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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

For 2-phosphonobutane-1,2,4-tricarboxylic acid there is no screening study for reproduction/developmental toxicity available.

A developmental study according OECD 414 (Renhof, 1984) with 2-phosphonobutane-1,2,4-tricarboxylic acid is available. Therefore a screening study (OECD 421 or 422) does not need to be conducted according REACH Regulation (EC) No 1907/2006, Annex VIII, column 2.

For 2-phosphonatobutane-1,2,4-tricarboxylic acid there is no EOGRTS (OECD 443) or a two-generation reproduction toxicity study available.

Based on the ECHA communication number: CCH-C-0000002299-67-03/F ECHA the registrant concludes, that an EOGRTS (Extended One-Generation Reproductive Toxicity Study) according to COMMISSION REGULATION (EU) 2015/282 of 20 February 2015 amending Annexes VIII, IX and X to Regulation (EC) No 1907/2006 is not necessary and should be omitted. Therefore a waiver for the EOGRTS is provided (see section 7.8.1).

Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate ("PBTCNa4") as a surrogate for 2-phosphonatobutane-1,2,4-tricarboxylic acid is of very low systemic toxicity after sub-chronic exposure with NOEL levels at or above 424 mg/kg/day (males) and 633 mg/kg/day (females) (Löser E, Kaliner G, 1976).

Toxicologically relevant information concerning reproduction can be deduced from this sub-chronic study because gravimetric and histopathologic investigation of the reproductive organs was reported. Testes and ovaries were weighted for all animals (15/sex/group). No compound related difference in organ weight was observed in any dose group. In addition histopathologic evaluation was performed in all dose groups (5/sex/dose). No compound related effect war observed in any reproductive organ investigated; seminal vesicle, prostate, testes and adrenals in males; uterus and ovaries in females.

Link to relevant study records
Reference
Endpoint:
reproductive toxicity, other
Remarks:
other: Sub-chronic oral repeated dose toxicity study with histopathologic investigation of the reproductive organs.
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted 1976 using scientifically accepted methods similar to OECD TG 408.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
other: similar to OECD TG 408 (repeated dose 90 day oral toxicity in rats)
Deviations:
not applicable
Remarks:
no analytical verification of the test substance was reported
Principles of method if other than guideline:
Sub-chronic repeated dose toxicity study in rats including gravimetric and histopathologic investigation of the reproductive organs.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- SPF
- Source: Winkelmann, Kirchborchen, Germany
- Age at study initiation: 28-32 days
- Weight at study initiation (male rats): Average 80-85 g
- Weight at study initiation (female rats): Average 78-81 g
- Housing: cages Makrolon, type II; 1 animals per cage
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +/-1°C
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on mating procedure:
No mating
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
NA
Duration of treatment / exposure:
3 month
Frequency of treatment:
daily (continuous in the feed)
Dose / conc.:
50 ppm
Remarks:
Doses / Concentrations:
50 ppm
Basis:
nominal in diet
approx. 4.2 mg/kg/day in males and 6.1 mg/kg/day in females
Dose / conc.:
200 ppm
Remarks:
Doses / Concentrations:
200 ppm
Basis:
nominal in diet
approx. 15 mg/kg/day in males and 13 mg/kg/day in females
Dose / conc.:
1 000 ppm
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
nominal in diet
approx. 84 mg/kg/day in males and 125 mg/kg/day in females
Dose / conc.:
5 000 ppm
Remarks:
Doses / Concentrations:
5000 ppm
Basis:
nominal in diet
approx. 424 mg/kg/day in males and 633 mg/kg/day in females
No. of animals per sex per dose:
test group: 15 animals / sex/ dose level
control group: 30 animals / sex
Control animals:
yes, plain diet
Details on study design:
Endpoints relevant for reproductive toxicity were included in this study as follows:



- Weights of reproductive organs (testes and ovaries) were investigated for all animals at the end of the experiment.



For histological examination of the following reproductive organs of five male and five female rats were reported:

- testes, prostate, seminal vesicle, ovaries and uterine horn ( highest dose group).

- testes and ovaries (all dose groups).



See cross reference to chapter 7.5.1. for a detailed description of all parameters investigated in this sub-cronic toxicity study similar to OECD TG 408.

CAGE SIDE OBSERVATION and DETAILED CLINICAL OBSERVATIONS: Yes
- The animals were observed for changes and symptoms daily (appearance, e.g. of fur, behaviour, e.g. concerning drinking and eating, motor activity ).

