Dialkyl C18 and C18-unsaturated phosphonates

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-05-30 to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-05-31 to 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation:10 to 12 weeks for males and 12 to 14 weeks for females
- Weight at study initiation: 280 to 350 g for,males and 190 to 240 g for females
- Adult animals will be fasted overnight before sampling for clinical laboratory determinations.
- Housing:
- Premating: 5 animals per cage
- One male + one female together
- Gestation: 5 males together and 1 female per cage
- Lactation: 1 female + litter in etha same cage
- Diet (e.g. ad libitum): pelleted complete diet ad libitum (Diet reference A04C-10), sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants.
- Water (e.g. ad libitum): softened and filtered (0.2 μm) mains drinking water ad libitum, analysed at least twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.
- Acclimation period: at least 5 days between animal arrival and start of pre-test estrous cycle smears (females) or start of treatment (males).
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): W 35%
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark (except when required for technical acts)
IN-LIFE DATES: From: 2017-05-30 To: 2017-09-09

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Weekly

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5mL/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 14 days.
- Proof of pregnancy: The day of mating will be confirmed by the presence of sperm in a vaginal smear or a vaginal plug and will be recorded and taken as day 0 of gestation (G 0).
- Mated females will be separated from the males once mating has been confirmed and smearing will cease or when the appearance of the female suggests pregnancy from an undetected mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Conditions
Instrument Acquity UPLC system (Waters, Milford, MA, USA)
Detector Xevo TQ-S mass spectrometer (Waters)
Column Acquity UPLC BEH C8, 50 mm x 2.1 mm i.d., dp =1.7 µm (Waters)
Column temperature 40°C ± 1°C
Injection volume 1 µL
Mobile phase 5 mM ammonium acetate in 90/10 (v/v) acetonitrile/water
Flow 0.9 mL/min
MS detection
+ Ionization source ESI+
+ Cone voltage 60 V
+ Collision energy 28 eV
+ Quantitation m/z 583.5 --> m/z 83.0

Preparation of Solutions
Stock and Spiking Solutions
Stock solutions of the test item were prepared in THF at concentrations of 2000 mg/L.
Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent of the spiking solutions was THF.

Calibration Solutions
Five calibration solutions in the concentration range of 0.01 – 1 mg/L were prepared from two stock solutions. The end solution of the calibration solutions was THF.
QC Samples
Approximately 400 – 500 mg vehicle was accurately weighed and spiked with the test item at a target concentration of 1 or 200 mg/g.
The QC samples were prepared in volumetric flasks of 20 or 50 mL.
The flasks were filled up to mark with THF. The solutions were further diluted to obtain an end solution of THF and concentrations within the calibration range.
The blank QC sample consisting of blank medium was treated similarly to the QC samples.

Formulas
Response (R) Peak area of the test item [units]

Response factor (Rf) Rf = (R / CN)
where:
CN: nominal concentration [mg/L]

Calibration curve R = a CN + b
where:
a: slope [units x L/mg]
b: intercept [units]

Analyzed concentration (CA) CA = ((R - b) / a ) x (( V x d) / w) [mg/g]
where:
w weight sample [mg]
V volume volumetric flask [mL]
d dilution factor

Accuracy (CA / CN) x 100 [%]

Duration of treatment / exposure:

For both sexes: 14 days before mating, throughout the mating period and up to the day before necropsy (i.e. minimum of 28 days for males).

For females:
- during gestation (the first day of gestation is designated as G 0) and at least 13 days after parturition, up to and including the day before scheduled necropsy (the first day of birth is designated as L 0).
- apparently unmated females for at least 24 days after the last day of the mating period.

Frequency of treatment:

Once daily

Details on study schedule:

- Age at mating of the mated animals in the study: 12-14 weeks old for males and 14-16 weekd old for males

Dose / conc.:

75 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

750 mg/kg bw/day

No. of animals per sex per dose:

10 animals per sex and per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Under the defined experimental conditions, daily oral (gavage) of Alkenyl phosphonate at doses of 500 and 1000 mg/kg/day in the female rat during 10 days was associated with a slight reduced body weigh gain and hypersalivation at 1000 mg/kg/day only. No macroscopic findings were noted at 500 and 1000 mg/kg/day.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: each weighing day

BODY WEIGHT: Yes
- Time schedule for examinations:
Male and females will be weighed on the first day of exposure (prior to the first exposure) and weekly thereafter during pre-mating and mating periods.
Mated females will be weighed:
− on Days 0, 6, 9, 12, 15, 18 and 20 of gestation
− on Days 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Food consumption of males will be recorded weekly during the pre-mating period.
Food consumption of females will be recorded for the following periods:
− weekly during the pre-mating period
− gestation: Days 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 20
− lactation: Days 1 to 4, 4 to 7 and 7 to 13.

