Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
The test substance was test for its reproductive properties in an OECD 422 study. Since there were no effects on fertility/reproduction/development the NOEAL was set at 450 mg/kg bw/d (highest dose tested).
Link to relevant study records
Reference
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:
Aug 2012 - Apr 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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: 235-271 g (mean: 248.25 g, ± 20% = 198.60-297.90 g),
females: 166-192 g (mean: 180.08 g, ± 20% = 144.06-216.09 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.
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
water
Details on exposure:
The test item was weighed into a tared plastic vial on a suitable precision balance and the sterile water was added to give the appropriate
final concentration of the test item based on the active ingredients. The formulation was placed on Vortex machine for short period to
ensure proper homogenistation of the formulation.
The vehicle has been selected as suggested by the sponsor and on the basis of the test item’s characteristics.
The test item formulation was prepared freshly on each administration day before the administration procedure.


The following doses were evaluated:
Control: 0 mg/kg body weight
Low Dose: 50 i.a mg/kg body weight
Medium Dose: 150 i.a mg/kg body weight
High Dose: 450 i.a mg/kg body weight

i.a = active ingredient

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 doses were selected on the basis of data from a Dose Range Finding Study.
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 pregnancy. 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 are minimised.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
EEach dosing concentration was analysed for nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for
the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the high dose and the low dose preparation in study week 1 and 5.
Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours after the
preparation (at room temperature), from high and low dose preparations.
All formulation samples were preserved at -20oC until the analysis. The samples were analyzed at BSL BIOSERVICE. The results are reported in
the annex of the final report.
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:
7 days / week
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.
Remarks:
Doses / Concentrations:
50 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
450 mg/kg bw/d
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 doses were selected on the basis of data from a Dose Range Finding Study.
Positive control:
None
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
Oestrous cyclicity (parental animals):
None
Sperm parameters (parental animals):
Parameters examined in male parental generations:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility]
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross external anomalies, weight gain]

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external abnormalities]
Postmortem examinations (parental animals):
SACRIFICE
All surviving male animals were sacrificed after the completion of the mating period (total dosing period: 28/29 days) on study day 29 or 30, while female animals were sacrificed on post-natal day 4 using an anaesthesia (ketamine/xylazin, 3:1, medistar Arzneimittel, lot no: 00212, expiry date: 03/2014 and Serumwerk, lot no: 00711, expiry date: 08/2013) was used.

GROSS NECROPSY
Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Females showing no evidence of copulation up to 14 days of the mating period were sacrificed on day 26 after the last day of the mating period.
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.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved in 10 % neutral buffered formalin, except for eyes, testes and epididymides which were preserved in modified Davidson’s Solution.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was not recorded for any females sacrificed 26 days after the end of the pairing period with no evidence of mating and for any females sacrificed on day 25 post-coitum due to non-delivery. At terminal sacrifice female no. 71 of HD group showed no sign of implantation. However, a fetal head was detected in the cage. Considering the no implantation sites in the uterus, the animals was put in the category of non pregnant and evaluated histopathologically. The histopathological examination of uterus indicated previous pregnancy.

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 animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved
for a histopathological examination.
Additional organs of animals which died during the course of the study were preserved and examined histopathologically (on addition cost) in order to determine the cause of death (Table 8). Animal no. 77 of HD group was euthanised for animal welfare reason. Inadvertantly the animal was wrongly communicated as terminal sacrificed and non pregnant animal to the pathologist. Hence, full histopathology (except reproductive organs) was not performed for this animal.


All organs and tissues listed above were evaluated from randomly selected males and females of the control and high dose group:
Males Nos.: 3, 4, 6, 8, 10, 32, 35, 37, 38, 39; Females Nos.: 41, 43, 45, 46, 49, 71, 73, 74, 76, 78.
Kidney, trachea, thymus and stomach (nonglandular and glandular) were also evaluated from randomly selected males and females of the
low and medium dose group:
Males Nos.: 11, 12, 15, 16, 18, 24, 26, 27, 28, 29; Females Nos.: 51, 52, 55, 57, 59, 61, 62, 66, 67, 70.
One Testis, epididymides (one complete organ and the leftover from sperm analysis), ovaries, uterus with cervix, vagina, accessory sex organs
(prostate, seminal vesicle with coagulating gland) and all organs showing gross lesions were examined in all animals.
All non-pregnant female animals (including animal 71 and 77) were examined histopathologically.
All decedents Nos. 33, 72, 75 and 80 (excluding animal no. 77) were examined histopathologically.
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).
Reproductive indices:
Copulation index; fertility index, delivery index, as well as viability index were calculated.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: see below
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Mortality
One male (No. 33) and three females (Nos. 72, 75 and 80) of HD group were found dead during the treatment period. Animal no. 77 was killed prematurely during the study.
Histopathologically, the death of animals 33, 72 and 75 was considered to be related to erroneous instillation/regurgitation of test item formulation into the airways. For animals 80, its death was considered to be due to prominent lesions in the kidney and heart (this could be considered incidental). Animal no. 77 was killed prematurely during the study and full histopathology could not be performed to verify the cause of death. However considering the clinical sign it is assumed that at some point during the study the animals was gavaged wrongly, which deteriorated the general health condition of the animal.

