Octylphosphonic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 14/08/2015 to 18/03/2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octylphosphonic acid

EC Number:

225-218-5

EC Name:

Octylphosphonic acid

Cas Number:

4724-48-5

Molecular formula:

C8H19O3P

IUPAC Name:

octylphosphonic acid

Test material form:

other: waxy solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, le Genest-Saint-Isle, France.
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: 249 g to 365 g
- Fasting period before study: No
- Housing: The animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2 ) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen since it is preferable for pregnant animals. Each cage contained a rat hut for the environmental enrichment of the animals. The cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 3413447 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From01/09/2015 to 07/10/2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared as a suspension in the vehicle according to the procedure described below:
+ weigh the required quantity of test item,
+ground the test item using a mortar and pestle,
+ add a few drops of vehicle and mix with the test item to obtain a homogeneous mixture,
+ progressively add the vehicle and transfer the mixture into a gauged flask,
+ rinse the mortar and pestle and add the rinsing liquid into the gauged flask,
+ homogenize the suspension by manual stirring before completing to final volume with vehicle.


VEHICLE: Corn Oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The results were describe in the report 42803VAA

Solvents and Reagents Reference Suppliers
Milli-Q water Not applicable CiToxLAB France
Ethanol absolute anhydrous 4125222 Carlo erba
Sulfuric acid (H2SO4) ACS 95.0-98.0% 320501-1L Sigma aldrich
Sodium Hydroxyde (pellets) anhydrous ≥ 98% S5881-500G Sigma aldrich
Diluent 1 and extraction solvent: Ethanol.
Diluent 2: sodium hydroxide at 17.5 mM.

Laboratory equipment and apparatus
Equipments Suppliers
High Performance Liquid Chromatography Ionic system (ICS5000) Thermo
Micro-balance; Balance Mettler-Toledo
Automatic pipettes Biohit
Centrifuge model Sigma 2-16 Bioblock
Ultrasonic bath model Branson 8510 Fischer
equipments for agitating which could be used (magnetic stirrers; vortex),
class A volumetric flasks,
PALL Syringe filters, Acrodisc GHP 0.45 μm, 25 mm (Waters Ref: WAT 200829),
glass pipettes.

Computerized systems
Critical computerized systems Description of data collected and/or analyzed
Empower 2 version Build 2154 Acquisition and management of chromatographic data
CIT Pharma version 2.2 Test item receipt and inventories
Documentum Management of study plan, report

Chromatographic conditions
Column: IonPac AS11 (Dionex)
Lenght = 250 mm, inner diameter = 4 mm
Guard Column: IonPac AG11 (Dionex) 4*50 mm
Mobile Phase: Mobile phase A: Sodium Hydroxyde at 350 mM
Mobile phase B: miliQ water
Elution mode: Gradient
Time (min) A (%) B (%)
0 5 95
2 5 95
8.5 17.5 82.5
9.5 17.5 82.5
9.6 5 95
14 5 95
Flow rate: 1 mL/min
Software: Empower 2 (Waters)
Column temperature: 25°C
Compartiment -TC: 25°C
Injector temperature: Not controlled
Injetion Volume: 50 µL
Needle wash: Milli-Q Water
Column Wash: Mobile phase
Regenberant: H2So4 at 2.5 mL/L
Regenerant Flow rate: about 5mL/min
Suppressor: AMMS III 4 mm
Dectection: Condutivity
Sensitivity: 2000 µS
Full Scale: 1.00 V
Polarity: Positive
CEll heater sel point: 35°C
Retention time: Octyl phosphonic acid: 7.9 min
Analysis time: 14 min

Quantification: The concentration of the test system is determined from the mean response of octyl phophonic acid in standard solutions.
The sample concentrations are determined using the equation:
[Concentration dasage form] = (Area sample / standard mean response factors) x dilution factor where:
- Area sample = Area of sample
- Standard mean response factors = Mean response factor of standard solutions 1 ans 2 (n = 10)
- Dilution factor (Also including conversion between units if required)
- Response factor = Area std / concentration Std

Results are expressed in mg/mL

Criteria for the accpetance of the analytical sequence are
- Precision of response factor for standars soluton STD 1: Coefficient of variation CV% ≤ 3.0%
- Precision of response factor for standars soluton STD 2: Coefficient of variation CV% ≤ 3.0%
- Precision of response factor for standars soluton STD 1 and STD 2 : Coefficient of variation CV% ≤ 3.0%
- accuracy of the response factor of the standards (ratio of mean response factor od STD1 with mean response factor of STD2) should be between 95.0% and 105.0 %

Details on mating procedure:

Mating: the females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as Day 0 post-coitum (p.c.).

