Magnesium bis(dihydrogenorthophosphate)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data available - studies not required in accordance with Regulation (EC) No. 1907/2006 (REACH).

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Section 8.7.1. Adaptation

According to Annex VIII, Section 8.7.1, Column 2 of Regulation (EC) No. 1907/2006, a screening for reproductive/developmental toxicity study does not need to be conducted if a prenatal development toxicity study is available. A developmental toxicity study on the analogous substance calcium bis(dihydrogenorthophosphate) monohydrate is included in IUCLID Section 7.8.2 of this dossier and therefore a screening study is not considered to be scientifically justified.

Section 8.7.2. Adaptation

The standard requirement for chemicals manufactured or imported into the EU in quantities of >100 tpa includes a two-generation reprotoxicity study (OECD 416). According to the Integrated Testing Strategy (ITS) proposed in the ECHA guidance document on the information requirements and chemical safety assessment, Chapter R. 7a: Endpoint specific guidance, Section 7.6.6, if there is sufficient data to permit a robust conclusion on reproductive toxicity testing then no further testing will be required. The justification for not conducting a two-generation is based on the evaluation of existing data in laboratory animals, available data on similar or parent compounds, historical data and general characteristics of the test material.

 

For the purpose of assessing the risk of reprotoxicity the following substances are considered to be similar enough to facilitate read across:

 

-         calcium bis(dihydrogenorthophosphate): EC No. 231-837-1

-         calcium hydrogenorthophosphate: EC No. 231-826-1

-         tricalcium bis(orthophosphate): EC No. 231-840-8

-         pentacalcium hydroxide tris(orthophosphate) : EC No. 235-330-6

-         magnesium hydrogenorthophosphate: EC No. 231-823-5

-         trimagnesium bis(orthophosphate): EC No. 231-824-0

-         magnesium bis(dihydrogenorthophosphate) : EC 236-004-6

 

Laboratory studies:

 

-          Developmental toxicity: Calcium bis(dihydrogenorthophosphate) showed no maternal toxicity or teratogenic effects at dose levels up to 465 mg/kg bw in mice and 410 mg/kg bw in rats (See Section 7.8.2 of this dossier).Although the studies were conducted prior to the implementation of Good Laboratory Practice, the study design and content were of sufficient standard to allow meaningful evaluation. The results showed no evidence of developmental toxicity to the foetus of any species or any other adverse effects to the foetus or mother at any dose level. It can therefore be concluded that calcium orthophosphates are not developmental toxicants at relatively high levels.

The lack of effects on maternal toxicity or offspring development at dose levels well in excess of normal human exposure suggests that that calcium orthophosphates are not a significant risk to the reproductive process in females or males and not a significant risk to the developing foetus. Further studies are unlikely to show any significant effects on reproductive performance or development. 

 

In addition, negative in vitro mutagenicity and genotoxicity evidence suggests a low potential for germ cell mutagenicity. The following studies have been conducted on Ca and Mg orthophosphates: Bacterial Reverse Mutation Assay;(OECD 471) In vitro cytogenicity study in mammalian cells (OECD 473) and In vitro gene mutation in mammalian cell lines (OECD Method 476). Whilst these studies look specifically at the effects in somatic cell lines the results are still relevant to the potential of the test material to induce mutagenicity in germ cell lines.

Negative in vivo genotoxicity data also exists; the results of a dominant lethal assay are presented in Section 7.8.2 of this dossier. This study looks at the potential of a chemical substance to induce chromosomal aberrations in the germ cells of rats. As no effects are noted this can be used as part of the evidence against performing studies to investigate the effects on reprotoxicity.

 

 

General discussion;

Calcium and magnesium orthophosphates are approved for use as food additives (the EU food reference / INS number for magnesium hydrogenorthophosphate is E343 ii). No evidence exists to show that calcium or magnesium orthophosphates are likely to pose a risk of reproductive or developmental toxicity.

In addition, Both the Ca²⁺and the Mg²⁺cation have a similar and essential biological functions and excess of these ions results in well documented toxicity; this does not include toxicity to reproduction or developmental toxicity.

