4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-05-25 - 2017-11-21 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals, No.421: “Reproduction/Developmental Toxicity Screening Test” (adopted 28 July 2015)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008

Version / remarks:

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 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:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin

EC Number:

277-633-6

EC Name:

4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin

Cas Number:

73912-21-7

Molecular formula:

C27H35O3P

IUPAC Name:

7,13-dicyclohexyl-5,15-dimethyl-9,11-dioxa-10-phosphatricyclo[10.4.0.0³,⁸]hexadeca-1(12),3(8),4,6,13,15-hexaen-10-ol

Test material form:

solid: pellets

Remarks:

pale yellow

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4°C, in the dark; used/formulated in light

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 12 weeks
- Weight at study initiation: males weighed 281 to 345g, the females weighed 196 to 245g
- Fasting period before study: no
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male:one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited Oxon, UK.) ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage ad libitum.
The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
The animals were housed in a single air-conditioned room within the Barrier Maintained Rodent Facility. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records.
- Temperature (°C): The Study Plan target ranges for temperature were 22 ± 3 °C.
- Humidity (%): The Study Plan target ranges for relative humidity were 50 ± 20%.
Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
IN-LIFE DATES: The in-life phase of the study was conducted between 13 June 2016 (first day of treatment) and 06 August 2016 (final day of necropsy).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations in Propylene Glycol. Test item formulations spanning the concentration ranges used in this study were previously shown to be homogenously prepared and stable for at least twenty-one days when stored refrigerated at approximately 4ºC, in the dark. Formulations were therefore prepared, aliquoted for use, and stored as above before use within the stability period.

VEHICLE
- Concentration in vehicle: 0, 12.5, 37.5, and 125 mg/ml
- Amount of vehicle (if gavage): 8 mL/kg
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Animals were paired on a 1 male:1 female basis within each dose group, for a period of up to fourteen days. Following evidence of successful mating, the males were returned to their original cages.
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing).
- After successful mating each pregnant female was caged (how): Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test item formulations were taken on three occasions and analyzed for concentration of the test item at the laboratories Analytical Services. The method used for analysis of formulations and the results obtained are given in the attachment. The results indicate that the prepared formulations were within 101 to 111% of the nominal concentration.

Duration of treatment / exposure:

Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45.
Surviving females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum.
Any females which failed to achieve pregnancy were killed on or after Day 25 post coitum.

Frequency of treatment:

daily

Details on study schedule:

Chronological Sequence of Study
i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.
ii. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). During the pre-pairing period, vaginal smears were performed for females. The first day of dosing was designated as Day 1 of the study.
iii. On Day 15, animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex, ano-genital distance and visible nipple counts (male offspring) and clinical signs were also recorded during this period.
vi. The male dose groups were killed and examined macroscopically on Day 44 or 45.
vii. At Day 13 post partum, all surviving offspring were killed and examined macroscopically. All surviving females were sacrificed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for females in the morning of the day of necropsy.
viii. Where possible, blood samples were taken from two offspring on Day 4 post partum and one male and one female offspring on Day 13 post partum. In addition, blood samples were taken from all adult surviving animals at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 / sex / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels and dose volume were selected in collaboration with the Sponsor Representative and were based on available toxicity data including a 28 Day toxicity study in the rat (see chapter repeated dose toxicity). In the 28 Day study, administration of the test item to animals of either sex at dose levels of up to 1000 mg/kg bw/day was well tolerated. A high dose level of 1000 mg/kg bw/day was therefore considered to be suitable for investigation in the present study together with 100 and 300 mg/kg bw/day as the low and intermediate dose levels, respectively. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Positive control:

not required

Examinations

Parental animals: Observations and examinations:

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

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

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1, 4, 7 and 14 post partum.
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.

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

OTHER: Thyroid Hormone Analysis
Where possible, blood samples were taken, allowed to clot, centrifuged and the serum from each blood sample was stored frozen at lower than -60 °C for possible evaluation of thyroid hormones from the following:
Two offspring from each litter at Day 4 post partum (where the litter contained six or fewer offspring, blood sampling was not performed in order to ensure the availability of adequate number of offspring for other evaluations); see deviations from Study Plan.
One male and one female offspring from each litter at Day 13 post partum.
All adult males at termination.
All adult females at termination.
All samples were dispatched to the Test Site where the serum from adult males and Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator.