Duration of treatment / exposure:

The dose formulations were administered daily from Day 6 to Day 20 p.c., inclusive.

Frequency of treatment:

Once a day

Duration of test:

21 days

No. of animals per sex per dose:

24 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, on the basis of the results of the following previous CiToxLAB France/Study No. 42805 RSR in which the test item, Octyl phosphonic acid (batch No. OPSS21C1), was administered daily by gavage to pregnant female rats from implantation to the day before scheduled hysterectomy (Day 6 to Day 20 post-coitum inclusive) at dose-levels of 30, 100 or 300 mg/kg/day.
No mortality was observed. At 300 mg/kg/day, severe findings were noted from Day 11 p.c. (piloerection, round back, dyspnea, cold to the touch, emaciated appearance), associated with body weight loss followed by low body weight gain (-29% mean body weight value on Day 21 p.c. when compared to controls), reduced food intake (up to -76% during the Day 9 to 12-period, when compared to controls). At necropsy, macroscopic lesions were noted in kidney and thymus, and higher number of resorptions together with lower number of live fetuses and lower mean fetal body weight (-32%) when compared to controls.
At 100 mg/kg/day, piloerection and half closed-eyes were observed from Day 11 p.c.. When compared to controls, mean lower body weight gain (-33% during the treatment period) (with body weight loss in a few animals) and lower food consumption (up to -48% during the Day 9 to 12 period) were noted. Macroscopic lesions were observed on kidney and thymus, associated with lower mean fetal body weight (-14%) when compared to controls.

Therefore, 100 mg/kg/day was selected as the high-dose level. The low-dose and mid dose were selected using a ratio representing approximately a 3-fold in exposure (i.e. 10 and 30 mg/kg/day).

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From arrival, each animal was observed once a day as part of the routine examinations.
From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c..

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each female was recorded for the following intervals:
-Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: principal thoracic and abdominal organs

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

The ovaries and uterus of the females were examined to determine:
 number of corpora lutea,
 number and distribution of dead and live fetuses,
 number and distribution of early and late resorptions,
 number and distribution of uterine scars,
 number and distribution of implantation sites.

The following classification was used to record:
 uterine scar: uterine implantation without implant,
 early resorption: evidence of implant without recognizable embryo,
 late resorption: dead embryo or fetus with external degenerative changes,
 dead fetus: dead fetus with no external degenerative changes.

For apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

A gross evaluation of placentas was also undertaken.

Fetal examinations:

The body weight of each fetus was recorded.
The sex of each fetus was determined at the time of hysterectomy by visual assessment of anogenital distance and was confirmed by internal examination of sexual organs at detailed dissection of the soft tissues or at evisceration.

- External examinations: Yes: Each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.

- Soft tissue examinations: Yes: As soon as possible after sacrifice, approximately half of the fetuses in each litter were subjected to a detailed dissection of the soft tissues, which included the observation of all the organs and structures of the neck, thorax and abdomen. The fetuses were then eviscerated and were fixed with Harrison's fluid for examination of the structures of the head.

- Skeletal examinations: Yes: The remaining fetuses per litter were eviscerated and then fixed with ethyl alcohol.
A detailed examination of the skeleton (bones + cartilages) was performed after staining with alizarin red S and alcian blue. This examination included the observation of all the bone and cartilage structures of the skull, spine, rib cage, pelvis and limbs.

- Head examinations: Yes: structures of the head.