WATER CONSUMPTION: No

OTHER: Functional Tests
Animals examined: 5 randomly selected animals/sex/group (for females, only those with live pups will be selected).
Frequency:
– Males: at the end of their treatment period, shortly before scheduled kill.
– Females: during lactation, shortly before scheduled kill.
The following tests will be performed:
– auditory reflex
– pupillary reflex
– righting reflex
– fore-limb grip strength
– locomotor activity in an open field test. Activity will be monitored by a video image analysis system (Videotrack supplied by Viewpoint, Lyon, France). The arena is divided into nine equal invisible regions; the time spent by the animal in each type of region (i.e. corner, centre or lateral) will be recorded. Motor activity is divided into three categories: ambulatory activity (in which the centre of the image moved at more than 7 cm/sec), small movements (including grooming etc.) and inactivity. The proportion of time spent engaged in each type of activity and the total distance travelled by the rat will be calculated.

Haematology, coagulation, chemistry for F0
Hormone Thyroxine (T4) and TSH

Oestrous cyclicity (parental animals):

Daily vaginal smears will be taken to determine the cycle stage beginning 14 days prior to treatment, the first 14 days of treatment and during mating until evidence of copulation is observed. Vaginal smears will continue for those females with no evidence of copulation until termination of the mating period.
On the day of scheduled necropsy, a vaginal smear will be taken to determine the cycle stage and allow correlation with histopathology of female reproductive organs.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
number of pups born (live and dead);
– external abnormalities of the pups;
– number, weight and sex of pups alive on PND 1, 4, 7 and 13.
– anogenital distance (AGD) will be measured on PND 1. The AGD will be
normalized to the cube root of body weight.
– all males in each litter will be examined for the number of areola/nipples on PND 13.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.

Hormone (T4) for PND 13

Blood analysis for PND4 and PND13

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
All adult males and females will be weighed before necropsy (except any found dead or killed moribund).
Animals will be killed by carbon dioxide inhalation and exsanguination
All animals (including any found dead or killed moribund and females showing signs of parturition difficulties or total litter death) will be submitted to necropsy procedures including an examination of following:
– external surface
– all orifices
– cranial cavity
– thoracic and abdominal cavities and organs and their contents
– the carcass.
Special attention should be paid to the organs of the reproductive system.

For females sacrificed before parturition (including any found dead or killed moribund after the first day of pairing), the ovaries and uterus will be removed and examined including examination of the placentae. The following data will be recorded:
 pregnancy status
 number of corpora lutea
 number of intrauterine implantations
 number of live embryos
 number of intrauterine deaths (resorption sites).
The uterus of all adult females will be placed in ammonium sulphide solution in order to stain any previously undetected implantation sites. The number of corpora lutea will be counted.
Any foetuses will be examined externally where possible and discarded.


HISTOPATHOLOGY / ORGAN WEIGHTS
See table 1 and 2

Postmortem examinations (offspring):

SACRIFICE
Pups (extra pups on PND4, any moribund pups and surviving pups on PND13) will be killed by intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
- Pups (including any found dead or killed moribund) will be necropsied. Any external abnormalities observed will be recorded but not preserved. For any pups found dead or killed moribund, the stomach will be examined for the presence of milk and defects or cause of death will be evaluated, if possible.
Each pup will be sexed and examined for external defects with special attention being paid to the external reproductive organs.