Oestrous cyclicity (parental animals):

Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment.
Vaginal smears were taken daily throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Sperm parameters (parental animals):

Parameters examined in male parental generation:
After necropsy, detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.

Litter observations:

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data) (see deviations from study plan)

Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.

Thyroid Hormone Analysis
Two offspring from each litter at Day 4 post partum (where the litter contained six or fewer offspring, blood sampling was not performed in order to ensure the availability of adequate number of offspring for other evaluations)
One male and one female offspring from each litter at Day 13 post partum.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals. Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45.
- Maternal animals: All surviving animals. Surviving females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum. Any females which failed to achieve pregnancy were killed on or after Day 25 post coitum.

GROSS NECROPSY
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ Weights
The following organs were dissected free from fat and weighed before fixation from all animals (where applicable) from each dose group.
Cowpers glands
Epididymides
Glans penis
LABC (levator ani-bulbocavernous) muscle
Testes
Thyroid (weighed post-fixation with Parathyroid)

Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Epididymides ♦
Glans penis
LABC (levator ani-bulbocavernous) muscle
Mammary gland (females only)
Ovaries
Pituitary
Prostate
Seminal vesicles (with coagulating gland)
Testes ♦
Thyroid/Parathyroid
Uterus/Cervix
Vagina
♦ Preserved in Modified Davidsons fluid

All tissues were dispatched to the histology processing Test Site for processing. The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to achieve a pregnancy, and any gross lesions were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.
At the request of the Sponsor, thyroid/parathyroid from control and high dose adult females were routinely processed to paraffin wax, sectioned and stained with Hematoxylin and Eosin, and examined.

Postmortem examinations (offspring):

SACRIFICE
Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation.

GROSS NECROPSY
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. For offspring used for blood sampling, a more limited gross necropsy, including internal confirmation of the sex of the offspring, was performed following blood sampling.

Thyroid Hormone Analysis
Two offspring from each litter at Day 4 post partum (where the litter contained six or fewer offspring, blood sampling was not performed in order to ensure the availability of adequate number of offspring for other evaluations); see deviations from Study Plan.
One male and one female offspring from each litter at Day 13 post partum.

Statistics:

Due to limitations of this free-text field, please see "Any other information on materials and methods"

Reproductive indices:

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

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

Offspring viability indices:

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).
i. Implantation Losses (%)
Group mean post-implantation loss was calculated for each female/litter as follows:
Post-implantation loss (%) = ((Number of implantation sites – Total number of offspring born) / Number of implantation sites) * 100
ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) * 100
Viability Index 1 (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) * 100
Viability Index 2 (%) = (Number of offspring alive on Day 13 / Number of offspring alive on Day 4) * 100
Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post partum.
iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1, 4 and 13 post partum, using the following formula: (Number of male offspring / Total number of offspring) * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs considered to be related to the toxicity of the test item.
At 1000 mg/kg bw/day, animals of either sex showed sporadic instances of increased post-dose salivation from Days 2 (females) or 3 (males) which persisted towards the end of the dosing period. Such observations are common in this type of study and may reflect an irritant nature of the test item and/or formulation and as such are considered to be of no toxicological significance.
One female each from the 100 or 300 mg/kg bw/day dose groups showed clinical signs of noisy respiration on Days 22 and 4, respectively. These observations may be due to the dosing procedure and in the absence of similar findings in the remaining animals, they were considered unlikely to be related to treatment with the test item.
There were no clinical signs for any other animals throughout the treatment period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

