Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-08-03 to 2013-03-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-oxybis[5,5-dimethyl-1,3,2-dioxaphosphorinane] 2,2'-disulphide
EC Number:
223-829-1
EC Name:
2,2'-oxybis[5,5-dimethyl-1,3,2-dioxaphosphorinane] 2,2'-disulphide
Cas Number:
4090-51-1
Molecular formula:
C10H20O5P2S2
IUPAC Name:
2,2'-oxybis[5,5-dimethyl-1,3,2-dioxaphosphorinane] 2,2'-disulphide
Test material form:
solid: particulate/powder
Remarks:
migrated information: particulates

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 9-10 weeks old, females: 9-10 weeks old.
Body weight at the allocation of the animals to the experimental groups: males: 236 - 263 g; (mean: 252.08 g, ± 20% = 201.66 – 302.49 g)
females: 162 - 188 g; (mean: 176.35 g, ± 20% = 141.08 – 211.62 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3°C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0939)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control,
microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male),
type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 300512)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made.
Before the first administration all animals used for the study were weighed and assigned to the experimental groups
with achieving a most homogenous variation in body weight throughout the groups of males and females.

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
corn oil
Details on exposure:
The test item was weighed into a tarred plastic vial on a precision balance.
The dose formulations were prepared by adding the required volume of corn oil and further vortexing it for 2-3 minutes.
The vehicle was selected based on the test item’s characteristics. The test item formulations were prepared freshly on each administration day
before the administration procedure. The time of preparation and time of dosing was recorded for all dosing formulations.
The homogeneity was guaranteed by intensive stirring during application.

The following doses were evaluated:
Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 1000 mg/kg body weight

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle
using the same dose volume.

Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.
Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start
of the mating period to confirm the copulation. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimised. In case of unsuccessful mating,
re-mating of females with proven males of the same group was considered.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each dosing concentration was analyzed for nominal concentration.
Stability and homogeneity of the test item in the vehicle was analyzed for the LD, MD and HD dosing formulation.
Samples for the nominal concentration verification was taken in study week 1 (first week of pre mating period), 3 (first week of mating),
5 (gestation) and 7 (gestation/lactation) of control and all treatment groups.
Samples for homogeneity were taken from the top, middle and bottom of HD, MD, and LD preparation in study week 1 and 5.
All formulation samples were preserved at -20 oC until the analysis depending on the procedure. The samples were analyzed at
BSL BIOSERVICE Scientific Laboratories GmbH. The procedure followed for the sample analysis was mentioned in phase plan
(BSL phase study Nr. 122500).
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days,
i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and
up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
Daily
Details on study schedule:
The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible
after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum.
Live pups were identified by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg body weight/day
Basis:
actual ingested
No. of animals per sex per dose:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle
using the same dose volume.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and on lactation days in 5 randomly selected females (only lactating females were evaluated) outside the home cage using a functional observational battery of tests.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups.

FOOD CONSUMPTION:
- Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the
dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: yes
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
All male animals were sacrificed after the completion of the mating period (total dosing period: 28/ 29 days) on study day 29/ 30, while female
animals were sacrificed on post-natal day 4 using an anaesthesia (ketamine/xylazin, 3:1, medistar Arzeneimittel, lot no: 00212, expiry date:
03/2014, Actavis, lot no: 146066, expiry date: 10/2014, Serumwerk, lot no: 00711, expiry date: 08/2013 and Narcoren®, Merial; lot no.:
221022; expiry date: 28/02/2015) was used. Pups were killed on PND 4 by decapitation.

GROSS NECROPSY
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices
´and the cranial, thoracic and abdominal cavities and their contents.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.


HISTOPATHOLOGY / ORGAN WEIGHTS
The wet weight of the organs (liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/parathyroid glands,
testes, spleen, epididymides, brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries, heart) of 5 males and 5 females
randomly selected from each group was recorded as soon as possible. Paired organs were weighed separately.
In addition reproductive organs of all animals were weighed.

The following tissues (brain (cerebrum, cerebellum and pons), ovaries (females), spinal cord, uterus with cervix (females),
liver, vagina (females), kidneys, testes (males), adrenal glands, epididymides (males), stomach, prostate and seminal vesicles
with coagulating glands as a whole (males), small and large intestines (including Peyer´s patches), urinary bladder, thymus,
lymphnodes (mesentric and axillary), Thyroid, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, spleen,bone with bone marrow (sternum),
lung and trachea, pituitary gland, mammary glands, oesophagus, heart, gross lesions) of the same selected animals from each group were preserved in 10% neutral buffered formalin except eyes, testes and epididymides that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 10% neutral buffered formalin.