Clinical Observations
The clinical signs observed in male and female animals during the study period were mostly piloerection, salivation, nasal discharge, moving
the bedding observed transiently in LD or MD groups. These finding were observed profoundly in HD group animals in a dose related manner.
Besides these signs there were few occational or transient appearance of clinical signs namely abnormal breathing, aggressive behavior, eschar
or injury, exophthalmos, half eyelid closure, vocalization and alopecia noted in MD or HD group animals.
In found dead or killed animals, in addition to above findings the clinical signs recorded were kyphosis, apathy, dehydration and hypothermia.
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 males, there were slight decrease in mean body weight gain noted in 1st week (MD and HD groups) and 2nd week (LD and HD groups) of
premating. The body weight changes were very minor and within the normal body weight range. Hence, no relevance due to treatment was
considered. In females, there was statistically significant decrease noted in mean body weight in HD group during the 2nd and 3rd week of study. In addition there were slight to moderate decrease noted in HD group during gestation and lactation period without attaining statistical
significance. There was slight decrease in body weight gain noted in HD group animals during the study period. These changes in body weight
gain were not considered to have biological relevance and therefore not considered to be due to treatment.

Food Consumption
In males, there were no statistically significant differences noted for food intake in treated groups when compared to corresponding control.
However, there was slight decrease noted in treated groups during the 1st week of treatment. In 2nd week the decrease was noted in LD and HD
groups indicating no dose response pattern.
In females, there was statistically significant decrease in food intake noted in HD group during 2nd week. There was slight decrease in food
intake noted during gestation and lactation days in treated groups without statistical significance.
The changes in food intake noted in male and female animals of treatment groups did not appear to have biological relevance and hence were
not associated with treatment.

Sperm analysis
No test item related changes were noted for epididymal sperm motility and testicular sperm count. However, there were very slight decrease
(without statistical significance) noted for testicular sperm count in MD and HD groups, but in the absence of histological changes in testes the
toxicity relevance was not considered.

Pathology
At terminal sacrifice, macroscopic organ findings noted were few, and none of them was considered to be test item-related.
Among the surviving females, one control rat (No. 49) and one rat of HD group (No. 77) were found not to be pregnant. Hyalinized mural arterial
walls were seen in the uterus of No. 71, which indicated a precedent gravid state of this animal. Hence, this animal was considered pregnant.
The two other females showed physiological sexual cycling and their non-pregnant state were therefore not considered treatment-related.
One male (No. 33) and three females (Nos. 72, 75 and 80) of HD group were found dead during the treatment period. Female No. 80 showed small
spleen and red discoloured axillary lymph node and thymus at necropsy, and it was non-pregnant. Based on the results of histopathological
evaluation, its death was considered to be due to prominent lesions in the kidney and heart. Based on macroscopic findings noted in the trachea
(foamy content) and/or lung (dark discoloured or not collapsed) and as confirmed histologically, death of the other three decedents was considered to be related to erroneous instillation/regurgitation of test item formulation into the airways.

Organ Weight
In males and females, there was statistically significant increase noted for absolute Uterus (with cervix) weight noted in female LD group, statistically significant decrease in absolute thymus weight in female MD group. The calculated relative (to terminal weight) weight indicated statistically
significant increase in liver weight in male MD and HD groups, statistically significant decrease in left adrenal weight in male LD group, s
tatistically significant increase in left kidney weight in MD group, statistically significant increase in uerus (with cervix) weight in all treated groups.
The changes noted for absolute or relative weight liver, uterus, thymus, adrenal and kidney in male or female treated groups did not show either
dose response pattern or there were no histological changes that were considered to be associated with test item.
Hence, the above changes in organ weight were not considered to have toxicological relevance.

Histopathology
At terminal sacrifice, no test item-related effects were noted on male and female reproductive organs.
In the kidney, nephropathy, mainly comprising basophilic tubules in the medulla and cortex and papillary edema, was seen in a dose-related
manner in females of all three dose-groups. The renal changes indicate tubular damage and regeneration as a direct effect of the test item.
In view of the type and severity of changes observed in MD and HD groups, they are considered adverse in these dose groups. In the forestomach,
a number of degenerative and/or inflammatory changes were seen in females of all three dose groups, in a dose-related manner. In the males,
differences to the control group were very small and seen in MD and HD groups, only. The gastric changes are indicative of a local irritant effect
of the test item, in the females probably secondarily exacerbated by the observed prominent renal pathology and associated stress situation.
They are therefore considered to be irrelevant for risk evaluation in humans. In the trachea, there was indication of a local irritant effect of the
test item formulation in some males and females of MD and HD groups, corroborating observations in the decedents, which was not considered
to be of toxicological relevance to humans.
No test item-related histopathological findings were noted in the other organs evaluated in this study.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
mortality
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 specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
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. Statistical analysis of data revealed no significant changes between treated and
corresponding controls.