The World Health Organisation, reports that the maximum tolerable daily intake (MTDI) for phosphates for all individuals is 70 mg P/kg bw (1), this value is considered to be well below that observed for developmental toxicity and as such human exposure is likely to be considerably less that the level required for reprotoxicity testing. No effects on development were observed at the highest does tested in animal studies. Reference values for the intake of calcium and magnesium are considerably higher than that for phosphorus: Adequate intake (AI) value for calcium in 19-30 year olds is 1000 mg/day and the Estimated Average Requirement (EAR) for magnesium in males aged 19-30 is 330 mg/day and for females aged 19-30 is 255 mg/day (2). These values indicate that humans will generally be exposed to relatively high daily levels with no adverse effects.

 

The main toxicological finding in feeding studies with high levels of phosphates is nephrocalcinosis (the rat is known to be more susceptible to these effects than humans (1, see also IUCLID section 7.5.), moreover these renal effects are not considered to be relevant to reproductive toxicity.

In conclusion, an additional two generation reproduction study in the rat is unlikely to result in providing further evidence of reproductive toxicity as the existing studies have demonstrated a lack of effect at dose levels well in excess of expected human exposure. A study would therefore be scientifically and ethically unjustified.

 

 

 (1)Evaluation of certain food additives and contaminants. Twenty-sixth report of the joint FAO/WHO expert committee of food additives. World Health Organisation. Technical Report Series 683. 1982. ISBN92 4 120683 7

 

(2)Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board, of. Dietary Reference Intakes for Calcium, Phosphorus,Magnesium, Vitamin D and Fluoride.,:Press, 1997.

Short description of key information:
No laboratory studies are provided for the endpoint 'toxicity to reproduction'. The justifications for the deviation from the standard testing requirements are detailed below.

Effects on developmental toxicity

Description of key information

Three key studies (One study report, three different test animals) are available to assess the teratogenic potential of calcium bis(dihydrogenorthophosphate) (Morgariedge, 1974) in mice, rats and rabbits. This study is considered to be adequate to fulfil this endpoint. 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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. Both substances are inorganic salts of a monovalent cation from Group 2 of the periodic table, calcium or magnesium, and phosphoric acid. Thus, they all share the Ca2+ or Mg2+ cation and the PO43- anion as common functional groups.
2. Both substances will ultimately dissociate into the common breakdown products of the Ca2+ or Mg2+ cations and the PO43- anion.
3. Calcium and magnesium orthophosphates have been shown to have a similar toxicological profile and physicochemical nature and therefore this data is considered to be adequate and reliable for use in read-across. In accordance with the provisions set out in Annex XI, Section 1.5, the results of the studies used for assessment and read-across are adequate for the purpose of classification and labelling and/or risk assessment; have adequate and reliable coverage of the key parameters addressed in the corresponding test method; cover an exposure duration comparable to or longer than the corresponding test method; and adequate and reliable documentation of the applied method is provided in the technical dossier. Orthophosphates are not considered to be genotoxic and are essential micronutrients. As such the reproductive toxicity will be predominantly determined by the presence of the Mg2+ cation.

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:

reference to same study

Reason / purpose for cross-reference:

reference to same study

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

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

Dose descriptor:

NOAEL

Effect level:

> 465 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no effects observed

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

> 465 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

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:

key study

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

Remarks:

Meets generally accepted scientific standards with acceptable restrictions. Deficiencies: Food consumption not reported Uterine weights not determined Test substance identification (Batch etc) missing No details on housing conditions/source of animals Administration only during periods of organogenesis, not until day before pregnancy The reliability has been amended in accordance with 'practical guide 6: How to report read-across and categories' which states that the maximum reliability for a read-across study is 2. JUSTIFICATION FOR READ ACROSS; calcium and magnesium orthophosphates. The following substances are considered to be similar enough to facilitate read across for systemic toxicity endpoints: Calcium bis(dihydrogenorthophosohate, EC No: 231-837-1 Calcium hydrogenorthophosphate. EC No: 231-826-1 Tricalcium bis(orthophosphate), EC No: 231-840-8 Pentacalcium hydroxide tris(orthophosphate), EC No: 235-330-6 Magnesium hydrogenorthophosphate, EC No: 231-823-5 Trimagnesium bis(orthophosphate), EC No. 231-824-0 Magnesium bis(dihydrogenorthophosphate), EC No. 236-004-6 All of the above substances have exhibited similar toxicity in acute oral and dermal studies. Read across is justified on the following basis: 1. Low systemic toxicity in acute oral studies. A number of studies are provided to show that 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. 2. Substance similarities All substances are ionic and will readily dissociate to their ionic forms in aqueous environments. The orthophosphate ion can under go 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 dependant upon the associated cation and the ambient pH (if in solution). The ionic form of the Group 1ii alkali metals is M2+.Calcium, magnesium 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.

