O,O,O',O'-tetraoctyl-(branched and linear) S,S'-{methylenebis[imino(3-oxopropane-3,1-diyl)]} bis(phosphorothioate)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 2014 to 17 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline with no major deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Target value of relative humidity was exceeded and post dose observations were not recorded on certain days. (See below)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Deviations:

yes

Remarks:

Target value of relative humidity was exceeded and post dose observations were not recorded on certain days. (See below)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of methylenebis(acrylamide) and (O,O)-diisooctyl dithiophosphoric acid

EC Number:

700-945-5

Molecular formula:

Complex UVCB substance

IUPAC Name:

Reaction product of methylenebis(acrylamide) and (O,O)-diisooctyl dithiophosphoric acid

Details on test material:

- Physical state: Yellow liquid
- Analytical purity: 100%
- Expiration date of the lot/batch: 01 August 2016
- Storage condition of test material: Room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: (P) 12 weeks
- Weight at study initiation: (P) Males: 283-345 g; Females: 185-230 g
- Fasting period before study: overnight
- Housing: initially housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. Transfer to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis during pairing. If evidence of successful mating, males were returned to their original cages. Mated females were housed individually in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes during gestation and lactation.
- Diet (e.g. ad libitum): Rodent 2018C Teklad Global Certified Pelleted Diet, Harlan Laboratories U.K Ltd., Oxon, UK
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3 °C
- Humidity (%): 50+/-20 %
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hours continuous light/dark

IN-LIFE DATES: From: 02 September 2014 To: 28 October 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

Arachis oil BP

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was diluted in Arachis oil BP

VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis oil was used as the substance formed stable homogenous solutions in this vehicle
- Concentration in vehicle: Low – 25 mg/mL
Intermediate I – 62.5 mg/mL
Intermediate II – 125 mg/mL
High – 250 mg/mL
- Amount of vehicle (if gavage): 4mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the substance in the test material formulations was determined by chemical analysis and all formulations were within +/- 11% of nominal.

Details on mating procedure:

- M/F ratio per cage: animals paired on a 1 male:1 female basis within each dose group at Day 15.
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: Presence of sperm in vaginal smear and/or vaginal plug in situ referred to as day 0 of gestation.
- After successful mating each pregnant female was caged (how): After evidence of mating (designated as Day 0 post coitum), the males were returned to their original cages. Mated females were transferred to individual cages during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

Duration of treatment / exposure:

Test duration:
- 42 days for males
- Until 5 days post partum for females
- Recovery group males and females were maintained without treatment for a further 14 days after sacrifice of the non-recovery males

Frequency of treatment:

The substance was administered daily, for 42 consecutive days, by gavage (except for females during parturition where applicable).

Duration of test:

Approximately 6 weeks

No. of animals per sex per dose:

12 animals of each sex (m/f) were allocated to each dose group. In addition, 5 animals of each sex (m/f) were allocated to each control group.

Control animals:

yes, concurrent vehicle

other: recovery control (concurrent vehicle) ... (see attached file)

Details on study design:

- Dose selection rationale: Based on results of previous toxicity study
- Rationale for animal assignment (if not random): Random
- Other: Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment
- Control animals were treated in an identical manner with 4 ml/kg/day of Arachis oil BP
- The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

- Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum.
-The male dose groups were killed and examined macroscopically on Day 43.
-All females and surviving offspring were killed and examined macroscopically on Day 5 post partum.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, soon after dosing and 1 hour after dosing (except for females during parturition where applicable).
-All animals were examined for overt signs of toxicity, ill-health and behavioral change.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing, daily for females until mating was confirmed. (Days 0, 7, 14 and 20 post coitum and Days 1 and 4 post partum for females). Day 1 (prior to dosing) and weekly thereafter for recovery group animals.

FOOD CONSUMPTION:
Food consumption was recorded during the pre-pairing period for each cage group at weekly intervals throughout the study. For males after the mating phase. For females showing evidence of mating during the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters during the lactation period (Days 1-4)
-Food efficiency (body weight gain/food intake) was calculated retrospectively for all animals of each cage group during the pre-pairing phase at weekly intervals throughout the study.
-Not accurately calculated for females during gestation and lactation due to offspring growth and milk production for lactation.