All organs and tissues listed above were evaluated from five randomly selected males and females of the control and high dose group:
Males Nos.: 2, 3, 4, 8, 9, 34, 35, 36, 38, 40; Females Nos.: 41, 42, 43, 45, 46, 72, 74, 77, 79, 80.
Reproductive organs (ovary, uterus, cervix, vagina, testis, epididymis, prostate gland, seminal vesicle and coagulating gland) and macroscopic
changes were evaluated in all study animals. For the testes, a detailed qualitative examination was made; taking into account the tubular stages
of the spermatogenic cycle for the evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd. (test site for
tissue processing), Willow Court, Netherwood Road, Hereford HR2 6JU, England. Histopathological evaluation was performed at the GLP-certified
contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking,
embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
Postmortem examinations (offspring):
Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and
clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control a
nimals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism
5.01 software (p<0.05 was considered as statistically significant).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

Clinical Observations
There were clinical signs recorded in male and female animals of treated groups, which were isolated and transient.
These findings were not considered to be of toxicological relevance.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.
There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observations
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.
There were no biologically relevant differences in body temperature between the groups.

Body Weight Development
In both males and females, no treatment related changes were noted for body weight and body weight gain during the study period.
Statistically there were no significant diferences noted between the treated and corresponding controls.

Food Consumption
In both males and females, no treatment related changes were noted for treated group when compared to corresponding control.
The statistical evaluation of data revealed significant increase in food intake in male HD group.
This change was not considered to have toxicological relevance.

Haematology and Coagulation
There were no statistically significant changes noted for haematological and coagulation parameters in male and female treated groups
when compared to the corresponding control. However, there was slight decrease in mean monocyte and eosinophil values in male
HD group and slight increase in mean WBC value in female HD group. In the absence of statistical significance and the values being within
the historical control range the changes were not considered to have toxicological relevance.

Clinical Biochemistry
There were no treatment related changes considered for measured clinical biochemistry parameters of male and female treated
groups when compared to corresponding control. However, there was statistically significant increase noted for mean TP value female LD
and HD groups.
In the absence of dose response pattern no relevance to treatment was considered.
There was also increase in mean ALP values in male treated groups (LD, MD and HD groups) and female HD group; increase in
mean GLU values in male MD and HD groups and increase in mean Crea values in male HD group. These changes did not show statistical
significance and in addition the changes noted for GLU and Crea of male and ALP of female lacked dose response pattern. These changes were not
of toxicological relevance. The changes noted for ALP values in males of HD group were not associoated with test item due to absence of relevant
histological changes.

Urinalysis
The urinalysis performed in male animals revealed no treatment related effect in treated groups when compared to the control
group.

Pathology
One control female (No. 50), one female LD group (No. 60), one female of MD group (No. 70) and one female of HD group (No. 73) were found not to be pregnant at terminal sacrifice, but in view of the group distribution this was not considered to be treatment-related.
Other macroscopic organ findings noted were very few, and all of them were considered to be incidental and not to be test item-related. This included a yellow spot on the epididymis in one control and two treated males, which were confirmed histologically to correspond to spermatic granuloma,
a change observed spontaneously in male rats of this strain and age.

Organ Weight
There were no changes noted for organ weight in both males and females considered to be related to treatment when compared to corresponding
control. However, the statistical analysis of data revealed significant increase in relative (to terminal body weight) of thyroid (+parathyroid) gland in male LD group. In the absence of dose response this change was not considered to be related to treatment.

Histopathology
Reproductive organs
There was no indication of test item-related histopathological findings in reproductive organs of male or female rats of this study.
In the males, some minor degenerative testicular and associated epididymal changes were seen in two rats of MD group, but were not considered
test item-related as they lacked dose relationship. Spermatic granuloma(s) were noted in a low number of treated rats. They were considered to be
spontaneous in nature as they are occasionally observed in untreated male rats of this strain and age.
Reproductive organs of most female study animals showed typical post-partum histomorphology without any relevant inter-group difference. There was a tendency towards a lower number of large corpora lutea in the ovary of females of HD group, but in the absence of any effect on litter size this was not considered a test item-related effect.
The reproductive organs of the control and treated females found not to be pregnant at terminal sacrifice showed normal reproductive sexual cycle. The female treated at 100 mg/kg/day had a mild mixed cell endometritis of the uterus which might have contributed to the lack of gravidity in this
animal but was not considered treatment-related in view of its lack of dose relationship and isolated occurrence in this study.

Other organs
No test item-related histopathological findings were noted in the other organs evaluated in this study.
All histopathological changes seen at terminal sacrifice were considered to be incidental in origin and/or within the range of expected changes
for rats of this age and strain kept under laboratory conditions.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at highest dose

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Litter Data
No treatment-related changes were noted for number of still births, number of runts, total number of pups born on PND 0 and number of
male and female pups, sex ratio, live pups on PND 0 and PND 4. The statistical evaluation of data revealed no significant differences between
the values of treated and control groups.

Litter Weight Data
No treatment related changes were noted for the mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 in treated
groups when compared to corresponding control. However, there was slight decrease in male litter weight in HD group on PND 0 and 4, but this was compensated by increase in female litter weight in HD group on PND 0 and 4. Hence, there was no toxicological relevance considered for this finding.

Precoital Interval and Duration of Gestation
No treatment related changes were noted for the precoital interval and duration of gestation in treated groups when compared to control.
All pregnancies resulted in normal births.
Successful mating resulted 90% pregnancy rates in C, LD, MD and HD groups.