Statistics:

Statistical analyses will be performed by the Provantis data acquisition system, where appropriate, as follows:
The best transformation for the data (none, log or rank) will be determined depending upon
- the kurtosis of the data
- the probability of the Bartlett's test for homogeneity of the variances and
- an assessment of whether the size of the groups are approximately equal or not.
Non- or log-transformed data will be analysed by parametric methods.
Rank transformed data will be analysed using non-parametric methods.
Data will be then analysed to test for a dose-related trend to detect the lowest dose at which there is a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means will be assessed by analysis of variances (ANOVA) for parametric
data or Kruskal-Wallis test for non parametric data.
If no trend is found and means are not homogeneous, the data will be analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Selected incidence data will be analysed using the Provantis data acquisition system and/or a SAS software package. A chi2 test will be used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 is significant.
Functional test, estrous cycles and pre-coital interval data will be analysed using a SAS software package.
Levene’s test will be used to test the equality of variance across groups and Shapiro-Wilk's test will be used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups will be analysed using ANOVA followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group will be analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.

Reproductive indices:

The reproductive indices will be calculated as follows:
• Pre-coital interval (in days) = sum of days until successful insemination / number of inseminated females
• Male and female copulation index (in %) = (number of inseminated females / number of paired animals) x100
• Male and female fertility index (in %) = (number of pregnant females / number of inseminated females) x 100
• Pre-birth loss (%) = (number of implantations - number of offspring born / number of implantations )x 100
• Sex ratio (proportion of male pups in %) = (number of males / number of pups) x 100

Offspring viability indices:

• Live birth index (in %) = (number of pups born alive / number of pups born) x 100
• Viability index (in %) = (number of pups alive on PND 4 (before culling) / number of pups alive at birth) x 100
• Lactation index (%) = number of pups alive on PND 13 / number of pups alive on PND 4 (after culling)) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was a higher incidence of hypersalivation associated or not with abnormal foraging and/or pedalling from Day 18 of treatment up to the end of the dosing period for both sexes in the 250 and 750 mg/kg/day groups. These signs were considering not adverse due to their nature, minor severity and transience (i.e. just after dosing).

Dilated pupils were noted for one male treated at 750 mg/kg/day on Day 18. This isolated finding was considered incidental.

One female (no. 151) treated at 250 mg/kg/day had piloerection and irregular/rapid breathing, was pale, subdued and recumbent on the day of mating (G0).These isolated clinical signs were noted only in the mid dose group and occurred at the time of mating while the female was housed with the male and were therefore considered incidental.

Other incidental findings were observed such as a torn claw, scab or localized hairloss.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Control female no. 120 was found dead on Gestation Day 18 (G18). Dark thymus, enlarged dark adrenal gland, distended urinary bladder and dilated ureters were noted at the macroscopic examination.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect on mean body weight gain in any group.

Mean body weight gain for males during the dosing period or females during the premating and gestation periods was comparable with, or superior to, that noted in the control group.

During the lactation phase, there was a slightly lower mean body weight gain for the females in all treated groups when compared with the control. Since this change was not dose-related and had no adverse effect on terminal body weights (-3.1, -4.4 and -1.2% in the 75, 250 and 750 mg/kg/day groups, respectively, when compared with control), it was considered not toxicologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect on mean food consumption in any group for either sex during the dosing period.

Mean food consumption was slightly lower during gestation in the 75 and 750 mg/kg/day groups (- 3.9 and -3.4%, respectively) and during the lactation period in the 250 and 750 mg/kg/day groups (-4.4 and -2.9% respectively). However, all values remained above the historical control data means (22.5 and 45.8 g/day from G0 to G20 and from L1 to L14, respectively). These findings were therefore considered as incidental.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences noted in haematological and coagulation parameters between control and treated rats of either sex that were considered to be toxicologically relevant.
Although differences in mean values attained statistical significance for males compared with the concurrent control, the decrease in mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, fibrinogen concentration and platelet concentration were considered of no toxicological significance since there was no dose-related trend and/or in view of the low magnitude. In addition, mean values were within the historical control range.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences noted in serum clinical chemistry parameters between control and treated rats of either sex that were considered to be toxicologically relevant.
Although differences in mean values attained statistical significance for males compared with the concurrent control, the decrease in sodium, urea, inorganic phosphorus, globulin and bile acid concentrations and the increase in creatinine concentration were considered of no toxicological significance since there was no dose-related trend and/or in view of the low magnitude. In addition, mean values were within the historical control range.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed.
Fore-limb grip strength mean values were lower than controls for males at
750 mg/kg/day and females at 75 mg/kg/day. The effect was not dose-related since the mean value in the 250 mg/kg/day group was comparable or higher to that of control for either sex and the mean value in the 750 mg/kg/day was higher to that of control for females.
This was therefore considered to be due to intra-group variability and therefore incidental.
The variation in motor activity (open field) did not indicate a relation with treatment.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment with the test item in any group.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no test item-related effect on mating performance in any group. All animals mated with the exception of one pair in each of the control and 250 mg/kg/day groups (female nos. 113 and 160 paired with male nos. 103 and 150, respectively). All mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle). The mean pre-coital interval was therefore normal in all groups.