Statistics:

Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

Historical control data:

HCD: Historical Control Data (Sprague-Dawley rat, Janvier Le Genest – France, March 2013 to June 2014).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Macroscopic findings were observed on stomach (thickened or colored mucosa) at 100 mg/kg/day and on kidneys (irregular color/granular surface/cystic/paleness/enlarged and/or with dilated pelvis) from 30 mg/kg/day.
Findings recorded on skin, ureter, uterus or placenta were observed in isolated animals and without any dose relationship. They are thus considered not to be test item treatment-related.

Mean gravid uterus weights and carcass weights of test item-treated animals were similar to control values.
Net body weight change (body weight change over the treatment period adjusted for the gravid uterus weight) was significantly decreased at 100 mg/kg/day when compared with controls (-32%, p<0.05).This finding was considered to be test item treatment-related and of toxicological importance.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were no test item-related effects on mean fetal body weight or sex ratio.

There were no fetal external variations.

There were no test item treatment-related external malformations.

The few external malformations recorded without any dose relationship in control, low- and mid-dose levels groups are common observations in this species and strain. There were no external malformations in fetuses from the high-dose group.

There were no test item treatment-related soft tissues variations.

There were no soft tissues malformations in fetuses from the test item-treated groups.
One fetus from a control litter (E26707-2) had an anophtalmia (left eye).

When compared with controls, there were no test item treatment-related cartilage findings.

At 100 mg/kg/day, an increased number of litters with fetuses with unossified or incomplete ossification of various part of the skeleton was noted, when compared to controls and Historical Control Data.
From 30 mg/kg/day, there was a dose-related increase in unossified distal phalanx when compared with controls. This was considered to be non adverse test item treatment-related.
These developmental variations were considered to be non adverse test item treatment-related and associated to low the body weight gain.

When compared with controls and Historical Control Data, there were increased fetal and litter incidences of squeletal malformations at 100 mg/kg/day (cervical ribs and absent of thoracic vertebra), and from 30 mg/kg/day (absent ribs). These malformations were considered to be related to the test item treatment and of toxicological importance.
At 10 and 30 mg/kg/day and when compared with the control group, there were increased percentages of fetuses and litters with skeletal malformations. However, each individual malformation was not observed at the high dose-level. Therefore, a test item treatment relationship was considered unlikely or of non toxicological significance.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

All the tables below summurize the maternal data

The pregnancy statusof females

Dose-level (mg/kg/day)	0	10	30	100
Number of females	24	24	24	24
Females with live fetuses	20	21	23	21
Females with total resorptions	0	0	0	0
Non-pregnant females	4 (E26692- E26693- E26711- E26712)	3 (E26728- E26730- E26738)	1 (E26739)	3 (E26763- E26773- E26783)

 

Clinical signs

Dose-level (mg/kg/day)	0	10	30	100
Piloerection	0	0	0	2
Ptyalism	0	1	0	4
Reddish vaginal discharge	1	0	2	3
Reflux at dosing	0	0	0	1
Enophthalmos	0	0	1	0
Cutaneous lesions (neck - ventral or dorsal area, head, ear)	1	0	1	2
Number of affected animals	2/24	1/24	4/24	10/24

 

Body weight and body weight change

Dose-level (mg/kg/day)	0	10	30	100
Body weight (g)
Day 6p.c.	307	307	309	310
		(0)	(+1)	(+1)
Day 21p.c.	463	453	462	448
		(-2)	(-0)	(-3)
Body weight change (g)
Days 6 - 9p.c.	+13	+12	+13	+12
Days 9 - 12p.c.	+22	+22	+22	+17*
Days 12 - 15p.c.	+20	+19	+21	+19
Days 15 - 18p.c.	+46	+44	+45	+46
Days 18 - 21p.c.	+55	+49	+52	+45
Days 6 - 21p.c.	+156	+147 (-6)	+153 (-2)	+139 (-11)

 