At 100 mg/kg bw/day, Female 47 was killed in extremis on Day 1 post partum (Day 41 relative to the start of dosing). Prior to death, clinical signs for this animal included decreased respiratory rate, ataxia, wet fur and increased post-dose salivation but there were no macroscopic observations at necropsy. In the absence of any premature decedents in the 300 or 1000 mg/kg bw/day dose groups, this death was considered unlikely to be treatment-related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no adverse effect of treatment with the test item on body weight development in animals of either sex.
At all dose levels, males occasionally showed marginally higher body weight gains than controls which resulted in slightly higher overall body weight gains for these animals. There was generally no dose-dependence and the intergroup differences were not statistically significant. It is worth noting that a body weight increase of this nature in this type of study is generally considered to be of no toxicological significance.
During the pre-pairing phase of the study, females treated with the test item at all dose levels also showed slightly higher body weight gains than controls achieving statistical significance for the 1000 mg/kg bw/day females during the second week (p<0.05). During the gestation period, group mean body weight gains in the test item-treated females were slightly lower than controls resulting in slightly lower overall body weight gains across these dose groups. The intergroup differences were, however, neither strictly dose-related nor they achieved statistical significance and may be due to the slightly lower litter size, i.e. less fetuses, for these females and as such were considered not to be of any toxicological importance. During the first week of lactation, group mean body weight gains in test item-treated females remained lower than controls but there was no dose-relationship and statistical significance was only achieved for the 100 and 300 mg/kg bw/day females over Days 1 to 4 (p<0.05). This resulted in statistically significantly lower overall body weight gains for females treated with 300 or 1000 mg/kg bw/day over the first week of lactation (p<0.05), but without depicting any dose-dependence. Subsequent improvement was evident and group mean body weight gains for test item-treated females during the second week of lactation were comparable with controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At all dose levels, dietary intake for the males was comparable with controls throughout the study. The corresponding females also showed similar food consumption to controls during pre-pairing and early gestation. Over Days 14 to 20 of gestation, food intake for females receiving 100 or 300 mg/kg bw/day was slightly lower than controls (p<0.05) but a dose-relationship was not evident and the corresponding values in females from the 1000 mg/kg bw/day dose group was similar to controls. Fluctuations in dietary intake were also evident during the lactation phase of the study attaining occasional statistical significance for the 100 or 300 mg/kg bw/day females (p<0.01-0.05) but the corresponding values for the 1000 mg/kg bw/day dose group were comparable with controls.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no effect of treatment with the test item on food conversion efficiency (where calculated) for animals of either sex receiving the test item. Any fluctuations were deemed to be reflective of minor intergroup differences in body weight gain and/or food consumption.

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

no effects observed

Description (incidence and severity):

Visual inspection of water bottles did not indicate any overt differences for animals of either sex receiving the test item in relation to controls.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis: An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic abnormalities at any dose level.
All of the histopathological findings encountered were considered to have occurred spontaneously or post mortem. In addition to the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment-related abnormalities were noted.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the pre-pairing phase of the study.
There were also no intergroup differences in the stage of estrous cycle on the day of necropsy.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In addition to the routine evaluation of the testes, the seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No treatment-related abnormalities were noted.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating: There was no effect of treatment on mating performance. With the exception of one female from the 100 mg/kg bw/day dose group which mated on Day 14 after pairing, the remaining animals mated within four days after pairing.
Fertility: Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level.
One female each from the control, 100 and 300 mg/kg bw/day dose groups (Females 18, 39 and 68, respectively) and two females from the 1000 mg/kg bw/day dose group (Females 92 and 95) did not achieve pregnancy following positive evidence of mating. Male 27 (paired with Female 39) had severe unilateral tubular atrophy and a subsequent unilateral aspermia in the epididymis which may have prevented pregnancy. There were no histopathological changes in the tissues examined to account for non-pregnancy in the remaining animals and these non-pregnancies were deemed to be due to biological variation.
Gestation length: Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for test item-treated females appeared to be similar to controls.

Effect levels (P0)