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of water bottles for any overt changes.

SACRIFICE
- Male animals: All surviving animals at Day 43
- Maternal animals: All surviving animals at Day 5 post partum for females that have offspring and at Day 25 post coitum or after for females without offspring
-Recovery group animals (m/f) killed at Day 57

GROSS NECROPSY
Full external and internal examination, any macroscopic abnormalities were recorded

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathology: Yes Samples from the following tissues were removed from all animals and preserved in 10% buffered formalin except where stated
- Organ weight: Yes. For males.
The following organs were weighted: epididymides and testes
The following tissues were examined: Coagulating gland, Epididymes, Ovaries, Mammary gland (females only), Pituitary, Prostate, Seminal vesicles, Testes, Uterus/Cervix, Vagina

Ovaries and uterine content:

-For all non-recovery females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. The corpora lutea were also counted.

PREGNANCY AND PARTURITION
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female: Date of pairing, Date of mating, Date and time of observed start of parturition, Date and time of observed completion of parturition

Fetal examinations:

LITTER DATA
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number of offspring born, number of offspring alive recorded daily and reported on Days 1 and 4 post partum, sex of offspring on Days 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum, individual offspring weights on Day 1 and 4 post partum.
PHYSICAL DEVELOPMENT
- All alive offspring were assessed for surface righting reflex on Day 1 post partum.


GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal examination and any macroscopic abnormalities; possible cause of death was examined for pups born or found dead.

SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann- Whitney U test (non-parametric).
Probability values (p) are presented as follows:

p<0.01
p<0.05
p≥0.05 (not significant)

Indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
- Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Fertility Indices
For each group the following were calculated:
Mating Index (%) = Number of animals mated/Number of animals paired x 100
Pregnancy Index (%) = Number of pregnant females / Number of animals mated x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
- Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition Index
The following was calculated for each group:
Parturition Index (%) = Number of females delivering live offspring / Number of pregnant females x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects. Remark: No unscheduled deaths. No significant clinical signs evidence

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Mortality: There were no further unscheduled deaths.
- Clinical signs: There were no toxicologically significant clinical signs evident in treated animals.
Animals of either sex treated with 1000 mg/kg bw/day and two females and one male treated with 500 mg/kg bw/day showed isolated incidences of increased salivation post dosing. One male treated with 500 mg/kg bw/day also had noisy respiration on day 5 only. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to represent adverse effects of treatment. The left hind foot of one male treated with 250 mg/kg bw/day had a scab present and was swollen between Days 37 and 42. This was considered to be due to a physical injury and unrelated to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Body weight: There were no treatment-related effects on body weight development.
Recovery males treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain during the first week of the treatment free period. In the absence of a similar effect detected in non-recovery animals throughout the treatment period the intergroup difference was considered not to be of toxicological importance. Recovery females treated with 1000 mg/kg bw/day showed a statistically significant increase in body weight gain during the first week of treatment. An increase in body weight gain is not considered to represent an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance.
- Food consumption: No adverse effects on dietary intake were noted for males during the study or for females during the pre-pairing, gestation or lactation phases of the study.
Statistical analysis of the data for females during gestation and lactation did not reveal any significant intergroup differences.
- Water consumption: No adverse effect on water consumption was evident in treated animals when compared to controls.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Mating: No treatment-related effects were detected in mating performance.
- Fertility: There were no treatment-related differences in fertility.
Three control females and one female treated with 250 mg/kg bw/day showed positive evidence of mating but were not pregnant. No histopathological abnormalities were observed to explainthe failure of two of the control females and the female treated with 250 mg/kg bw/day to breed successfully however one control male had severe hypospermia in both epididymides and marked tubular atrophy in both testes. At necropsy this male had small epididymides and testes.
These effects were considered sufficient to render the male infertile.
One female treated with 250 mg/kg bw/day showed positive evidence of mating but failed to give birth to any observed live offspring. Although this female had a mass present in the left horn of the uterus at necropsy, histopathological evaluation was unable to confirm if this was the cause of the no litter. In the absence of a similar effect at 1000 mg/kg bw/day this was considered incidental and unrelated to treatment.
- Gestation length: Gestation lengths were between 22 and 24 days and the distribution of gestation lengths for treated females was essentially similar to control.

