Registration Dossier

Toxicological information

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Key value for chemical safety assessment

Effects on fertility

Description of key information

No data available; a data waiver is submitted.

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

Justification for data waiving; reproductive toxicity

The standard requirement for chemicals manufactured or imported into the EU in quantities of >1,000 tpa includes an extended one generation reproductive toxicity test (EOGRTS). 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:  

- disodium dihydrogenpyrophosphate / sodium acid pyrophosphate (EC No. 231-835-0) 

- trisodium hydrogen diphosphate (EC No. 238-735-6) 

- tetrasodium pyrophosphate (EC No. 231-767-1) 

- tetrapotassium pyrophosphate (EC No. 230-785-7)  

LABORATORY STUDIES

Negative In vitro mutagenicity and genotoxicity evidence suggests a low potential for germ cell mutagenicity. The following studies exist on either sodium or potassium pyrophosphates or sodium orthophosphate: Bacterial Reverse Mutation Assay; AMES (OECD 471), In vitro cytogenicity study in mammalian cells (OECD 473), in vitro gene mutation in mammalian cells (OECD 490). 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; 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 reprotoxicity. 

Developmental toxicity / teratogenicity studies similar to OECD 414 have been conducted on the following substances on behalf of the United States Food and Drug Administration; disodium dihydrogenpyrophosphate (sodium acid pyrophosphate) and tetrasodium pyrophosphate. The studies were conducted in four laboratory animal models; rat, mouse, hamster and rabbit. A range of dose levels up to a high dose of 335 mg/kg bw (mouse, disodium dihydrogenpyrophosphate), 169 mg/kg bw (rat, disodium dihydrogenpyrophosphate), 166 mg/kg bw (hamster, disodium dihydrogenpyrophosphate), 128 mg/kg bw (rabbit, disodium dihydrogenpyrophosphate), 130 mg/kg bw (mouse, tetrasodium pyrophosphate) and 138 mg/kg bw (rat, tetrasodium pyrophosphate) were used. The material was administered by oral intubation for ten consecutive days for rats and mice covering the gestation days 6 to 15. 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 sodium and potassium 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 sodium and potassium pyrophosphates are not a significant risk to the reproductive process and further studies are unlikely to show any significant effects.   

GENERAL DISCUSSION  

The pyrophosphate ion is the simplest form of a condensed phosphate group. A condensed phosphate anion has one or several P-O-P bonds. As the pyrophosphate only contains two phosphate groups, both of the phosphorus ions are classed as terminal phosphorus. The pyrophosphate can undergo ionisation with loss of H+ from each of the two –OH group groups on each P and can therefore occur in the -1, -2, -3 or -4 state. The degree of ionisation is dependent on upon the associated cations and the ambient pH (if in solution). As pyrophosphates are ionic in nature they will readily dissociate into their cations and anions in solution. Sodium and potassium cations are not considered to pose a risk of reprotoxicity as they are essential micronutrients that are well regulated by the homeostatic mechanisms in the body. In addition, the recommended daily intakes of sodium and potassium for young adults are 1500 mg/day/person and 4700 mg/day/person respectively (1). These levels are well above the levels that would be expected in a reprotoxicity study and as such the sodium or potassium cation can be discounted from further discussion. The pyrophosphate anion is hydrolysed to orthophosphate by ubiquitous alkaline phosphatase activity. Orthophosphate then goes on to take part in various physiological processes. The World Health Organisation, reports that the maximum tolerable daily intake (MTDI) of phosphates is 70 mg/kg bw (2).  

Sodium and potassium pyrophosphates are frequently used as food additives (the EU food reference INS number for disodium dihydrogenpyrophosphate / sodium acid pyrophosphate is E450 i). The contribution of food supplements to phosphorus intake is low. No evidence exists to show that sodium or potassium pyrophosphates are likely to pose a risk of reproductive or developmental toxicity. 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 (2). Such effects are not considered to be relevant to reproductive toxicity.  

In conclusion, reproductive toxicity testing is unlikely to result in 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) Dietary Reference Intakes for Water, Potassium, Sodium, Chloride and sulphate. www.nap.edu. The National Academies. U.S.A. 