Pre- and Post-Natal Data
No treatment related changes were noted for number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and
percentage of pre and post implantation loss in treated groups when compared to control. However, there was slight increase in mean values
of percent post implantation loss in treated groups, but in the absence of dose response pattern and statistical significance the finding was
not considered to have toxicological relevance.

Reproductive Indices
There were no treatment related changes noted for copulation index (%), fertility index (%) and delivery index (%) in treated groups
when compared to corresponding control group.
One control female (No. 50), one female of LD group (No. 60), one female of MD group (No. 70) and one female of HD group (No. 73) were
found not to be pregnant at terminal sacrifice, but in view of the group distribution this was not considered to be treatment-related.

Pup Survival Data
No treatment related changes were noted for survival of the pups from PND 0 to PND 4 in any treated group when compared to controls.
However, there was 1 pup (animal 69) found dead in MD group. This was considered to be incidental. No treatment related changes were
considered for viability index (%).

Pup External Findings
No treatment related gross external findings were observed in any of the treated groups. However, there were few isolated findings namely
hematoma on shoulder, dark snout, white spot on head, dark spot on shoulders and injury on limb noted in few isolated pups of treated or
control groups, which was considered to be incidental.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Concentration analysis of formulation samples was determined in study week 1, 3, 5, and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 105.1%, 100.0%, and 94.6% of the nominal concentration, respectively.

Homogeneity of formulation samples was determined in study week 1 and 5 for all dose groups. The mean recoveries observed for LD group were 105.3% and 106.1%, for MD group 97.4% and 98.9%, and for HD group 94.3% and 95.7% of the nominal value. The coefficients of variation of the different sampling locations (top, middle, bottom) were in LD group 0.5% and 4.6%, in MD group 1.0% and 2.5%, and in HD group 0.7% and 0.7%.

Applicant's summary and conclusion

Conclusions:
In conclusion, the repeated dose administration of the test item to the male (28 days) and female (maximum 54 days)
Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight/ day revealed neither mortalities nor findings of toxicological relevance in male and female animals. There were also no toxicologically relevant findings noted for reproductive and developmental parameters.
Based on the data generated from this “Combined Repeated Dose Oral Toxicity Study with the Reproduction/ Developmental Toxicity Screening
Test the no observed adverse effect level (NOAEL) for general and reproductive and developmental toxicity
is considered to be 1000 mg/kg body weight/ day.
Executive summary:

The aim of this study was to assess the possible effects of the test item on male and female fertility and embryofetal development after repeated dose administration in Wistar rats (10 animals per sex and group). The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of approx. 54 days. Animals of control group were handled identically as the dose groups but received same dose volume of the vehicle used in this study. The groups received the test item in concentrations of 0, 100, 300 and 1000 mg/kg body weight/day

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The males were sacrificed after completion of the mating period on day 29 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on GD 26. Pups were sacrificed on postnatal day 4. A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically. Reproductive organs were evaluated in all study animals.

No mortality occurred in the control or any of the dose groups during the treatment period of this study. There were no clinical signs considered to be of toxicological relevance recorded in male and female animals of treated groups. During the weekly detailed clinical observation, no significant changes or differences between the groups were found. There were no ophthalmoscopic findings in any of the animals of this study.

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.

There were no biologically relevant differences in body temperature between the groups. In both males and females, no treatment related changes were considered for body weight, body weight change and food consumption in treated groups when compared to control.

No treatment-related changes were noted for number of still births, number of runts, total number of pups born on PND 0 and number of male and female pups, sex ratio, live pups on PND 0 and PND 4. No treatment related changes were noted for the mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 in treated groups when compared to corresponding control. No treatment related changes were noted for the precoital interval and duration of gestation in treated groups when compared to control. All pregnancies resulted in normal births. No treatment related changes were noted for number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and percentage of pre- and post-implantation loss in treated groups when compared to control. There were no treatment related changes noted for copulation index (%), fertility index (%) and delivery index (%) in treated groups when compared to corresponding control group. No treatment related changes were noted for survival of the pups from PND 0 to PND 4 treated group when compared to controls. No treatment related changes were considered for viability index (%). No treatment-related gross external findings were observed in any of the treated groups.

There were no statistically significant changes considered to be of toxicological relevance noted for haematological, clinical biochemistry and coagulation parameters in male and female treated groups when compared to the corresponding control. The urinalysis performed in male animals revealed no test- item related effect in any of the treated groups when compared to control. One control female, one female of the low dose group, one female of mid dose group and one female of high dose group were found not to be pregnant at terminal sacrifice, but in view of the group distribution this was not considered to be treatment-related.

The macroscopic organ findings noted were very few, and all of them were considered to be incidental and not to be test item-related. There were no changes noted for organ weight in both males and females considered to be related to treatment when compared to corresponding control. No test item-related histopathological findings were noted in the reproductive organs and in the other organs evaluated in this study.

The no observed adverse effect level (NOAEL) for general and reproductive and developmental toxicity of this study is considered to be 1000 mg/kg body weight/day.