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. Both substances are inorganic salts of a monovalent cation from Group 2 of the periodic table, calcium or magnesium, and phosphoric acid. Thus, they all share the Ca2+ or Mg2+ cation and the PO43- anion as common functional groups.
2. Both substances will ultimately dissociate into the common breakdown products of the Ca2+ or Mg2+ cations and the PO43- anion.
3. Calcium and magnesium orthophosphates have been shown to have a similar toxicological profile and physicochemical nature and therefore this data is considered to be adequate and reliable for use in read-across. In accordance with the provisions set out in Annex XI, Section 1.5, the results of the studies used for assessment and read-across are adequate for the purpose of classification and labelling and/or risk assessment; have adequate and reliable coverage of the key parameters addressed in the corresponding test method; cover an exposure duration comparable to or longer than the corresponding test method; and adequate and reliable documentation of the applied method is provided in the technical dossier. Orthophosphates are not considered to be genotoxic and are essential micronutrients. As such the reproductive toxicity will be predominantly determined by the presence of the Mg2+ cation.

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:

reference to same study

Reason / purpose for cross-reference:

reference to same study

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

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

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOAEL

Effect level:

> 217 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no effects observed

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

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

Dose descriptor:

NOAEL

Effect level:

> 217 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Abnormalities:

no effects observed

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:

key study

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. Both substances are inorganic salts of a monovalent cation from Group 2 of the periodic table, calcium or magnesium, and phosphoric acid. Thus, they all share the Ca2+ or Mg2+ cation and the PO43- anion as common functional groups.
2. Both substances will ultimately dissociate into the common breakdown products of the Ca2+ or Mg2+ cations and the PO43- anion.
3. Calcium and magnesium orthophosphates have been shown to have a similar toxicological profile and physicochemical nature and therefore this data is considered to be adequate and reliable for use in read-across. In accordance with the provisions set out in Annex XI, Section 1.5, the results of the studies used for assessment and read-across are adequate for the purpose of classification and labelling and/or risk assessment; have adequate and reliable coverage of the key parameters addressed in the corresponding test method; cover an exposure duration comparable to or longer than the corresponding test method; and adequate and reliable documentation of the applied method is provided in the technical dossier. Orthophosphates are not considered to be genotoxic and are essential micronutrients. As such the reproductive toxicity will be predominantly determined by the presence of the Mg2+ cation.

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:

reference to same study

Reason / purpose for cross-reference:

reference to same study

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

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

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOAEL

Effect level:

> 410 mg/kg bw/day

Basis for effect level:

other: no effects observed

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
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.

Dose descriptor:

NOAEL

Effect level:

> 410 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Abnormalities:

no effects observed

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:

subchronic

Species:

rabbit

Quality of whole database:

Three key studies (One study report, three different test animals) are available to assess the teratogenic potential of calcium bis(dihydrogenorthophosphate) (Morgariedge, 1974) in mice, rats and rabbits. This study is considered to be adequate to fulfil this endpoint

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

JUSTIFICATION FOR READ ACROSS;calcium and magnesium orthophosphates.

The following substances are considered to be similar enough to facilitate read across for systemic toxicity endpoints:

Calcium bis(dihydrogenorthophosphate), EC No: 231-837-1

Calcium hydrogenorthophosphate. EC No: 231-826-1

Tricalcium bis(orthophosphate), EC No: 231-840-8

Pentacalcium hydroxide tris(orthophosphate), EC No: 235-330-6

Magnesium hydrogenorthophosphate, EC No: 231-823-5

Trimagnesium bis(orthophosphate), EC No. 231-824-0

Magnesium bis(dihydrogenorthophosphate), EC No. 236-004-6

All of the above substances have exhibited similar toxicity in acute oral and dermal studies.

Read across is justified on the following basis:

1. Low systemic toxicity in acute oral studies.

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.

2. Substance similarities

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).

 

The ionic form of the Group 1ii alkali metals is M²⁺.Calcium, magnesium 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.

Justification for selection of Effect on developmental toxicity: via oral route:
Endpoint record relating to the lowest NOAEL in the 3 species tested. NOAEL was > 217 mg/kg bw/ day.

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 magnesium bis(dihydrogenorthophosphate) on reprotoxicity, developmental or maternal toxicity and as such no classification is proposed for this endpoint and no further studies are deemed necessary.