BODY WEIGHT: Yes
- The bodyweights of all involved animals were recorded weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal was determined weekly. Based on that the mean daily consumption was calculated and given as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One and three months after start of the feeding study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 5 per sex and dose level
- Parameters examined: See "study design"

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: One and three months after start of the feeding study
- Animals fasted: No data
- How many animals: 5 per sex and dose level
- Parameters examined: See "study design

URINALYSIS: Yes
- Time schedule for collection of urine: Not indicated
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: See "study design

NEUROBEHAVIOURAL EXAMINATION: No data
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
5 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at any parameter in the highest dose tested (ca. 424 mg/kg/day in males and 633 mg/kg/day in females)
Remarks on result:
other: Generation not specified.
Key result
Dose descriptor:
NOEL
Remarks:
reproductive organs
Effect level:
5 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect on weight or histopathology of reproductive organs up to the highest dose tested (ca. 424 mg/kg/day in males and 633 mg/kg/day in females)
Remarks on result:
other: Generation not specified.
Critical effects observed:
no
Remarks on result:
not measured/tested
Remarks:
sub-chronic oral repeated dose toxicity study with histopathologic investigation of the reproductive organs.
Critical effects observed:
not specified
Key result
Reproductive effects observed:
no

Organ weights:

control/50/200/1000/5000ppm group

Males

Absolute testes weights:

3305/3415/3306/3328/3295 mg

Relative testes weight

972/1020/958/956/966 mg/100g body weight

Females

Absolute ovary weight:

92/94/97/97/105/89 mg

Relative ovary weight:

47/48/49/53/44

Histopathology:

dose 0 50 200 1000 5000

Seminal vesicles: no effect at any group

Prostate

No effect:        0/4 3/5

Epithelial proliferation                                    2/5

Testes/adrenals

Without findings                                   4/5 4/5 4/5 2/5 4/5

Cellular infiltration (very slight)                 1/5 1/5 1/5 3/5 1/5

Uterus/Ovaries

Without findings                                   3/5 0/5 1/5 2/5 2/5

Cellular dilation; endometrium                          1/5                                                  2/5

Dilation (unilateral)                                                             1/5                               1/5

Dilation (bilateral)                                             1/5             1/5              3/5

Cellular infiltration (slight)                                                    1/5             1/5                             2/5

Cellular infiltration (very slight)                                    2/5                                                1/5

Conclusions:
Doses of up to 5000 ppm tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate applied over 3 months were tolerated without any effects.
The NOAEL of the tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate is equal or higher than 5000 ppm (equivalent to about 424 mg/kg bw for male rats and 633 mg/kg bw for female rats).
Executive summary:

The available information on repeated dose toxicity gave no evidence of any compound related effect in an early but comprehensive sub-chronic toxicity study similar to OECD TG 408 in which technical tetrasodium hydrogen 2-phosphonobutane-1,2,4-tricarboxylate was administered to 15 female and 15 male Wistar rats in diet, at dose levels of 0, 50, 200, 1000, and 5000 ppm (Löser E, Kaliner G, 1976).


 


 


All doses were tolerated without any compound related effect; for further details see chapter Repeated Dose Toxicity.


 


 


Consequently, it can be concluded that tetrasodium hydrogen 2-phosphonobutane-1,2,4-tricarboxylate is of very low systemic toxicity after sub-chronic exposure with NOEL levels at or above 424 mg/kg/day (males) and 633 mg/kg/day (females).


 


Toxicologically relevant information concerning reproduction can be deduced from this sub-chronic study because gravimetric and histopathologic investigation of the reproductive organs was reported. Testes and ovaries were weighted for all animals (15/sex/group). No compound related difference in organ weight was observed in any dose group. In addition histopathologic evaluation was performed in all dose groups (5/sex/dose). No compound related effect war observed in any reproductive organ investigated; seminal vesicle, prostate, testes and adrenals in males; uterus and ovaries in females.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
424 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Data on developmental toxicity are available in a prenatal developmental toxicity study similar to OECD TG 414 (Renhof, 1984). Under the experimental conditions, the test item is considered to have no maternal or embryonic toxic effects and no teratogenicity effects in rats, even at the highest dose tested 1000 mg/kg/day (limit dose).

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984-01 until 1984-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No analytical verification of the test substance was performed.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
, the test substance was administered daily from day 6-15 of pregnancy (organogenesis period) instead of day 5-19 of pregnancy
Principles of method if other than guideline:
See above, deviations.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: sexually mature
- Weight at study initiation: Males-above 300 gr. Females-189-231 gr.
- Fasting period before study: Not indicated
- Housing: Makrolon cages type III (males), type II (females)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Not directly mentioned. At least 6 days before exposure to the test substance ( the test substance is given in the 6th day of pregnancy)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 40%
- Air changes (per hr): Not mentioned
- Photoperiod (hrs dark / hrs light): 12 / 12 hours rhythm


Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): NA
- Mixing appropriate amounts with (Type of food): NA
- Storage temperature of food: NA


VEHICLE: Water
- Justification for use and choice of vehicle (if other than water): NA
- Concentration in vehicle: Test substance was dissolved in water and given in volume of 10 mL/kg to a final concentrations of 100 mg/kg bw, 300mg/kg bw, 1000 mg/kg bw
- Amount of vehicle (if gavage): See above
- Lot/batch no. (if required): Not required
- Purity: NA
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
NA
Details on mating procedure:
Cohoused:
- M/F ratio per cage: 1 male / 2 females
- Length of cohabitation: over night
- Proof of pregnancy: sperm in vaginal smear, referred to as day 0 of pregnancy
Duration of treatment / exposure:
BAYHIBIT-AM was administrated from the 6th-15th day of pregnancy
Frequency of treatment:
Daily treatment
Duration of test:
20 days. At day 20 of pregnancy, embryos were taken out by abdominal delivery (cesarian surgery).
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control group
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
25 Wistar rats per group (4 groups)
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: NA
- Rationale for animal assignment (if not random): Random
Maternal examinations:
- Time schedule: Daily examination for changes in appearance and behaviour of the pregnant rats in all the four groups (control and other 3 different test substance concentration groups)
- Time schedule for examinations: During the whole pregnancy period
- Time schedule for examinations: Daily, by gavage.