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, except for one isolated female (no. 71) of HD group where the gestation days lasted for 25 days.
Successful mating resulted in 90% pregnancy rates in C, 100% pregnancy rate in LD and MD. 100% pregnanacy rate was also recorded in HD
group (excluding 2 premature deaths, animal 77 and 80).
In one female (Animal 71), no implantation sites could be detected at necropsy. However there was just a fetal head noticed in cage.
Considering no implanation site in uterus during the terminal sacrifice, the animal was put in category of non pregnant females.
Histopathological examination showed hyalinized mural arterial walls in the uterus, which indicated a precedent gravid state of this animal.

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. Statistical analysis of data revealed no significant
changes between the treated and control group.

Reproductive Indices
There were no treatment related changes noted for copulation, fertility, delivery and viablity indices in treatment groups when compared to control.

Pup Survival Data
No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.

Pup External Findings
No treatment-related gross external findings were observed in any of the treated groups. Black spot on snout and reddish snout was noticed in 1
pup each of female 46 (pup no. 1) and 64 (pup no. 1), respectively. These findings were considered incidental in nature.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on reproductive/developmental parameters observed up to highest dose tested
Reproductive effects observed:
not specified
Conclusions:
In conclusion, the repeated dose administration of Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate to the male (28/29 days) and female (maximum 54 days) Wistar rats at
dosages of 50, 150, and 450 a.i mg/kg body weight day revealed findings of toxicological relevance at 150 and 450 a.i mg/kg/day.
There were 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 with this test item, a dose of 50 mg/kg/day was considered to be the NOAEL (maternal) and of 450 mg/kg bw/d the NOAEL (male) .
The NOAEL for reproductive and developmental toxicity is considered to be 450 a.i mg/ kg body weight/day.
Executive summary:

The aim of this study was to assess the possible effects of Reaction mass of potassium ethyl octyl phosphonate and diethyl octylphosphonate on male and female fertility and embryofetal development after repeated dose administration inWistar rats.

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 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 is completed. Animals of an additional control group were handled identically as the dose groups but received sterile water, the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistar rats.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except the food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

Haematological and clinical biochemistry evaluations were performedon blood samples collected at terminal sacrifice from five males and five randomly selected females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment and at the end of the study.

After 14 days of treatment to both male and female animals were mated (1:1) for a maximum of 14 days. From subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. 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, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

The males were sacrificed after completion of the mating period on treatment days 29 and 30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26 from the day of mating.

Pups sacrificed on postnatalday 4 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically. The examinations ofKidney, trachea, thymus and stomach (nonglandular and glandular)were extended to 5 selected animals of low and mid dose groups.

The following doses based on the purity of the test item (85.1%) were evaluated:

Control:                       0        mg/kg body weight

Low Dose:                   50       mg/kg body weight

Medium Dose:             150     mg/kg body weight

High Dose:                  450     mg/kg body weight

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in sterile water and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28/29 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministration volume was 5 mL/kg body weight.

Summary Results

One male (No. 33) and three females (Nos. 72, 75 and 80) treated at 450 mg/kg/day were found dead during the treatment period. Animal no. 77 was killed due to severe clinical signs. The cause of death of this animal was not attributed to treatment.

Slight clinical signs were observed during the treatment period in the treated groups (LD and MD groups) and the control group of this study. However, findings namelypiloerection, salivation, nasal discharge, moving the bedding were observed profoundly in HD group animals. Besides there were few occational or trancient appearance of clinical signs namely abnormal breathing, aggressive behavior, eschar or injury, exophthalmos, half eyelid closure, vocalization and alopecia noted in MD or HD group animals.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

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 treatment related changes considered for body weight, body weight gain and food intake in male and female animals of treated groups when compared to corresponding 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 except for one isolated female (animal 71) of HD group, where the duration of gestation was longer than the normal and the pups delivered were cannibalised. This isolated finding was assumed to be incidental in origin.

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.

No changes in reproductive indices and on thesurvival of the pups from PND 0 to PND 4 were observed in any treatment group when compared with controls.

No treatment-related gross external findings were observed in any of the treated groups.

There were no treatment related changes considered for hematology, blood coagulation and clinical biochemistry parameters measured at the end of the study.

There were no considerable test item-related differences noted in any of the urinary parameters tested.

Organ weight (absolute and relative to terminal body weight) data revealed no changes considered to be of toxicological relevance.

No test item related changes were noted for epididymal sperm motility and testicular sperm count.

 

At terminal sacrifice, no test item-related effects were noted on male and female reproductive organs examined histopathologically.

In the kidney, nephropathy, mainly comprising basophilic tubules in the medulla and cortex and papillary edema, was seen in a dose-related manner in females of all three dose-groups. The renal changes indicate tubular damage and regeneration as a direct effect of the test item. In view of the type and severity of changes observed in MD and HD groups, they are considered adverse in these dose groups. In theforestomach, a number of degenerative and/or inflammatory changes were seen in females of all three dose groups, in a dose-related manner. In the males, differences to the control group were very small and seen in MD and HD groups, only. The gastric changes are indicative of a local irritant effect of the test item, in the females probably secondarily exacerbated by the observed prominent renal pathology and associated stress situation. They are therefore considered to be irrelevant for risk evaluation in humans. In thetrachea, there was indication of a local irritant effect of the test item formulation in some males and females of MD and HD groups, corroborating observations in the decedents, which was not considered to be of toxicological relevance to humans.