Maternal necropsy findings

Dose-level (mg/kg/day)	0	10	30	100
Stomach: thickened mucosa	0	0	0	1
Stomach: colored mucosa	0	0	0	1
Kidney: granular surface	0	0	0	3
Kidney: cystic	0	0	1	0
Kidney: dilated pelvis	1	0	1	0
Kidney: enlarged	0	0	1	1
Kidney: irregular color	0	0	0	4
Kidney: paleness	0	0	0	2
Ureter: dilatation	0	0	1	0
Uterus: serous contents in uterine horn	0	1	0	0
Placenta: enlarged placenta(s)	1	0	0	0
Placenta: small placenta(s)	0	0	1	0
Placenta: paleness	0	1	0	0
Skin: cutaneous lesion	1	0	0	0
Number of affected animals	3/24	2/24	2/24	6/24

 

Gravid uterus weight, carcass weight and net body weight change

Dose-level (mg/kg/day)	0	10	30	100
Number of pregnant females	20	21	23	21
Gravid uterus weight	106.1	98.4	104.4	104.9
Variation (%)		(-7)	(-2)	(-1)
Carcass weight	356.7	355.1	357.2	343.5
Variation (%)		(0)	(0)	(-4)
Net body weight change from Day 6p.c.	49.6	48.2	48.7	33.7*
		(-3)	(-2)	(-32)

 

Hysterectomy data

Dose-level (mg/kg/day)	0	10	30	100
Number of females with live fetuses at termination	20	21	23	21
Mean number ofcorpora lutea	16.0	15.1	15.4	15.8
Mean number of implantation sites	14.5	13.3	13.9	14.3
Mean pre-implantation loss (%)	8.5	13.6	9.0	8.7
Mean number of fetuses	13.6	12.3	12.8	13.3
Dead fetuses (%)	0.0	0.0	0.0	0.0
Mean number of implantation scars	0.0	0.0	0.0	0.0
Mean number of early resorptions	0.9	1.0	1.1	0.9
Mean number of late resorptions	0.1	0.0	0.0	0.1
Mean post-implantation loss (%)	6.6	7.3	8.3	6.6

Applicant's summary and conclusion

Conclusions:

The test item, Octylphosphonic acid (batch No. OPSS21C1, 93% purity), was daily administered as suspension in corn oil at dose-levels of 10, 30 or 100 mg/kg/day, from Day 6 to Day 20 p.c., by oral route (gavage), to mated female Sprague-Dawley rats.
At 100 mg/kg/day, non adverse excessive salivation and piloerection were observed. A significantly reduced mean body weight gain was recorded between Days 9 and 12 p.c., and between Days 18 and 21 p.c.. Macroscopic findings were observed on stomach and on kidneys, and net body weight change was decreased when compared with controls. Fetal skeletal variations and malformations were observed.
At 30 mg/kg/day, macroscopic findings were observed on kidneys, and non adverse fetal skeletal variations were noted.
At 10 mg/kg/day, there were no effects of the test item.
On the basis of the results obtained in this study, the No Observed Adverse Effect Level (NOAEL) was considered to be 30 mg/kg/day for both maternal parameters and for embryo-fetal development.
At 100 mg/kg/day, in the context of maternal toxicity, the test item treatment was associated with fetal dysmorphogenic potential.

Executive summary:

Octylphosphonic acid was daily administered as suspension in corn oil at dose-levels of 10, 30 or 100 mg/kg/day, from Day 6 to Day 20 p.c., by oral route (gavage), to mated female Sprague-Dawley rats. At 100 mg/kg/day, non adverse excessive salivation and piloerection were observed. A significantly reduced mean body weight gain was recorded between Days 9 and 12 p.c., and between Days 18 and 21 p.c.. Macroscopic findings were observed on stomach and on kidneys, and net body weight change was decreased when compared with controls. Fetal skeletal variations and malformations were observed. At 30 mg/kg/day, macroscopic findings were observed on kidneys, and non adverse fetal skeletal variations were noted. At 10 mg/kg/day, there were no effects of the test item. On the basis of the results obtained in this study, the No Observed Adverse Effect Level (NOAEL) was considered to be 30 mg/kg/day for both maternal parameters and for embryo-fetal development. At 100 mg/kg/day, in the context of maternal toxicity, the test item treatment was associated with fetal dysmorphogenic potential.