Ovaries and uterine content:
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: Yes
- Other: Weight of placenta
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No data
Statistics:
For the statistic calculations of the loss of weight, number of implantation and resorptions the WILCOXON-MANN-WHITNEY-U-TEST was used.
The Chiquadrat-Test was used for the statistic calculations of the embryotoxicity parameters
If not specified differently, the significant difference to control is under 5%.
Indices:
Examination for visceral modifications was according the modification of the WILSON technique.
The examination of the bones was done according to the DAWSON technique
Historical control data:
NA
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
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
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
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:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
NA - no effects
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
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
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
NA - no effects
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: teratogenicity
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: embryotoxicity
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: fetotoxicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Weight gain during pregnancy

 Group

    Mean weight gain of pregnant animals in gramm during

   Application  Total pregnancy
 Control  24.7 83.7 
 100 mg/kg bw  20.9  72.3
 300 mg/kg bw  23.0  82.3
 1000 mg/kg bw  22.2  81.7

Results on insemination and fertilisation

   Inseminated females  Fertilised total  Females in % of insemination  Pregnant total  Females in % of fertilisation
 Control  25  19  76.0  19  100.0
 GRP. 1  25  20  80.0  20 100.0 
 GRP. 2  25  24  96.0  22  91.7
 GRP. 3  25  23  92.0  22  95.7

Number of fetuses and examination method

   Total number of fetuses  Examination according to Wilson  Bone staining
 Control  198 60   138
 GRP. 1 100 mg/kg  183  55  128
 GRP.2 300 mg/kg  255  77  178
 GRP.3 1000 mg/kg  232  67  165

Effect of Bayhibit AM on pregnant rats and their fetuses

Group     Weight gain during (G)        Number (per dam) of fetuses           Mean weight (G)   No. of fetuses examined by   Fetuses with    Runts
   Pregnancy  Treatment  Implant  Male  Female  Sum Losses   Foetus  Placent.  Wilson  Dawson

 Minor skeletal

deviat.

 Malfor-

mations

 < 3 G

Control

83.7

13.8 

24.7

4.7

10.8

2.4 

4.9

1.7 

5.5

1.7 

10.4

2.3 

0.4

0.6 

3.61

0.50 

0.59

0.05 

3.16

0.83 

 7.26

1.59

2.32

1.77 

0.0

0.0 

0.68

1.45

GRP.1

72.3

22.4

20.9

7.6

10.8

1.6

4.9

2.0 

4.2*

1.6 

9.1

2.8 

 1.7

3.0

 3.47

0.34

0.50

0.06 

2.75

0.91 

6.40

1.93 

2.10

1.89 

0.05

0.22 

0.40

0.68 

GRP.2

82.3

21.7 

23.0

5.5

11.4*

2.6 

5.2

2.4 

5.5

2.4 

10.6

3.7 

0.8

1.3 

3.39

0.28 

0.59

0.08 

3.50

0.74 

8.09

1.11 

3.82*

2.34 

0.09

0.29

1.18

2.65 

 GRP.3

81.7

10.8

22.2

5.4

10.9

2.6 

5.3

2.0 

4.8

2.0 

10.1

2.8 

0.8

1.0 

3.52

0.22 

0.58

0.06 

3.05

0.79

7.50

1.10 

2.95

1.99 

0.05

0.21 

0.32

0.78 

* Significant difference to control (P < 0.05)

Conclusions:
No maternal toxicity, no teratogenicity, no embryotoxicity under study conditions.
Executive summary:

After oral application of BAYHIBIT-AM (2 -phosphonobutane-1,2,4 -tricarboxylic acid) up to a maximal dosage of 1000 mg/kg no signs of maternal toxicity were found (by means of death, weight loss, changes in appearance and behaviour). Moreover, female mother rats were proved later to be fertile. No influence was observed in embryo and foetus development (resorption, placenta weight, any skeletal and internal malformation). The NOEL value for these effects is therefore determined as 1000 mg/kg bw/day.

Under the experimental conditions, the test item is considered to have no maternal and embryonic toxic effects and no teratogenic effects in rats.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The materials/methods and results are described in detail und are sufficient for evaluation
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

Based on the low toxicity profile and the available data on reproduction, there is sufficient data available to conclude that 2-phosphonobutane-1,2,4-tricarboxylic acid is not a reproduction toxicant.


 


According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.

Additional information