No test item-related histopathological findings were noted in the other organs evaluated in this study.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable witouth restrictions
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The aim of this OECD 422study was to assess the possible effects of Reaction mass of potassium ethyl octyl phosphonate and diethyl octylphosphonate on male and female fertility and embryofetal development after repeated dose administration inWistar rats. 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 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 is completed. Animals of an additional control group were handled identically as the dose groups but received sterile water, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats. During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly, except the food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals. Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five males and five randomly selected females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group. Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment and at the end of the study. After 14 days of treatment to both male and female animals were mated (1:1) for a maximum of 14 days. From subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. 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, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. The males were sacrificed after completion of the mating period on treatment days 29 and 30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26 from the day of mating. Pups sacrificed on post natal day 4 and those found dead, were carefully examined for gross external abnormalities. A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically. The examinations ofKidney, trachea, thymus and stomach (nonglandular and glandular) were extended to 5 selected animals of low and mid dose groups. The following doses based on the purity of the test item (85.1%) were evaluated:

Control:                       0        mg/kg body weight

Low Dose:                   50       mg/kg body weight

Medium Dose:             150     mg/kg body weight

High Dose:                  450     mg/kg body weight

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in sterile water and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28/29 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.

 

Summary Results

One male (No. 33) and three females (Nos. 72, 75 and 80) of HD group were found dead during the treatment period. Animal no. 77 was killed prematurely during the study. 

Histopathologically, the death of animals 33, 72 and 75 was considered to be related to erroneous instillation/regurgitation of test item formulation into the airways. For animals 80, its death was considered to be due to prominent lesions in the kidney and heart (this could be considered incidental). Animal no. 77 was killed prematurely during the study and full histopathology could not be performed to verify the cause of death. However considering the clinical sign it is assumed that at some point during the study the animals was gavaged wrongly, which deteriorated the general health condition of the animal.

Slight clinical signs were observed during the treatment period in the treated groups (LD and MD groups) and the control group of this study. However, findings namely piloerection, salivation, nasal discharge, moving the bedding were observed profoundly in HD group animals. Besides there were few occasional or trancient appearance of clinical signs namely abnormal breathing, aggressive behavior, eschar or injury, exophthalmos, half eyelid closure, vocalization and alopecia noted in MD or HD group animals.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

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 treatment related changes considered for body weight, body weight gain and food intake in male and female animals of treated groups when compared to corresponding 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 except for one isolated female (animal 71) of HD group, where the duration of gestation was longer than the normal and the pups delivered were cannibalised. This isolated finding was assumed to be incidental in origin.

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.

No changes in reproductive indices and on thesurvival of the pups from PND 0 to PND 4 were observed in any treatment group when compared with controls.

No treatment-related gross external findings were observed in any of the treated groups.

There were no treatment related changes considered for hematology, blood coagulation and clinical biochemistry parameters measured at the end of the study.

There were no considerable test item-related differences noted in any of the urinary parameters tested.

Organ weight (absolute and relative to terminal body weight) data revealed no changes considered to be of toxicological relevance.

No test item related changes were noted for epididymal sperm motility and testicular sperm count.  

At terminal sacrifice, no test item-related effects were noted on male and female reproductive organs examined histopathologically.
In the kidney, nephropathy, mainly comprising basophilic tubules in the medulla and cortex and papillary edema, was seen in a dose-related manner in females of all three dose-groups. The renal changes indicate tubular damage and regeneration as a direct effect of the test item. In view of the type and severity of changes observed in MD and HD groups, they are considered adverse in these dose groups. In the forestomach, a number of degenerative and/or inflammatory changes were seen in females of all three dose groups, in a dose-related manner. In the males, differences to the control group were very small and seen in MD and HD groups, only. The gastric changes are indicative of a local irritant effect of the test item, in the females probably secondarily exacerbated by the observed prominent renal pathology and associated stress situation. They are therefore considered to be irrelevant for risk evaluation in humans. In the trachea, there was indication of a local irritant effect of the test item formulation in some males and females of MD and HD groups, corroborating observations in the decedents, which was not considered to be of toxicological relevance to humans. No test item-related histopathological findings were noted in the other organs evaluated in this study.

In conclusion, the repeated dose administration of Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate to the male (28/29 days) and female (maximum 54 days) Wistar rats at

dosages of 50, 150, and 450 a.i mg/kg body weight day revealed findings of toxicological relevance at 150 and 450 a.i mg/kg/day. There were 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 with this test item, a dose of 50 mg/kg/day was considered to be the NOAEL (maternal) and of 450 mg/kg bw/d the NOAEL (male).The NOAEL for reproductive and developmental toxicity is considered to be 450 a.i mg/ kg body weight/day


Short description of key information:
In conclusion, the repeated dose administration of Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate to the male (28/29 days) and female (maximum 54 days) Wistar rats at
dosages of 50, 150, and 450 a.i mg/kg body weight day revealed findings of toxicological relevance at 150 and 450 a.i mg/kg/day. There were 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 with this test item, a dose of 50 mg/kg/day was considered to be the NOAEL (maternal) .The NOAEL for reproductive and developmental toxicity is considered to be 450 a.i mg/ kg body weight/day

Justification for selection of Effect on fertility via oral route:
The OECD 422 study was selected as relevant available reproductive toxicity screening study due to its reliability and since it provides a sensitive NOEL.

