2-phosphonobutane-1,2,4-tricarboxylic acid

Endpoint summary

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.