(2)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

Effects on developmental toxicity

Description of key information
Four key studies (One study report, four different test animals) are available to assess the teratogenic potential of disodium dihydrogenpyrophosphate (Morgariedge, 1973) in mice, rats, hamsters and rabbits. This study is considered to be adequate to fulfil this endpoint. In addition supporting data on the analogous substance tetrasodium pyrophosphate is provided to support a lack of developmental toxicity potential for sodium and potassium pyrophosphates.
Link to relevant study records

Referenceopen allclose all

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 or copora lutea not determined One third used for visceral examination; should be 50% 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
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: no data
Deviations:
not specified
Principles of method if other than guideline:
Adult female golden hamsters were mated with mature males (1:1). Observation of motile sperm in the vaginal smear 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 1.66, 7.71, 35.8 and 166.0 mg/kg was carried out daily on Days 6 to 10 of gestation. Observations of body weight, appearence, behaviour, and food consumption were performed. On Day 14 of gestation all dams underwent Caesarean section. Implantation sites, resorption sites and live/dead foetuses recorded. Body weights of live pups recorded. Genital 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:
hamster
Strain:
other: Golden
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Outbred
- Age at study initiation: No data
- Weight at study initiation: 122.2 - 133.4 g
- 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: 1:1 mating
- Proof of pregnancy: appearence of motile sperm in vaginal smear
Duration of treatment / exposure:
5 days (Day 6 to Day 10 of gestation)
Frequency of treatment:
Daily
Duration of test:
14 days
No. of animals per sex per dose:
Table 1 Number of animals dosed
Material Dose (mg/kg) Total
Mated Pregnant
Sham 0.0 22 20
Aspirin 250 22 21
FDA 71-81 1.66 22 20
7.71 24 20
35.8 22 21
166.0 22 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, 8, 10 and 14.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 14
- Organs examined: uterus and genital 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
- 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:
> 166 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEL
Effect level:
> 166 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects noted
Abnormalities:
no effects observed
Developmental effects observed:
no
Table 2 Reproduction data

Dose (mg/kg)

Sham

Aspirin

1.66

7.71

35.8

166.0

Pregnancies

 

 

 

 

 

 

Total No.

20

21

20

20

21

22

Died or aborted (before Day 14)

0

0

0

0

1

1

To term (on Day 14)

20

21

20

20

20

21

Live litters

 

 

 

 

 

 

Total No.*

20

21

20

20

20

21

Implant Sites

 

 

 

 

 

 

Total No.

298

311

292

289

290

300

Average/dam*

14.9

14.8

14.6

14.5

14.5

14.3

Resorptions

 

 

 

 

 

 

Total No*

1

8

1

1

1

8

Dams with 1 or more sites resorbed

1

6

1

1

1

5

Dams with all sites resorbed

--

--

--

--

--

--

Per cent partial resorptions

5.00

28.6

5.00

5.00

5.00

23.8

Per cent complete resorptions

--

--

--

--

--

--

Live foetuses

 

 

 

 

 

 

Total No

295

303

291

288

289

292

Average/dam*

14.8

14.4

14.6

14.4

14.5

13.9

Sex ratio (M/F)

0.89

0.94

0.81

0.78

0.75

0.75

Dead Foetuses

 

 

 

 

 

 

Total No.*

2

--

--

--

--

--

Dams with 1 or more dead

2

--

--

--

--

--

Dams with all dead

--

--

--

--

--

--

Per cent partial dead

10.0

--

--

--

--

--

Per cent all dead

--

--

--

--

--

--

Average foetus weight (g)

1.70

1.72

1.72

1.82

1.82

1.75

* Includes only those dams examined at term

** Positive control: 250 mg/kg

 

Table 3 Summary of skeletal findings

Findings

Dose (mg/kg)

Sham

Aspirin

1.66

7.71

35.8

166.0

Live foetuses examined (at term)

204/20

209/21

200/20

199/20

202/20

202/21

Sternebrae

 

 

 

 

 

 

Incomplete oss.

131/20

85/20

124/20

69/17

88/18

80/20

Scrambled

 

 

 

 

 

 

Bipartite

18/9

16/11

22/14

20/11

15/9

25/13

Fused

 

 

 

 

 

 

Extra

8/2

 

1/1

6/3

5/3

 

Missing

53/14

48/13

49/12

39/10

33/10

51/14

Other

 

 

 

 

 

 

Ribs

 

 

 

 

 

 

Incomplete oss.