Justification for selection of Effect on fertility via inhalation route:
In accordance with column 2 of REACH Annexes VIII and IX, the reproductive toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the inhalation route because exposure of human via inhalation, especially in a higher extent than via oral application as performed in the animal studies, is considered unlikely taking into account the vapour pressure of the substance and the physical form (paste).

Justification for selection of Effect on fertility via dermal route:
In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the dermal route because
- no systemic effects or other evidence of absorption were observed in skin and eye irritation studies in rabbits
- due its irritant properties only local skin effects are expected to occur; however, since the edema/erythema were reversible these are considered to have no impact on the absorption of the test substance via skin. Due to the combination of its polar (ionic) character and the long extent of the alcoholic chain it is unlikely that higher amounts than tested in a repeated oral toxicity study will be systemically available via the skin barrier.

Effects on developmental toxicity

Description of key information

Based on the effects observed in course of an OECD 414 study, it was concluded that the NOAEL for

- maternal toxicity is 50 mg/kg bw/d due to treatment related significant reduction in maternal body weight, corrected body weight gain and food consumption at 200 and 800/450 mg/kg bw/d and significant reduction in uterine weight at 800/450 mg/kg bw/d.

- fetal developmental toxicity is 800/450 mg/kg bw/d as there were no signs of teratogenicity in any of the tested leveks up to the highest dose.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311, Gajwel, Mandal, Medak District, Telangana

- Age at study initiation: 12 to 13 weeks
- Weight at study initiation:
Mean body weight Body weight range
G1 : 215.166 ± 15.01 191.21 to 244.60g
G2 : 215.756 ± 15.72 191.21 to 241.87g
G3 : 214.956 ± 13.81 189.93 to 239.41g
G4 : 215.421 ± 14.94 184.21 to 238.16g

- Housing:
Rats were housed in standard polysulfone rat cages (size: Length 425 mm x Breadth 266 mm x Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube.

i. Pre mating / Acclimatization: Two rats of the same sex per cage were housed.
ii. Mating: Female rats were cohabited with males in a 1:1 ratio in same cage.
iii. Post-mating / Treatment: After mating confirmation, females were housed individually.

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories, P.O.Box 44220, Madison Wi 53744-4220, was provided ad libitum to the animals.

- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 65 – 68 %,
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.

IN-LIFE DATES: From: 14 April 2016 To: 13 May 2016
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dose formulations were prepared once in 3 – 4 days based on the stability results. The prepared dose formulations were divided into daily aliquots and stored in the experimental room (19 to 25 ºC) when not in use.

Required quantities of the test item were weighed in pre-calibrated beakers for each dose level separately and about 3 to 8 mL of vehicle [Milli-Q water] was added and stirred using a glass rod till a uniform solution was obtained and the volume was made up to the mark using the vehicle to get the final desired concentration.

The homogeneity of the Leomin AN Liq formulation during treatment/sampling was maintained by constant stirring using a magnetic stirrer.

Pre-calibration of the beaker to desired volume: Milli-Q water was measured in a graduated cylinder to the final volume of 150 mL. The measured water was transferred into a clean beaker (to be pre-calibrated) and upper and lower meniscus of water was marked on the beaker. Once these lines had been marked, the water was discarded and the beaker was dried. The upper meniscus was used to make up the volume while preparing the dose formulations.

For vehicle control groups, vehicle [Milli-Q water] was administered.

The weight of the test item, the volume of preparation and volume of administration varied depending on the requirement. The unused dose formulations were sent for disposal.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
by Liquid Chromatograph with Mass Spectrometer (LC-MS/MS)
Details on mating procedure:
During the mating period, female rats were cohabited with males in a 1:1 ratio. When sperm was detected in a vaginal smear or vaginal plug was observed in the morning, the animal was considered to be mated. This day was considered as day 0 of gestation.

The mated female rats obtained each day were assigned to the treatment groups and vehicle control groups by body weight stratification. This procedure was continued till the required numbers of Day 0 mated females were obtained (24 per group).
Duration of treatment / exposure:
The dose formulations of test substance was administered orally by gavage using disposable plastic syringe attached with a metal feeding/intubation cannula to rats of low dose (G2), mid dose (G3) and high dose (G4) groups once daily from GD 5 to GD19 of presumed gestation, at approximately the same time each day (varying by± 2 hours).