There was no test item-related effect on fertility. Most mated females became pregnant with the exception of one female in each of the control and 75 mg/kg/day groups (female nos. 112 and 135, respectively).

Dose descriptor:

NOAEL

Effect level:

>= 750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

- Litter data

a/ Parturition
There were 7, 9, 8 and 10 females in the control, 75, 250 and 750 mg/kg/day groups, respectively, that successfully completed delivery of liveborn pups.
One female given 250 mg/kg/day (no. 154) lost the entire litter on Lactation Day 0. This isolated finding in the intermediate dose group was considered incidental.
The mean duration of gestation was comparable (approximatetly 22 days) in all groups.

b/ Number of implantation sites
The mean number of implantation sites was comparable or higher to that of control in all treated groups.

c/ Pre-birth loss
There was no test item-related effect on the percentage pre-birth loss in any group.
The value was slightly higher in the 250 mg/kg/day group (12.8%) compared with other groups (6.3, 5.7 and 7.3% in the control, 75 and 750 mg/kg/day groups, respectively) but remained within the historical control data range (1.2 – 12.9%). This difference in the intermediate dose group was considered incidental.

d/ Pup viability and litter sizes
There were no test item-related differences in pup viability and litter size.
The mean litter size at birth was incidentally higher in the 75 and 750 mg/kg/day groups (13.1 and 14.2 pups/litter, respectively) than in the control and 250 mg/kg/day groups (12.6 and 12.0 pups/litter, respectively).
The number of live pups on Day 4 before culling compared to the number of pups alive at birth was lower in the 750 mg/kg/day group (92.9%) when compared with other groups (97.7, 99.2 and 100% in the control, 75 and 250 mg/kg/day groups, respectively). However, the value was exacerbated by one female (no. 174) given 750 mg/kg/day with an incidental high litter size (20 pups delivered) which lost seven pups between L0 and L4. After excluding this female, the viability index was comparable with other groups (97.5%) and was within the historical control data range (94.1 to 100%).
The number of live pups on PND 13 compared with the number of offspring on Day 4 (post culling) was not affected by treatment. There was no pup found dead or missing in any treated group.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed

Reproductive effects observed:

no

Conclusions:

Under the experimental conditions of the study, the daily oral (gavage) administration of the test item, Alkenyl phosphonate, to the rat during pre-mating (males and females), during pregnancy and lactation (females) at doses of 75, 250 and 750 mg/kg/day induced no reproduction or developmental toxicity.
Based on these results, a reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 750 mg/kg was derived

Executive summary:

Objective

The objectives of the study was to evaluate the potential toxic effects of the test item, Alkenyl phosphonate, when exposed for a minimum of 28 days and the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. Parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No-Observed-Adverse-Effect-Levels (NOAELs) were evaluated.

 

Procedure

Alkenyl phosphonate was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 75, 250 and 750 mg/kg/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, 1 % (w/v) Carboxymethylcellulose in water, alone. Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females that delivered were treated for at least 51 days, i.e. during 2 weeks prior to mating, during mating, during assumed pregnancy, and during 13 days of lactation. Females which failed to deliver healthy offspring were treated for at least 40 days.

The following observations and examinations were evaluated: mortality / morbidity, clinical signs, functional observations and locomotor activity, body weight, food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4, macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: copulation and fertility indices, pre-coital time, number of implantation sites, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy). Formulations were analyzed during the study to assess accuracy, homogeneity and stability.

 

Results

The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations and the formulations of Group 2 and Group 4 were homogeneous.

No differences in Total T4 levels were noted among the different groups of adult males or among the different groups of PND 13 pups.

There was no test item-related mortality in any group.

