	37.0	39.3	39.7	36.0	39.7

* Includes only those dams examined at term

** Positive control: 2.5 mg/kg 6-AN dosed on Day 9

 

Table 3 Summary of skeletal findings

Findings	Dose (mg/kg)
	Sham	6-AN	2.17	10.10	46.7	217.0
Live foetuses examined (at term)	51/10	49/8	49/10	76/13	43/9	44/8
Sternebrae
Incomplete oss.	1/1			1/1	1/1
Scrambled
Bipartite
Fused
Extra					3/1	1/1
Missing						1/1
Other
Ribs
Incomplete oss.
Fused/split
Wavy
Less than 12
More than 13
Other
Vertebrae
Incomplete oss.
Scrambled
Fused
Extra ctrs. oss.
Scoliosis
Tail defects		1/1		1/1	1/1
Other
Skull
Incomplete closure				3/1
Missing
Craniostosis
Other; facial bones, inc
Extremities
Incomplete oss.
Missing
Extra
Miscellaneous

* Numerator = Number of foetuses affected; Denominator = Number of litters affected

** Positive control: 2.5 mg/kg 6-AN dosed on Day 9 No soft tissue abnormalities observed.

Conclusions:

Under the conditions of the study, the test material administered to pregnant rabbits for 13 days up to a dose level of 217 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and developmental toxicity is > 217 mg/kg bw.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

(1) All salts are inorganic phosphates, composed of a phosphate anion and a calcium cation.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Ca2+ cation and the PO43-anion.
(3) A number of studies are provided to show that monovalent calcium and/or magnesium inorganic orthophosphates exhibit low acute oral toxicity. These data are provided in Section 7.2.1 of this dossier. The information provided in these records is considered to be of suitable relevance and reliability to underpin the read across for the acute dermal and inhalation endpoints. All substances are ionic and will readily dissociate to their ionic forms in aqueous environments. The orthophosphate ion can undergo ionisation with loss of H+ from each of the three –OH groups and therefore can occur in the -1, -2 or -3. The degree of ionisation is dependent upon the associated cation and the ambient pH (if in solution). Calcium and phosphate are key elements in various cellular processes their import and export over cell membranes is regulated via pore systems and usually tightly regulated. Orthophosphate salts of these types are not considered to differ in their systemic toxicity profiles. Differences arise in their local effects profile due differences in pH and buffering capacities. This does not have an effect on systemic toxicity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: no data

Deviations:

not specified

Principles of method if other than guideline:

Adult female albino (Wistar derived stock) rats were mated with young adult males. Observation of a vaginal sperm plug was considered as Day 0 of gestation. Dosing by oral intubation with a control (Vehicle at level equivalent to group receiving the highest dose or aspirin at 250 mg/kg) or test article in a water suspension at 4.1, 19.1, 88.5 and 410.0 mg/kg was carried out daily on Days 6 to 15 of gestation. Observations of body weight, appearence, behaviour, and food consumption were performed. Daily room temperature was recorded. On Day 20 of gestation all dams underwent Caesarean section. Number of implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Urogenital tract of each dam examined for anatomical normality. All foetuses examined grossly for presence of external congenital abnormalities. One third foetuses of each litter underwent detailed visceral examination and the remaining two thirds were cleared in potassium hydroxide, stained with alizarin red S dye and examined for skeletal defects.

GLP compliance:

no

Remarks:

Study predates GLP

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Outbred
- Age at study initiation: No data
- Weight at study initiation: 216 - 224 g
- Fasting period before study: No data
- Housing: Individual housing in mesh bottom cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: No data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not recorded
- Humidity (%): Not recorded

IN-LIFE DATES: From: 12/01/1975 To: 06/02/1975

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE: Water
- Amount of vehicle (if gavage): 1 mL/kg bw at doses equal to or below 250 mg/kg bw and 2 mL/kg at doses up to 500 mg/kg bw

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: No data
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

10 days (Day 6 to Day 15 of gestation)

Frequency of treatment:

Daily

Duration of test:

20 days

Remarks:

Doses / Concentrations:
4.1 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
19.1 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
88.5 mg/kg
Basis:
nominal in water

Remarks:

Doses / Concentrations:
410.0 mg/kg
Basis:
nominal in water

No. of animals per sex per dose:

Table 1 Number of animals dosed
Material Dose (mg/kg) Total
Mated Pregnant
Sham 0.0 25 21
Aspirin 250.0 25 21
FDA 71-81 4.1 28 22
19.1 29 21
88.5 25 19
410.0 25 22

Control animals:

yes, sham-exposed

other: positive control: 250 mg/kg aspirin

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Appearence, behaviour, food consumption and weight observed daily.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights recorded on days 0, 6, 11, 15 and 20.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus and urogenital tract

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of resorptions: Yes

Fetal examinations:

- External examinations: Yes: one third per litter
- Soft tissue examinations: Yes: one third per litter
- Skeletal examinations: Yes: two thirds per litter
- Head examinations: Yes: two thirds per litter

Statistics:

No data

Indices:

No data

Historical control data:

No

Clinical signs:

not specified

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Description (incidence and severity):

Urogenital tract was observed but no data regarding this point were described in the report.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

> 410 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

mortality

Key result

Dose descriptor:

NOAEL

Remarks:

development toxicity

Effect level:

> 410 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

dead fetuses

number of abortions

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weights after Caesarean section were determined. The average body weights of fetuses were similar in all groups except the group with aspirin which showed decreased body weig

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The results indicate that the number of wavey ribs (47 foetuses affected/ 12 litters) observed at the highest dose level in the study (410.0 mg/kg) might be significant in relation to the sham control. However, the same result is not observed in either of the other two species tested (mice and rabbit) so can be discounted.