The dose volume ( at 10 mL/kg) to be administered was calculated based on the body weight of individual animals on first day of treatment
(on GD 5) and was adjusted according to the most recently recorded body weights recorded till GD 19. The animals in the vehicle control group (G1) were handled in an identical manner to the treatment group and were administered vehicle only.
Frequency of treatment:
Gestation days: 5 to 19
Duration of test:
14 April 2016 to 27 October 2016
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
due to mortality at 800 mg/kg bw/d at high dose of 800 mg/kg bw/d the dosing was reduced to 450 mg/kg bw/d
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Remarks:
reduced to 450 mg/kg bw/d on gestation day
- 10: rats no. Rs3891 to Rs3898 (8 rats)
- 9: rats no. Rs3899 to Rs3903 (5 rats)
- 8: rats no Rs3904 to Rs3913 (10 rats)
- 7: rat no. Rs3914
No. of animals per sex per dose:
24 day '0' pregnant rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:

As per the Sponsor’s suggestion based on the results of a combined repeated dose oral toxicity study with reproduction/developmental toxicity screening test the following doses are selected for definitive embryo-fetal developmental toxicity study with Leomin AN Liq in Wistar rats by oral route:
• G1 - Vehicle control - 0 mg/kg/day
• G2 - Low dose - 50 mg/kg/day
• G3 - Mid dose - 200 mg/kg/day
• G4 - High dose - 800/450 mg/kg/day

- Rationale for animal assignment (if not random): The mated female rats obtained each day were assigned to the treatment groups and vehicle control groups by body weight stratification. This procedure was continued till the required numbers of Day 0 mated females were obtained (24 per group).
Maternal examinations:
Observations for clinical signs were performed twice a day - pre dose and post dose (within 1-2 hours of administration) during treatment days and once on non-treatment days.

Each rat was observed twice daily for morbidity and mortality i.e., once in the morning and once in the afternoon. Based on the assessment, as there were toxic signs of concern, the observation was carried out twice during weekends and public holidays.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [half per litter ]
- Skeletal examinations: Yes: [ half per litter ]
- Head examinations: Yes: [half per litter ]
Statistics:
The data on maternal body weight, body weight change, gravid uterine weight, corrected body weight gain, maternal food consumption, number of corpora lutea , number of implantations, total number of fetuses, male and female fetus number and weight was analyzed using ANOVA model, after testing for homogeneity for intra group variance using Levene’s test.

Incidence of pre-implantation loss, post implantation loss, Number of early, late and total resorptions were analyzed using Kruskal Wallis test.

Overall percentage of minor external, visceral and skeletal malformations, Sex ratio and number of dams with any resorptions were analyzed using
2 X 2 Contingency Table.

Statistically significant differences (p < 0.05), indicated by the aforementioned tests were designated by symbol ‘*’ throughout the report.
Indices:
Maternal Parameters:
- Mean no. of corpora lutea/group = total no. of corpora lutea/total no. of pregnant animals
- Mean no. of implantations/group = total no. of implantations/total no. of pregnant animals
- embryonic resorption index (%) = no. of early resorptions/no. of implantations x 100
- Fetal resorption index (%) = no. of late resorptions/no. of implantations x 100
- Pre-implantation loss/group (%) = no. of CL - no. of implantations/ no. of CL x 100
- Post-implantation loss/group (%) = no. of (early + late) resorptions/total no. of implantations x 100
- Implantation index (%) = no. of implantation sites/ no. of corpora lutea x 100

Litter Paramenters:
- Mean litter size/group = total no. of fetuses/total no. of pregnant animals
- Percentage of abnormal fetuses = no. of abnormal fetuses/total no. of fetuses x 100
- Percentage of live fetuses/group (live fetus index) = no. of live fetuses/total no. of fetuses x 100
- Percentage of dead fetuses/group (dead fetus index) = no. of dead fetuses/total no. of fetuses x 100
- Sex ratio (F:M) = no. of females/no. of males


Other:
- Corrected body weight (carcass weight) = terminal body weight (body weight on GD 20) - unopened uterine weight
- Correctred body weight gain = Corrected body weight - body weight on GD 5
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In high dose (800/450 mg/kg/day) group, there was treatment related clinical sign of hypoactivity during post dose observation in few rats (8/23) during GD 7- 9 and one rat Rs 3891 was found dead on GD 9 (when treated at 800 mg/kg/day). After the dose was reduced to 450 mg/kg/day, there were no clinical signs and all the rats were normal.
Mortality:
mortality observed, treatment-related
Description (incidence):
One animal at 800 mg/kg bw/d.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg/day, there was a significant reduction in mean body weights on GD 20 (-6%) as compared to vehicle control group. At 800/450 mg/kg/day, there was a significant decrease in mean body weight on GD 8 (-9%), GD 11 (-11%), GD 14 (-9%), GD 17 (-8%) and GD 20 (-9 %) as compared to vehicle control group.