There were no adverse test item-related clinical signs. There was a higher incidence of hypersalivation associated or not with abnormal foraging and/or pedalling for both sexes in the 250 and 750 mg/kg/day groups.

There were no test item-related changes in parental body weight and food consumption, or on the haematological and serum clinical chemistry parameters.

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex, grip strength or open field tests were observed for the males or females at any dose level.

There were no test item-related effects on mating performance of the males and females or on fertility in any group.

There were 7, 9, 8 and 10 females in the control, 75, 250 and 750 mg/kg/day groups, respectively, that successfully completed delivery of liveborn pups.

There were no test item-related effects on the number of implantation, pre-birth loss, litter size, pup viability or pup body weight.

There were no test item-related effects on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

There was no test item-related effect on anogenital distance (normalized for body weight) for the male and female pups.

There was no evidence of any test item-related parental histological findings.

 

Conclusion

Under the experimental conditions of the study, the daily oral (gavage) administration of the test item, Alkenyl phosphonate, to the rat during pre-mating (males and females), during pregnancy and lactation (females) at doses of 75, 250 and 750 mg/kg/day induced no reproduction or developmental toxicity.

Based on these results, a reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 750 mg/kg was derived.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dialkyl C18 and C18-unsaturated phosphonates

EC Number:

701-298-1

Cas Number:

64051-29-2

Molecular formula:

Not applicable for a UVCB Substance

IUPAC Name:

Dialkyl C18 and C18-unsaturated phosphonates

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation:10 to 12 weeks for males and 12 to 14 weeks for females
- Weight at study initiation: 280 to 350 g for,males and 190 to 240 g for females
- Adult animals will be fasted overnight before sampling for clinical laboratory determinations.
- Housing:
- Premating: 5 animals per cage
- One male + one female together
- Gestation: 5 males together and 1 female per cage
- Lactation: 1 female + litter in etha same cage
- Diet (e.g. ad libitum): pelleted complete diet ad libitum (Diet reference A04C-10), sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants.
- Water (e.g. ad libitum): softened and filtered (0.2 μm) mains drinking water ad libitum, analysed at least twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.
- Acclimation period: at least 5 days between animal arrival and start of pre-test estrous cycle smears (females) or start of treatment (males).
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): W 35%
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark (except when required for technical acts)
IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Conditions
Instrument Acquity UPLC system (Waters, Milford, MA, USA)
Detector Xevo TQ-S mass spectrometer (Waters)
Column Acquity UPLC BEH C8, 50 mm x 2.1 mm i.d., dp =1.7 µm (Waters)
Column temperature 40°C ± 1°C
Injection volume 1 µL
Mobile phase 5 mM ammonium acetate in 90/10 (v/v) acetonitrile/water
Flow 0.9 mL/min
MS detection
+ Ionization source ESI+
+ Cone voltage 60 V
+ Collision energy 28 eV
+ Quantitation m/z 583.5 --> m/z 83.0

Preparation of Solutions
Stock and Spiking Solutions
Stock solutions of the test item were prepared in THF at concentrations of 2000 mg/L.
Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent of the spiking solutions was THF.

Calibration Solutions
Five calibration solutions in the concentration range of 0.01 – 1 mg/L were prepared from two stock solutions. The end solution of the calibration solutions was THF.
QC Samples
Approximately 400 – 500 mg vehicle was accurately weighed and spiked with the test item at a target concentration of 1 or 200 mg/g.
The QC samples were prepared in volumetric flasks of 20 or 50 mL.
The flasks were filled up to mark with THF. The solutions were further diluted to obtain an end solution of THF and concentrations within the calibration range.
The blank QC sample consisting of blank medium was treated similarly to the QC samples.

Formulas
Response (R) Peak area of the test item [units]

Response factor (Rf) Rf = (R / CN)
where:
CN: nominal concentration [mg/L]

Calibration curve R = a CN + b
where:
a: slope [units x L/mg]
b: intercept [units]

Analyzed concentration (CA) CA = ((R - b) / a ) x (( V x d) / w) [mg/g]
where:
w weight sample [mg]
V volume volumetric flask [mL]
d dilution factor

Accuracy (CA / CN) x 100 [%]

Duration of treatment / exposure:

For both sexes: 14 days before mating, throughout the mating period and up to the day before necropsy (i.e. minimum of 28 days for males).

