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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-07-2016 til 26-09-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide
EC Number:
221-088-9
EC Name:
3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide
Cas Number:
3001-98-7
Molecular formula:
C7H14O6P2
IUPAC Name:
3,9-dimethyl-2,4,8,10-tetraoxa-3λ⁵,9λ⁵-diphosphaspiro[5.5]undecane-3,9-dione
Test material form:
solid: particulate/powder
Details on test material:
AFLAMMIT® PCO 962

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
AFLAMMIT PCO 962 was administered by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day (Low, Mid and High dose levels, respectively)
Details on mating procedure:
Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male from the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 5 days except for a control animal (#1506) where mating occurred on the 13th mating day.
A vaginal smear was prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope. The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were housed individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed at Analytical Department of the Test Facility using a validated Liquid Chromatography -Mass Spectrometry (LC-MS) method. Duplicate samples were analysed from the top, middle and bottom of the test item formulations four times during the study.
All test item formulations were within the range of 92-109% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.
Duration of treatment / exposure:
Males were dosed for at least 28 days (14 days pre-mating and at least 14 days mating/post-mating);
Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13 days of post-partum dosing).
Frequency of treatment:
daily on a 7 days/week basis
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, concurrent vehicle
Positive control:
none

Examinations

Parental animals: Observations and examinations:
Clinical observations: twice daily, Body weight measurement: least weekly, Food consumption measurement: on the weekly body weight measurements days
- Clinical observations and functional observation battery (FOB) †
- Body weight and body weight gain †
- Food consumption
Oestrous cyclicity (parental animals):
Yes, and reproductive ability assessment and indices
Parental Females
- Oestrus cycle data
- Number of pairings
- Number of pregnant females †
- Number of sperm positive, but non-pregnant females †
- Number of non-mated females †
- * Female mating index †
- * Female fertility index †
- * Gestation index †
- Duration of pregnancy (days) †
- Number of corpora lutea / dams †
- Number of implantations / dams †
- Number of dams with live pups on PPD 0, 4 and 13 †
- * Pre-implantation mortality †
- * Pre-natal (“intrauterine”) mortality †
- * Total mortality (intra and extra uterine mortality) †
- Clinical pathology †
- Necropsy findings
- Organ weights (absolute and relative to the body and brain weights) †
- Histopathology findings

Sperm parameters (parental animals):
Yes, see terminal procedures as well as reproductive ability assessment and indices
Parental Males
- Number of pairings
- Number of fertile pairings †
- Number of infertile males †
- * Male mating index †
- * Male fertility index †
- Clinical pathology †
- Results of thyroid hormone analysis †
- Necropsy findings
- Organ weights (absolute and relative to the body and brain weights) †
- Histopathology findings
Litter observations:
Offspring
- Mean pup body weight (per pup within the group and per litter) on PND 0, 4 and 13 †
- Mean pup body weight gain (per litter) between PND 0-4, PND 4-13 and PND 0-13 †
- Number of live births per litter, and number of viable pups per litter on PND 0, 4 and 13 †
- * Survival Index of pups on PND 0, 4 and 13 †
- F*Sex ratio % (on PND 0, 4 and 13) †
- Results of thyroid hormone analysis †
- Anogenital distance, nipple retention †
- Necropsy findings (macroscopic)
- Organ weights (thyroid glands, absolute and relative to the body weight) †
Postmortem examinations (parental animals):
CLINICAL PATHOLOGY
All animals selected for blood sampling were fasted (overnight period of food deprivation, after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
For terminal blood sampling in all selected animals (5 males and 5 females/group, the same animals as used for neurotoxicity screening), 3 samples were taken from each animal: one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.
- Haematology and blood clotting times
- Clinical chemistry
- Urinalysis

THYROID HORMONE ANALYSIS
For thyroid hormone analysis, blood samples were taken by cardiac puncture or venepuncture (or decapitation in case of pups) into tubes containing K3-EDTA as anticoagulant as follows:
-from at least two pups per litter on PND 4,
-from all dams on PND 14 and at least two pups per litter on PND 13,
-from all adult males at termination.
Samples of adult males and PND 13 pups were assessed first for T4 (thyroxin) levels. Based on the observed results, additional analysis for T4 and/or TSH (thyroid-stimulating hormone) levels were not deemed necessary by the Study Director, thus no additional samples were analysed. The results of the evaluation in the form of a Phase Report is presented in Appendix 7.
Any sample not required for analysis will be discarded following acceptance of the results of the thyroid hormone analysis by the Principal Investigator and Study Director (after the finalization of the study report).

PATHOLOGY
Terminal procedures and macroscopic evaluation
At termination, the adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination.
Gross necropsy was performed on all adult animals, irrespective of the date of death. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.
The number of implantation sites and of corpora lutea were recorded in the females as applicable.
The pups found dead and intact (not cannibalized or autolyzed) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. Pups terminated on PND 4 and/or PND 13 were also carefully examined externally for gross abnormalities.

Organ weight measurements (adults only)
Tissue preservation and microscopic evaluation (adults only)
Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Postmortem examinations (offspring):
See post mortem examinations (parental animals)
Statistics:
Data were recorded on the appropriate forms from the relevant SOPs of Citoxlab Hungary Ltd. or collected using the software PROVANTIS v.9; and then tabulated using the Microsoft Office Word, Excel and/or PROVANTIS v.9, as appropriate. Group means and standard deviations were calculated from numerical data obtained in the study.
The statistical evaluation of data (labelled as † in the lists below) was performed with the program package SPSS PC+4.0 (SPSS Hungary Ltd., Budapest) or SAS v9.2
(built-in Provantis system).
Reproductive indices:
yes
Offspring viability indices:
yes

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Only minor clinical signs, not related to the test item treatment were observed during the study.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No endocrine disruptor effect of test item was observed in the study based on the results of thyroid hormone analysis and thyroid gland weights.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Results: P1 (second parental generation)

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
other: thyroid hormone analysis

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no observed adverse effect level (NOAEL) for the test item is considered to be 1000 mg/kg bw/day for the female and male parental/adult generation, and also for the F1/pups generation.