At 200 mg/kg/day, there was a significant reduction in maternal body weight gain during treatment period GD 5 to 20 (-23 %), and for entire period of gestation GD 0- 20 (-21%), as compared to vehicle control group.
At 800/450 mg/kg/day, there was a significant reduction in maternal weight gain during GD 5 - 8 (-314%), during treatment period GD 5-20 (-37 %), and for entire period of gestation GD 0-20 (-31 %), as compared to vehicle control group.
There was significant reduction in corrected body weight (-7 to -8 %) and weight gain (-111 % to -140 %) at 200 mg/kg/day and 800/450 mg/kg/day respectively as compared to vehicle control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg/day, there was a significant reduction in maternal food consumption during GD 11-14 (-23%), GD 14-17 (-13%), during treatment period GD 5-20 (-12%) and for entire gestation period GD 0-20 (-10 %) as compared to vehicle control group.
At 800/450 mg/kg/day, there was a significant reduction in maternal food consumption during GD 5-8 (-48 %), GD 8-11 (-24 %), GD11-14 (-17 %), and during treatment period GD5-20 (-19 %) and for entire gestation period GD 0-20 (-15 %) as compared to vehicle control group.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were gross pathological findings of thickening of stomach- non glandular mucosa - multifocal in 8/24 rats at 200 mg/kg/day and 17/23 at 800/450mg/kg/day. The rat found dead had scanty ingesta in stomach and intestine.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
no effects observed
Description (incidence and severity):
There were no treatment-related changes observed in litter parameters at all the doses tested. At 50 mg/kg/day there was a significant increase in fetal weight of total live fetuses and female fetuses and a significant decrease in number of female fetuses as compared to vehicle control group these findings were considered incidental and not attributed to treatment.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MATERNAL BODY WEIGHT AND WEIGHT GAIN

The maternal group mean body weights were unaffected by the administration of test item Leomin AN Liq at the dose of 50 mg/kg/day.

At 200 mg/kg/day, there was a significant reduction in mean body weights on GD 20 (-6%) as compared to vehicle control group. At
800/450 mg/kg/day, there was a significant decrease in mean body weight on GD 8 (-9%), GD 11 (-11%), GD 14 (-9%), GD 17 (-8%) and GD 20
(-9 %) as compared to vehicle control group.

At 200 mg/kg/day, there was a significant reduction in maternal body weight gain during treatment period GD 5 to 20 (-23 %), and for entire period of gestation GD 0- 20 (-21%), as compared to vehicle control group.

At 800/450 mg/kg/day, there was a significant reduction in maternal weight gain during GD 5 - 8 (-314%), during treatment period GD 5-20
(-37 %), and for entire period of gestation GD 0-20 (-31 %), as compared to vehicle control group.

There was significant reduction in corrected body weight (-7 to -8 %) and weight gain (-111 % to -140 %) at 200 mg/kg/day and 800/450 mg/kg/day respectively as compared to vehicle control group.

FOOD CONSUMPTION
At 50 mg/kg/day, the maternal food consumption was comparable to vehicle control group.

At 200 mg/kg/day, there was a significant reduction in maternal food consumption during GD 11-14 (-23%), GD 14-17 (-13%), during treatment period GD 5-20 (-12%) and for entire gestation period GD 0-20 (-10 %) as compared to vehicle control group.

At 800/450 mg/kg/day, there was a significant reduction in maternal food consumption during GD 5-8 (-48 %), GD 8-11 (-24 %), GD11-14 (-17 %), and during treatment period GD5-20 (-19 %) and for entire gestation period GD 0-20 (-15 %) as compared to vehicle control group.

The significant reduction in maternal body weights along with concomitant reduction in food consumption at 200 and 800/450 mg/kg/day was considered as treatment-related finding.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 50 mg/kg bw/day
Based on:
other: treatment-related significant reduction in maternal body weight, corrected body weight gain and food consumption
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 800 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 50 mg/kg/day there was a significant increase in fetal weight of total live fetuses and female fetuses and a significant decrease in number of female fetuses as compared to vehicle control group these findings were considered incidental and not attributed to treatment.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
There was an incidence of small fetus at 200 and 800/450mg/kg/day.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed during skeletal examination of fetuses of dams treated up to 800/450 mg/kg/day.
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed during visceral examination of fetuses of dams treated up to 800/450 mg/kg/day.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
MATERNAL DATA

No treatment-related changes were observed in maternal parameters at 50, and 200 mg/kg/day. There was significant decrease in mean gravid uterine weight (-15%) and non- significant increase in late resorptions, pre and post implantation loss at 800/450 mg/kg/day as compared to vehicle control group. No treatment-related changes were observed in other maternal parameters (mean numbers of corpora lutea, early resorptions, and dams with any resorption).

LITTER DATA
There were no treatment-related changes observed in litter parameters at all the doses tested. At 50 mg/kg/day there was a significant increase in fetal weight of total live fetuses and female fetuses and a significant decrease in number of female fetuses as compared to vehicle control group these findings were considered incidental and not attributed to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 800 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Key result
Developmental effects observed:
no
Conclusions:
Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

Maternal toxicity is 50 mg/kg/day due to treatment-related significant reduction in maternal body weight, corrected body weight gain and food consumption at 200 and 800/450 mg/kg/day and significant reduction in uterine weight at 800/450 mg/kg/day.

Fetal developmental toxicity is 800/450 mg/kg/day in Wistar rats as the litter data parameters were comparable to vehicle control group up to the high dose of 800/450mg/kg/day.

Teratogenicity is 800/450 mg/kg/day as there were no signs of teratogenicity in any of the tested dose levels up to the high dose of
800/450 mg/kg/day

in Wistar rats when Leomin AN Liq was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.
Executive summary:

The objective of this study was to evaluate theembryo-fetal developmentaltoxicity of test item Leomin AN Liq when administered to pregnant Wistar rats by oral route during gestation days (GD) 5 to GD19.The results of this study helped to establish the No Observed Adverse Effect Level (NOAEL) of the test item.