For females:
- during gestation (the first day of gestation is designated as G 0) and at least 13 days after parturition, up to and including the day before scheduled necropsy (the first day of birth is designated as L 0).
- apparently unmated females for at least 24 days after the last day of the mating period.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 per sex and dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Under the defined experimental conditions, daily oral (gavage) of Alkenyl phosphonate at doses of 500 and 1000 mg/kg/day in the female rat during 10 days was associated with a slight reduced body weigh gain and hypersalivation at 1000 mg/kg/day only. No macroscopic findings were noted at 500 and 1000 mg/kg/day.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Parental animals: Observations and examinations
CAGE SIDE OBSERVATIONS: Yes

- Time schedule: daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: each weighing day

BODY WEIGHT: Yes
- Time schedule for examinations:
Male and females will be weighed on the first day of exposure (prior to the first exposure) and weekly thereafter during pre-mating and mating periods.
Mated females will be weighed:
− on Days 0, 6, 9, 12, 15, 18 and 20 of gestation
− on Days 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption of males will be recorded weekly during the pre-mating period.
Food consumption of females will be recorded for the following periods:
− weekly during the pre-mating period
− gestation: Days 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 20
− lactation: Days 1 to 4, 4 to 7 and 7 to 13.

WATER CONSUMPTION: No

OTHER: Functional Tests
Animals examined: 5 randomly selected animals/sex/group (for females, only those with live pups will be selected).
Frequency:
– Males: at the end of their treatment period, shortly before scheduled kill.
– Females: during lactation, shortly before scheduled kill.
The following tests will be performed:
– auditory reflex
– pupillary reflex
– righting reflex
– fore-limb grip strength
– locomotor activity in an open field test. Activity will be monitored by a video image analysis system (Videotrack supplied by Viewpoint, Lyon, France). The arena is divided into nine equal invisible regions; the time spent by the animal in each type of region (i.e. corner, centre or lateral) will be recorded. Motor activity is divided into three categories: ambulatory activity (in which the centre of the image moved at more than 7 cm/sec), small movements (including grooming etc.) and inactivity. The proportion of time spent engaged in each type of activity and the total distance travelled by the rat will be calculated.

Haematology, coagulation, chemistry for F0
Hormone Thyroxine (T4) and TSH

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Other examinations:

Estrous cyclicity (parental animals)

Statistics:

Statistical analysis was performed, where appropriate, by the data acquisition software, as follows:
The best transformation for the data (none, log or rank) was determined depending upon:
- the kurtosis of the data
- the probability of the Bartlett's test for homogeneity of the variances and
- an assessment of whether the size of the groups are approximately equal or not.
Non- or log-transformed data were analyzed by parametric methods.
Rank transformed data were analyzed using non-parametric methods.
Data were then analyzed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by ANOVA for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analyzed by parametric or non-parametric Dunnett's test.
Selected incidence data were analysed using the Provantis data acquisition system and/or a SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
The locomotor activity in an open field, fore-limb grip strength, estrous cycle and pre-coital interval data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using ANOVA followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.
Microsoft Excel® (version 2003 or higher) was employed to present certain results and perform associated calculations.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was a higher incidence of hypersalivation associated or not with abnormal foraging and/or pedalling from Day 18 of treatment up to the end of the dosing period for both sexes in the 250 and 750 mg/kg/day groups (see text table 1). These signs were considering not adverse due to their nature, minor severity and transience (i.e. just after dosing).
Dilated pupils were noted for one male treated at 750 mg/kg/day on Day 18. This isolated finding was considered incidental.

One female (no. 151) treated at 250 mg/kg/day had piloecrection and irregular/rapid breathing, was pale, subdued and recumbent on the day of mating (G0).These isolated clinical signs were noted only in the mid dose group and occurred at the time of mating while the female was housed with the male and were therefore considered incidental.