 

 

 

 

 

 

Fused/split

 

 

 

 

 

2/1

Wavy

 

 

 

 

 

 

Less than 12

 

 

 

 

 

 

More than 13

62/17

56/13

46/16

69/17

38/13

60/16

Other

 

 

 

 

 

 

Vertebrae

 

 

 

 

 

 

Incomplete oss.

2/2

15/5

 

1/1

 

8/2

Scrambled

 

 

 

 

 

 

Fused

 

 

 

 

 

 

Extra ctrs. oss.

 

 

 

 

 

 

Scoliosis

 

 

 

 

 

 

Tail defects

 

 

 

 

 

 

Other

 

 

 

 

 

 

Skull

 

 

 

 

 

 

Incomplete closure

 

 

 

 

 

 

Missing

 

 

 

 

 

 

Craniostosis

 

 

 

 

 

 

Other

 

 

 

 

 

 

Extremities

 

 

 

 

 

 

Incomplete oss.

4/4

17/6

 

6/4

 

13/3

Missing

 

 

 

 

 

 

Extra

 

 

 

 

 

 

Miscellaneous

 

 

 

 

 

 

Hyoid; missing

6/6

14/6

5/4

4/2

2/1

13/3

Hyoid; reduced

7/7

6/5

9/7

7/3

6/5

8/6

* 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

Sham

0.0

S 5908

1

Meningoencephalocele

 

 

S 5914

1

Hydrocephalus

Aspirin

250.0

A 5912

1

Hepatomegaly

FDA 71-61

1.66

M 5013

1

Hydrocephalus

FDA 71-61

166.0

M 5092

4

Hydrocephalus

 

 

 

2

Hydromyelia

Conclusions:
Under the conditions of the study, the test material administered to pregnant hamsters for 5 days up to a dose level of 166 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 166 mg/kg bw.
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 and corpora lutea not determined One third used for visceral examination; should be 50% 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
Reason / purpose:
reference to same study
Qualifier:
according to
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 3.35, 15.6, 72.3 and 335.0 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 and environmental conditions:
TEST ANIMALS
- Source: Outbred
- Age at study initiation: No data
- Weight at study initiation: 26.3 - 29.3 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 25
Aspirin 150.0 24 21
FDA 71-61 3.35 25 23
15.6 25 23
72.3 24 23
335.0 25 22
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:
> 335 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects noted
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEL
Effect level:
> 335 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects noted
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 2 Reproduction data

Dose (mg/kg)

Sham

Aspirin**

3.35

15.6

72.3

335.0

Pregnancies

 

 

 

 

 

 

Total No.

25

21

23

23

23

22

Died or aborted (before Day 17)

0

0

0

0

0

0

To term (on Day 17)

25

21

23

23

23

22

Live litters

 

 

 

 

 

 

Total No.*

25

20

23

23

23

21

Implant Sites

 

 

 

 

 

 

Total No.

286

254

280

279

265

256

Average/dam*

11.4

12.1

12.2

12.1

11.5

11.6

Resorptions

 

 

 

 

 

 

Total No*

17

37

8

3

10

24

Dams with 1 or more sites resorbed

10

13

6

2

9

9

Dams with all sites resorbed

0

1

0

0

0

1

Per cent partial resorptions

40.0

61.9

26.1

8.70

39.1

40.9

Per cent complete resorptions

--

4.76

--

--

--

4.55

Live foetuses

 

 

 

 

 

 

Total No

264

217

271

274

255

229

Average/dam*

10.6

10.3

11.8

11.9

11.5

10.4

Sex ratio (M/F)

0.96

0.75

0.75

0.73

0.82

0.84

Dead Foetuses

 

 

 

 

 

 

Total No.*

5

0

1

2

0

3

Dams with 1 or more dead

4

--

1

2

--

3

Dams with all dead

0

--

0

0

--

0

Per cent partial dead

16.0

--

4.35

8.70

--

13.6

Per cent all dead

--

--

--

--

--

--

Average foetus weight (g)

0.88

0.84

0.84

0.89

0.86

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**

3.35

15.6

72.3

335.0

Live foetuses examined (at term)

183/25

154/20

188/23

190/23

176/23

159/21

Sternebrae

 

 

 

 

 

 

Incomplete oss.