ORGAN WEIGHTS (PARENTAL ANIMALS): No treatment-related changes were evident in the organ weights measured. Statistical analysis of the data didnot reveal any significant intergroup differences.

GROSS PATHOLOGY (PARENTAL ANIMALS): No treatment-related macroscopic abnormalities were detected.
One female treated with 250 mg/kg bw/day had a mass in the left horn of the uterus. In the absence of a similar effect detected at 1000 mg/kg bw/day this finding was considered of no toxicological significance. One control male had small testes and epididymides at necropsy. In the absence of treatment this was considered incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS): There were no treatment-related microscopic abnormalities detected.


In total nine females from the control group, ten females treated with 250 mg/kg bw/day and twelve females treated with 100, 500 and 1000 mg/kg bw/day gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

VIABILITY (OFFSPRING): No significant differences were detected for corpora lutea, implantation counts, implantation losses, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

CLINICAL SIGNS (OFFSPRING): No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, cold, weak, no milk in stomach, scab formation, missing or found dead were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

BODY WEIGHT (OFFSPRING): There were no toxicologically significant effects detected. Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.

SEXUAL MATURATION (OFFSPRING): Not examined

ORGAN WEIGHTS (OFFSPRING): Not examined

GROSS PATHOLOGY (OFFSPRING): No treatment-related macroscopic abnormalities were detected for offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

The oral administration of the test material to rats by gavage, at dose levels of 100, 250, 500 and 1000 mg/kg bw/day, did not result in any treatment related effects. A ‘No Observed Effect Level’ (NOEL) for limited systemic toxicity was considered to be 1000 mg/kg bw/day.
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods…….

The test item was administered by gavage to four groups, each of twelve males and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week preparing phase, pairing, gestation and early lactation for females), at dose levels of 100, 250, 500 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of non-recovery animals within each dose group was undertaken on a one male: one

female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Non-recovery adult males were terminated on Day 43, followed by the termination of all nonrecovery females and surviving offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25post coitum. Recovery animals were terminated on Day 57. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

 

Results…….

Adult Responses

Mortality

There were no unscheduled deaths.

Clinical Observations

No toxicologically significant clinical signs were detected in treated animals.

Body Weight

No adverse effects were detected in body weight gain for non-recovery males or recovery animals throughout the treatment period or for non-recovery females during maturation, gestation or lactation.

Food Consumption

No adverse effects were detected in food consumption or food conversion efficiency for nonrecovery males or recovery animals throughout the treatment period or for non-recovery females during maturation, gestation or lactation.

Water Consumption

No adverse effect on water consumption was evident in treated animals when compared to controls.

Reproductive Performance

Mating

No treatment-related effects were detected in mating performance.

Fertility

There were no treatment-related differences in fertility.

Gestation Length

Gestation lengths were between 22 and 24 days and the distribution of gestation lengths for treated females was essentially similar to control.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Of the litters born, litter size at birth and subsequently on Days 1 and 4post partumwere comparable to controls. Sex ratio in treated litters was comparable to controls.

Offspring Growth and Development

Offspring body weight gain and litter weights at birth and subsequently on Days 1 and 4post partumwere comparable to controls. Surface righting in treated litters was comparable to controls.

Offspring Observations

The clinical signs and necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 250, 500 or 1000 mg/kg bw/day.

Pathology

Necropsy

No treatment-related macroscopic abnormalities were detected.

Organ Weights

No treatment-related changes were evident in the organ weights measured.

Histopathology

No treatment-related microscopic abnormalities were detected.

Conclusion

The oral administration of the test material to rats by gavage, at dose levels of 100, 250, 500 and 1000 mg/kg bw/day, did not result in any treatment related effects. A ‘No Observed Effect Level’ (NOEL) for limited systemic toxicity was considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.