Other incidental findings were observed such as a torn claw, scab or localized hairloss.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Control female no. 120 was found dead on Gestation Day 18 (G18). Dark thymus, enlarged dark adrenal gland, distended urinary bladder and dilated ureters were noted at the macroscopic examination.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect on mean body weight gain in any group.
Mean body weight gain for males during the dosing period or females during the premating and gestation periods was comparable with, or superior to, that noted in the control group.
During the lactation phase, there was a slightly lower mean body weight gain for the females in all treated groups when compared with the control. Since this change was not dose-related and had no adverse effect on terminal body weights (-3.1, -4.4 and -1.2% in the 75, 250 and 750 mg/kg/day groups, respectively, when compared with control), it was considered not toxicologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no test item-related effect on mean food consumption in any group for either sex during the dosing period.

Mean food consumption was slightly lower during gestation in the 75 and 750 mg/kg/day groups (- 3.9 and -3.4%, respectively) and during the lactation period in the 250 and 750 mg/kg/day groups (-4.4 and -2.9% respectively). However, all values remained above the historical control data means (22.5 and 45.8 g/day from G0 to G20 and from L1 to L14, respectively). These findings were therefore considered as incidental.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences noted in haematological and coagulation parameters between control and treated rats of either sex that were considered to be toxicologically relevant.
Although differences in mean values attained statistical significance for males compared with the concurrent control, the decrease in mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, fibrinogen concentration and platelet concentration were considered of no toxicological significance since there was no dose-related trend and/or in view of the low magnitude. In addition, mean values were within the historical control range.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences noted in serum clinical chemistry parameters between control and treated rats of either sex that were considered to be toxicologically relevant.
Although differences in mean values attained statistical significance for males compared with the concurrent control, the decrease in sodium, urea, inorganic phosphorus, globulin and bile acid concentrations and the increase in creatinine concentration were considered of no toxicological significance since there was no dose-related trend and/or in view of the low magnitude. In addition, mean values were within the historical control range.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed.
Fore-limb grip strength mean values were lower than controls for males at
750 mg/kg/day and females at 75 mg/kg/day. The effect was not dose-related since the mean value in the 250 mg/kg/day group was comparable or higher to that of control for either sex and the mean value in the 750 mg/kg/day was higher to that of control for females.
This was therefore considered to be due to intra-group variability and therefore incidental.
The variation in motor activity (open field) did not indicate a relation with treatment.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No differences in Total T4 levels were noted among the different groups of adult males or among the different groups of PND 13 pups.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 750 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of the study, the daily oral (gavage) administration of the test item, Alkenyl phosphonate, to the rat during pre-mating (males and females), during pregnancy and lactation (females) at doses of 75, 250 and 750 mg/kg/day induced no parental toxicity.
Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of at least 750 mg/kg was derived.

Executive summary:

Objective

The objectives of the study was to evaluate the potential toxic effects of the test item, Alkenyl phosphonate, when exposed for a minimum of 28 days and the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. Parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No-Observed-Adverse-Effect-Levels (NOAELs) were evaluated.

 

Procedure

Alkenyl phosphonate was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 75, 250 and 750 mg/kg/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, 1 % (w/v) Carboxymethylcellulose in water, alone. Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females that delivered were treated for at least 51 days, i.e. during 2 weeks prior to mating, during mating, during assumed pregnancy, and during 13 days of lactation. Females which failed to deliver healthy offspring were treated for at least 40 days.

The following observations and examinations were evaluated: mortality / morbidity, clinical signs, functional observations and locomotor activity, body weight, food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4, macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: copulation and fertility indices, pre-coital time, number of implantation sites, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy). Formulations were analyzed during the study to assess accuracy, homogeneity and stability.

 

Results

The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations and the formulations of Group 2 and Group 4 were homogeneous.

No differences in Total T4 levels were noted among the different groups of adult males or among the different groups of PND 13 pups.

There was no test item-related mortality in any group.

There were no adverse test item-related clinical signs. There was a higher incidence of hypersalivation associated or not with abnormal foraging and/or pedalling for both sexes in the 250 and 750 mg/kg/day groups.

There were no test item-related changes in parental body weight and food consumption, or on the haematological and serum clinical chemistry parameters.

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex, grip strength or open field tests were observed for the males or females at any dose level.

There was no evidence of any test item-related parental histological findings.

 

Conclusion

Under the experimental conditions of the study, the daily oral (gavage) administration of the test item, Alkenyl phosphonate, to the rat during pre-mating (males and females), during pregnancy and lactation (females) at doses of 75, 250 and 750 mg/kg/day induced no parental toxicity.

Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of at least 750 mg/kg was derived.