23/10

34/13

19/11

45/13

33/13

29/9

Scrambled

 

 

 

 

 

 

Bipartite

5/4

6/5

9/7

10/9

5/3

9/6

Fused

 

 

 

 

 

 

Extra

 

1/1

 

 

 

1/1

Missing

35/14

26/11

27/11

39/8

30/10

14/6

Other

 

 

 

 

 

 

Ribs

 

 

 

 

 

 

Incomplete oss.

 

 

3/1

 

2/1

 

Fused/split

 

1/1

 

 

 

 

Wavy

 

 

 

 

 

 

Less than 12

 

 

 

 

 

 

More than 13

12/7

37/15

34/15

32/14

38/11

31/15

Other

 

 

 

 

 

 

Vertebrae

 

 

 

 

 

 

Incomplete oss.

4/3

9/5

12/4

8/3

4/2

4/2

Scrambled

 

 

 

 

 

 

Fused

 

 

 

 

 

 

Extra ctrs. oss.

 

 

 

 

 

 

Scoliosis

 

 

 

 

 

 

Tail defects

 

 

 

 

 

 

Other

 

 

 

 

 

 

Skull

 

 

 

 

 

 

Incomplete closure

 

 

1/1

 

 

 

Missing

 

 

1/1

 

 

 

Craniostosis

 

 

 

 

 

 

Other; facial bones, inc

 

 

 

1/1

 

 

Extremities

 

 

 

 

 

 

Incomplete oss.

4/3

6/4

12/6

7/3

7/3

4/2

Missing

 

 

 

 

 

 

Extra

 

 

 

 

 

 

Miscellaneous

 

 

 

 

 

 

Hyoid; missing

29/12

35/15

39/15

52/16

46/15

33/13

Hyoid; reduced

17/13

23/13

24/12

18/10

17/13

23/10

* 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 3911

1

Microblepharia

Gastroschisis

FDA 71-61

3.35

M 3010

1

Umbilical hernia

FDA 71-61

15.6

M3033

1

Umbilical hernia

 

 

M 3053

1

Hydrocephalus

FDA 71-61

72.3

M3078

1

Hydrocephalus

Conclusions:
Under the conditions of the study, the test material administered to pregnant mice for 10 days up to a dose level of 335 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 335 mg/kg bw.
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
Reason / purpose:
reference to same study
Qualifier:
according to
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 at 1.28, 5.95, 27.6 and 128.0 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 and environmental conditions:
TEST ANIMALS
- Source: Outbred
- Age at study initiation: No data
- Weight at study initiation: 2.17 - 2.51 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 17 11
6-AN 2.5 15 10
FDA 71-61 1.28 15 12
5.95 16 12
27.6 20 9
128.0 15 11
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:
> 128 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:
> 128 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

1.28

5.95

27.6

128.0

Pregnancies

 

 

 

 

 

 

Total No.

11

10

12

12

9

11

Died or aborted (before Day 29)

1

0

0

2

0

0

To term (on Day 29)

10

10

12

10

9

11

Corpora Lutea

 

 

 

 

 

 

Total no.

175

132

146

191

140

159

Average/dam mated

14.6

12.0

12.2

13.6

9.33

12.2

Live litters

 

 

 

 

 

 

Total No.*

10

6

12

9

9

11

Implant Sites

 

 

 

 

 

 

Total No.

53

42

70

64

44

80

Average/dam*

5.30

4.20

5.83

6.40

4.89

7.27

Resorptions

 

 

 

 

 

 

Total No*

--

18

3

9

1

7

Dams with 1 or more sites resorbed

--

6

2

3

1

5

Dams with all sites resorbed

--

4

--

1

--

--

Per cent partial resorptions

--

60.0

16.7

30.0

11.1

45.5

Per cent complete resorptions

--

40.0

--

10.0

--

--

Live foetuses

 

 

 

 

 

 

Total No

52

19

67

55

43

73

Average/dam*

5.20

1.90

5.58

5.50

4.78

6.64

Sex ratio (M/F)

0.73

5.00

0.63

1.04

1.26

0.92

Dead Foetuses

 

 

 

 

 

 

Total No.*

1

5

--

--

--

73

Dams with 1 or more dead

1

2

--

--

--

--

Dams with all dead

--

--

-

--

--

--

Per cent partial dead

10.0

20.0

--

--

--

--

Per cent all dead

--

--

--

--

--

--

Average foetus weight (g)

42.2

29.7

37.0

36.0

40.0

35.8

* 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

1.28

5.95

27.6

128.0

Live foetuses examined (at term)

52/10

19/6

67/12

55/9

43/9

72/11a

Sternebrae

 

 

 

 

 

 

Incomplete oss.