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

food efficiency

water consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

highest dose tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs detected in pups from all treated groups included small size, cold, weak, no milk in the stomach, physical injury, found dead or missing. Females 23 (control), 41 and 45 (100 mg/kg bw/day), 70 (300 mg/kg bw/day) and 88 (1000 mg/kg bw/day) showed total litter losses. Such findings are often seen in this type of study and were considered unlikely to be treatment-related.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Litter Responses: In total, eleven females each from the control, 100 and 300 mg/kg bw/day dose groups and ten females from the 1000 mg/kg bw/day dose group gave birth to a live litter. One litter each was lost in the control, 300 and 1000 mg/kg bw/day dose groups during the lactation phase of the study with the 100 mg/kg bw/day dose group showing two litter losses. The following assessment of litter response is based on all litters reared to termination on Day 13 of lactation/age.
Offspring Litter Size, Sex Ratio and Viability: There was considered to be no adverse effect of treatment with the test item at any dose level on the mean number of implantations and post-implantation losses. Of the litters born, there was also no adverse effect of treatment with the maternal treatment on litter size at birth and subsequently on Days 1, 4, 7 and 13 post partum indicating the lack of any adverse effect on offspring viability.
The mean number of implantation sites in females treated with the test item up to a dose level of 1000 mg/kg bw/day was slightly lower than controls. The majority of individual values were within the historical control data ranges and the intergroup differences were neither dose-related nor they attained statistical significance. Histopathological examination of the ovaries from the 1000 mg/kg bw/day did not identify any treatment-related findings and this observation was considered likely to be due to biological variation. Group mean post-implantation losses across all dose groups including controls were comparable, but due to the slightly lower number of implantation sites, litter size at birth and on subsequent days in the test item-treated dose groups remained lower than controls. These intergroup differences did not achieve statistical significance and were not dose-dependent, and as such were considered to be of no toxicological relevance.
There were no treatment-related intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated groups when compared with controls.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was considered to be no adverse effect of treatment with the test item on offspring body weights and body weight development.
At birth, group mean offspring body weights across all test item-treated dose groups were comparable with controls. Group mean offspring body weight gains from the 100 and 300 mg/kg bw/day dose groups remained slightly lower than controls over Days 1 to 7 of lactation which resulted in lower cumulative body weight gains for these pups, but improvement was noted over Days 7 to 13 of lactation. Consequently, the mean body weights on Day 4 and thereafter for these offspring were slightly lower than controls. It is worth noting that corresponding values for the 1000 mg/kg bw/day dose group were generally similar to controls and none of these intergroup differences achieved statistical significance. Due to the slightly lower litter size and lower body weight gains, litter weights for the 100 and 300 mg/kg bw/day dose groups remained lower than controls attaining statistical significance on Day 13 post partum for the latter. The corresponding values for the 1000 mg/kg bw/day dose group were, however, generally similar to controls, and these intergroup differences were considered not to be of any toxicological relevance.

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:

no effects observed

Description (incidence and severity):

An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

When compared with controls, evaluation of ano-genital distance on Day 1 post partum (male and female offspring) and visible nipple count on Day 13 post partum (male offspring) did not reveal any treatment-related intergroup differences.

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

clinical signs

mortality

body weight and weight gain

clinical biochemistry

gross pathology

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

highest dose tested

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:

The study was conducted under GLP according to OECD guideline 421 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as sufficiently reliable to assess the reproductive toxicity of the test item in rats (screening study). The oral (gavage) administration of the test item to Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 1000 mg/kg bw/day did not produce any adverse effects in adults or offspring. Based on the available results, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day within the confines of this study. Additionally, the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.

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 also includes some endocrine disruptor relevant endpoints. 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 28 July 2015).

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 three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to ten weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 or 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Propylene Glycol).

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. 

Pairing of 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 13 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 ano-genital distance and visible nipple count (male offspring only).

Blood samples were taken at termination from all surviving adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females.

Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from control and 1000 mg/kg bw/day dose groups was performed.

 

Results

Adult Responses

Mortality: There were no treatment-related deaths on the study.

Clinical Observations: There were no clinical signs considered to be related to the toxicity of the test item.

Body Weight: There was no adverse effect of treatment with the test item on body weight development for animals of either sex during the dose administration period.

Food Consumption: There was no effect of treatment with the test item on food consumption or food conversion efficiency for animals of either sex during the treatment period.

Water Consumption: Visual inspection of water bottles did not indicate any differences for the animals given the test item in comparison with controls.

Reproductive Performance

Mating: There was no effect of treatment with the test item on mating performance.

Fertility: There were no treatment-related effects in conception rates for test item-treated animals in relation to controls.

Gestation Length: There were no treatment-related differences in gestation lengths in animals receiving the test item when compared with controls.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability: There was no adverse effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 100, 300 or 1000 mg/kg bw/day.

Offspring Growth and Development: There was no adverse effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights, ano-genital distance on Day 1 post partum, visible nipple count in male offspring on Day 13 post partum or clinical signs up to Day 13 of age at 100, 300 or 1000 mg/kg bw/day.

Pathology

Necropsy: Neither the type, incidence or distribution of macroscopic observations in adult animals or offspring indicated any adverse effects up to a dose level of 1000 mg/kg bw/day.

Organ Weights: No adverse effects were detected in animals of either sex at any dose level.

Histopathology: Histopathological examination of the selected tissues from the 1000 mg/kg bw/day animals of either sex did not reveal any treatment-related abnormalities.

 

Conclusion

The oral (gavage) administration of4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12Hdibenzo[d,g][1,3,2]dioxaphosphocinto Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 1000 mg/kg bw/day did not produce any adverse effects in adults or offspring. Based on the available results, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day within the confines of this study. Additionally, the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.