Visceral malformations:

no effects observed

Description (incidence and severity):

one pup in the lowest dose group (4.1 mg/kg bw) had a hydrocephalus which was considered to be a spontanous effects since it was not observed in any other dose group or in the other two tested species mice and rabbits.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 410 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

skeletal malformations

visceral malformations

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: rib

Description (incidence and severity):

increased wavy ribs only in the highest dose group

Key result

Developmental effects observed:

no

  Table 2 Reproduction data

Dose (mg/kg)	Sham	Aspirin	4.1	19.1	88.5	410.0
Pregnancies
Total No.	21	21	22	22	19	22
Died or aborted (before Day 20)	0	0	0	0	0	0
To term (on Day 20)	21	21	22	22	19	22
Live litters
Total No.*	21	19	22	22	19	22
Implant Sites
Total No.	235	235	257	257	196	244
Average/dam*	11.2	11.2	11.7	11.7	10.3	11.1
Resorptions
Total No*	2	63	3	3	3	2
Dams with 1 or more sites resorbed	2	13	3	3	2	1
Dams with all sites resorbed	--	2	--	--	--	--
Per cent partial resorptions	9.52	61.9	13.6	13.6	10.5	4.55
Per cent complete resorptions	--	9.52	--	--	--	--
Live foetuses
Total No	233	170	253	253	192	242
Average/dam*	11.1	8.10	11.5	11.5	10.1	11.0
Sex ratio (M/F)	1.33	1.07	0.92	0.92	0.92	0.87
Dead Foetuses
Total No.*	--	2	1	1	1	--
Dams with 1 or more dead	--	2	1	1	1	--
Dams with all dead	--	--	--	--	--	--
Per cent partial dead	--	9.52	4.55	4.55	5.26	--
Per cent all dead	--	--	--	--	--	--
Average foetus weight (g)	3.62	2.43	3.85	3.85	3.56	3.65

* Includes only those dams examined at term

** Positive control: 250 mg/kg

 

Table 3 Summary of skeletal findings

Findings	Dose (mg/kg)
	Sham	Aspirin	4.1	19.1	88.5	410.0
Live foetuses examined (at term)	165/21	124/19	174/22	162/21	133/19	168/22
Sternebrae
Incomplete oss.	45/18	49/16	31/12	36/16	42/15	45/15
Scrambled		1/1
Bipartite	1/1	13/7				1/1
Fused		1/1
Extra
Missing	27/12	92/18	6/4	5/4	22/10	16/7
Other
Ribs
Incomplete oss.		1/1				9/3
Fused/split		8/4
Wavy	17/10	51/14	16/9	15/7	11/8	47/12
Less than 12		1/1
More than 13	5/5	71/17	2/2	1/1	2/2	4/3
Other
Vertebrae
Incomplete oss.	26/14	92/19	10/6	6/5	22/11	29/10
Scrambled
Fused
Extra ctrs. oss.
Scoliosis
Tail defects
Other
Skull
Incomplete closure	29/12	38/12	25/11	12/6	14/10	28/10
Missing		1/1
Craniostosis
Other; facial bones, inc		9/6
Extremities
Incomplete oss.		6/3
Missing
Extra
Miscellaneous
Hyoid; missing	15/8	51/16	13/7	14/9	8/6	22/12
Hyoid; reduced	22/11	11/7	24/10	12/7	9/8	30/14

* Numerator = Number of foetuses affected; Denominator = Number of litters affected

** Positive control: 250 mg/kg

 

Table 4 Summary of soft tissue abnormalities

Material	Dose level (mg/kg)	Dam	Number of pups	Description
Aspirin	250.0	A 7065	1	Encephalomyelocele
		A 7066	1	Gastroschisis
		A 7070	1	Meningoencephalocele
		A 7076	2	Meningoencephalocele
FDA 71-81	4.1	H 6028	1	Hydrocephalus

 

Conclusions:

Under the conditions of the study, the test material administered to pregnant rats for 10 days up to a dose level of 410 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and developmental toxicity is > 410 mg/kg bw.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

217 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The available information comprises studies which each alone are regarded insufficient for assessment from a reference substance with similar structure and intrinsic properties. Read-across is justified based on structural similarities of the calciumphosphate compounds and their similarities in PC/ECO/TOX properties (refer to read across justification for further details). However, the information from these independent sources is consistent and provides sufficient weight of evidence for hazard assessment leading to an endpoint conclusion in accordance with Annex XI, 1.2, of Regulation (EC) No 1907/2006. Therefore, the available information as a whole is sufficient to fulfil the standard information requirements set out in Annex X, 8.7.2, of Regulation (EC) No 1907/2006.

Justification for classification or non-classification

Results of the developmental toxicity study conducted on monocalcium phosphate monohydrate:

Under the conditions of the study, the test material administered to pregnant mice for 10 days up to a dose level of 465 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and developmental toxicity is > 465 mg/kg bw.

 

Under the conditions of the study, the test material administered to pregnant rats for 10 days up to a dose level of 410 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and developmental toxicity is > 410 mg/kg bw.

 

Under the conditions of the study, the test material administered to pregnant rabbits for 13 days up to a dose level of 217 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and developmental toxicity is > 217 mg/kg bw.

It is not considered to be scientifically justified to further investigate the effects of the reaction mass of calcium bis(dihydrogenorthophosphate) and calcium hydrogenorthophosphate on reprotoxicity, developmental or maternal toxicity and as such no classification is proposed for this endpoint and no further studies are deemed necessary.