2/2

1/1

 

1/1

2/2

 

Scrambled

 

 

 

 

 

 

Bipartite

 

1/1

 

1/1

 

1/1

Fused

 

3/2

 

 

 

 

Extra

 

1/1

2/1

 

 

6/4

Missing

 

2/2

 

 

 

 

Other

 

 

 

 

 

 

Ribs

 

 

 

 

 

 

Incomplete oss.

 

 

 

 

 

 

Fused/split

 

5/2

 

 

3/1

 

Wavy

 

 

 

 

 

 

Less than 12

 

 

 

 

 

 

More than 13

 

 

 

 

 

 

Other

 

 

 

 

 

 

Vertebrae

 

 

 

 

 

 

Incomplete oss.

 

 

 

 

 

 

Scrambled

 

4/2

 

 

 

 

Fused

 

 

 

 

 

 

Extra ctrs. oss.

 

 

 

 

 

 

Scoliosis

 

4/3

 

 

 

 

Tail defects

 

13/4

 

 

1/1

1/1

Other

 

 

 

 

 

 

Skull

 

 

 

 

 

 

Incomplete closure

 

1/1

 

 

 

 

Missing

 

 

 

 

 

 

Craniostosis

 

 

 

 

 

 

Other; facial bones, inc

 

1/1

 

 

 

 

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

a One pup lost in processing

 

Table 4 Summary of soft tissue abnormalities

Material

Dose level (mg/kg)

Dam

Number of pups

Description

6-AN

2.5

Z 6479

1

Medial rotation of hind limbs

 

 

Z 6488

1

Medial rotation of hind limbs

 

 

Z 6489

1

Siamese pups (combined stomach and head)

 

 

Z 6490

3

Anopia, Medial rotation of hind limbs

 

 

 

2

Anopia

FDA 71-61

27.6

M 6051

2

Anopia

Conclusions:
Under the conditions of the study, the test material administered to pregnant rabbits for 13 days up to a dose level of 128 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 128 mg/kg bw.
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 and corpora lutea not determined One third used for visceral examination; should be 50% 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
Reason / purpose:
reference to same study
Qualifier:
according to
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 1.69, 9.24, 42.95 and 169.0 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 and environmental conditions:
TEST ANIMALS
- Source: Outbred
- Age at study initiation: No data
- Weight at study initiation: 214 - 225 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: 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: 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 23
Aspirin 250.0 24 21
FDA 71-61 1.69 25 24
9.24 25 24
42.95 24 24
169.0 25 21

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:
> 169 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:
> 169 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects noted
Abnormalities:
no effects observed
Developmental effects observed:
no
Table 2 Reproduction data

Dose (mg/kg)

Sham

Aspirin

1.69

9.24

42.95

169.0

Pregnancies

 

 

 

 

 

 

Total No.

23

21

24

24

24

21

Died or aborted (before Day 20)

0

1

0

0

0

0

To term (on Day 20)

23

20

24

24

24

21

Live litters

 

 

 

 

 

 

Total No.*

23

18

24

24

24

21

Implant Sites

 

 

293

 

 

 

Total No.

283

234

12.2

284

287

254

Average/dam*

12.3

11.7

 

11.8

12.0

12.1

Resorptions

 

 

 

 

 

 

Total No*

4

35

4

4

4

--

Dams with 1 or more sites resorbed

2

5

4

4

3

--

Dams with all sites resorbed

--

2

--

--

--

--

Per cent partial resorptions

8.70

25.0

16.7

16.7

12.5

--

Per cent complete resorptions

--

10.0

--

--

--

--

Live foetuses

 

 

 

 

 

 

Total No

279

197

288

280

283

254

Average/dam*

12.1

9.85

12.0

11.7

11.8

12.1

Sex ratio (M/F)

1.08

0.80

1.27

1.00

0.98

1.03

Dead Foetuses

 

 

 

 

 

 

Total No.*

--

2

1

--

--

--

Dams with 1 or more dead

--

2

1

--

--

--

Dams with all dead

--

--

--

--

--

--

Per cent partial dead

--

10.0

4.17

--

--

--

Per cent all dead

--

--

--

--

--

--

Average foetus weight (g)

3.65

2.41

3.71

3.78

3.78

3.75

* Includes only those dams examined at term

** Positive control: 250 mg/kg

 

Table 3 Summary of skeletal findings

Findings

Dose (mg/kg)

Sham

Aspirin

1.69

9.24

42.95

169.0

Live foetuses examined (at term)

191/23

137/18

198/24

193/24

196/24

176/21

Sternebrae

 

 

 

 

 

 

Incomplete oss.

71/20

86/16

83/19

54/19

61/16

47/17

Scrambled

 

 

 

 

 

 

Bipartite

 

7/5

 

 

2/2

4/3

Fused

 

 

 

 

 

 

Extra

 

 

 

 

 

 

Missing

7/6

117/7

9/7

15/8

1/1

5/4

Other

 

 

 

 

 

 

Ribs

 

 

 

 

 

 

Incomplete oss.

1/1

3/2

3/2

 

 

1/1

Fused/split

 

14/4

 

 

 

 

Wavy

14/8

54/14

34/9

12/7

15/8

13/8

Less than 12

 

 

 

 

 

 

More than 13

1/1

89/15

6/5

3/3

 

6/5

Other

 

 

 

 

 

 

Vertebrae

 

 

 

 

 

 

Incomplete oss.

6/5

120/17

12/4

21/11

5/4

10/8

Scrambled

 

1/1

 

 

 

 

Fused

 

 

1/1

 

 

 

Extra ctrs. oss.

 

 

 

 

 

 

Scoliosis

 

3/3

 

 

 

 

Tail defects

 

1/1

 

 

 

 

Other

 

 

 

 

 

 

Skull

 

 

 

 

 

 

Incomplete closure

23/10

60/13

50/16

31/12

25/13

20/11

Missing

 

9/4

 

 

 

 

Craniostosis

 

 

 

 

 

 

Other

 

1/1

 

 

 

 

Extremities

 

 

 

 

 

 

Incomplete oss.

 

4/3

 

 

 

 

Missing

 

 

 

 

 

 

Extra

 

 

 

 

 

 

Miscellaneous

 

 

 

 

 

 

Hyoid; missing

19/11

69/17

37/15

14/9

26/13

7/6

Hyoid; reduced

7/5

20/8

38/14

27/12

35/16

28/12

* 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 4910

1

Encepalomyelocele

 

 

A 4913

1

Exencephaly; exophthalmos

 

 

A 4917

1

Renal agenesis

 

 

A 4921

1

Encephalomyelocele; gastroschisis

 

 

A 4923

3

Encephalomyelocele

 

 

 

1

Exophthalmos; gastroschisis

 

 

A 4925

2

Encepalomyelocele

FDA 71-61

42.95

M 4076

1

Gastroschisis

Conclusions:
Under the conditions of the study, the test material administered to pregnant rats for 10 days up to a dose level of 169 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity is > 169 mg/kg bw.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Under the conditions of the study on disodium dihydrogenpyrophosphate, the test material administered to pregnant mice for 10 days up to a dose level of 335 mg/kg bw showed no maternal or developmental toxicity. The NOAEL for both maternal and fetotoxicity in mice is > 335 mg/kg bw. When the test material was administered to pregnant rats for 10 days up to a dose level of 169 mg/kg bw no maternal toxicity or developmental toxicity was observed. The NOAEL for both maternal and fetotoxicity is > 169 mg/kg bw.

When the test material was administered to pregnant hamsters for 10 days up to a dose level of 166 mg/kg bw no maternal toxicity or developmental toxicity was observed. The NOAEL for both maternal and fetotoxicity is > 166 mg/kg bw.

When the test material was administered to pregnant rabbits for 10 days up to a dose level of 128 mg/kg bw no maternal toxicity or developmental toxicity was observed. The NOAEL for both maternal and fetotoxicity is > 128 mg/kg bw.

It is not considered to be scientifically justified to further investigate the effects of disodium dihydrogenpyrophosphate on reprotoxicity, developmental or maternal toxicity and as such no classification is proposed for this endpoint and no further studies are deemed necessary.