 

In this study, each group (G1, G2, G3 and G4) consisted of 24 presumed pregnant Wistar rats (gestation day 0). Day `0' of gestation for each individual female rat in the study was considered as the day on which vaginal smear was found positive for sperm.

 

The test item, Leomin AN Liq was dissolved in vehicle [Milli-Q water] and administered orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 50, 200 and 800/450 mg/kg/day for low (G2), mid (G3) and high(G4) dose group rats, respectively. The rats in the vehicle control (G1) group received the vehicle alone. A constant dose volume of 10 mL/kg body weight was administered to all groups.

 

High dose group (G4) rats were treated at the dose of 800 mg/kg/day initially (From 25 April 2016 to 29 April 2016). The rats exhibited treatment related clinical sign of hypoactivity, one rat was found dead on GD 9 and there was drastic reduction in body weight and food intake. Considering the treatment related findings and in consultation with the sponsor, the high dose was reduced from 800 to 450 mg/kg/day
(30 April to 12 May 2016). The dose for the high dose group is presented as 800/450mg/kg/day.
  

 

 The dose formulation solutions were analyzed for active ingredient concentrations at the initiation and termination of treatment. The results of analysis of formulations revealed that the analyzed concentrations were within the acceptable limits.

 

The mated females were observed twice daily for clinical signs, mortality and morbidity. Body weights were recorded on GD0, 3, 5, 8, 11, 14, 17 and 20. About 200 g (food input) was provided on Day ‘0’. The food left over was recorded and replenished to a known weight on GD 3, 5, 8, 11, 14 and 17. The food left over was also recorded on Day 20 of presumed gestation. The intermittent body weight gain and food intake was calculated and presented for rats found pregnant at caesarean section.

 

Caesarean section was performed for all the surviving rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the animal were removed (by laparotomy) and the contents were examined.The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. The number of corpora lutea was counted on each ovary. All the fetuses were sexed, weighed and examined for external malformations.Approximately half the number of fetuses from each dam was examined for visceral malformations and the remaining half was evaluated for skeletal malformations.

 

Results of the study are presented below:

 

·       There was no mortality and clinical signs in the low dose and mid dose groups. In the high dose group there was a treatment-related clinical sign of hypoactivity and in addition, one rat (Rs3891) was found dead on GD 9 (when treated at the dose of 800 mg/kg/day). After the dose was reduced to 450 mg/kg/day, there were no clinical signs and all the rats were normal.

·       There was treatment-related significant reduction in maternal body weights, corrected body weight gain and food consumption in mid and high dose groups as compared to vehicle control group.

·       Gross necropsy finding of thickening of stomach non-glandular mucosa multifocal was observed in 8/24 and 17/23 rats of mid and high dose groups respectively.

·       Maternal data parameter - uterine weight was significantly reduced in high dose group. The other maternal data parameters were comparable to vehicle control group up to the high dose.

·        Litter data parameters were comparable to vehicle control group at all the doses tested.

·       Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs ofteratogenicity in any of the tested doses.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

 

·          Maternal toxicity is 50 mg/kg/day due to treatment-related significant reduction in maternal body weight, corrected body weight gain and food consumption at 200 and 800/450 mg/kg/day and significant reduction in uterine weight at 800/450 mg/kg/day. 

 

·          Fetal developmental toxicity is 800/450 mg/kg/day in Wistar rats as the litter data parameters were comparable to vehicle control group up to the high dose of 800/450 mg/kg/day.

 

·          Teratogenicity is 800/450 mg/kg/day as there were no signs of teratogenicity in any of the tested dose levels up to the high dose of 800/450 mg/kg/day

 

in Wistar rats when Leomin AN Liqwas administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable without restriction
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In this study, each group (G1, G2, G3 and G4) consisted of 24 presumed pregnant Wistar rats (gestation day 0). Day `0' of gestation for each individual female rat in the study was considered as the day on which vaginal smear was found positive for sperm.

The test item, Leomin AN Liq was dissolved in vehicle [Milli-Q water] and administered orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 50, 200 and 800/450 mg/kg/day for low (G2), mid (G3) and high(G4) dose group rats, respectively. The rats in the vehicle control (G1) group received the vehicle alone.

Results of the study are:

• There was no mortality and clinical signs in the low dose and mid dose groups. In the high dose group there was a treatment-related clinical sign of hypoactivity and in addition, one rat (Rs3891) was found dead on GD 9 (when treated at the dose of 800 mg/kg/day). After the dose was reduced to 450 mg/kg/day, there were no clinical signs and all the rats were normal.

• There was treatment-related significant reduction in maternal body weights, corrected body weight gain and food consumption in mid and high dose groups as compared to vehicle control group.

• Gross necropsy finding of thickening of stomach non-glandular mucosa multifocal was observed in 8/24 and 17/23 rats of mid and high dose groups respectively.

• Maternal data parameter - uterine weight was significantly reduced in high dose group. The other maternal data parameters were comparable to vehicle control group up to the high dose.

• Litter data parameters were comparable to vehicle control group at all the doses tested.

• Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs of teratogenicity in any of the tested doses.

Toxicity to reproduction: other studies

Description of key information

no further data mandatory

Justification for classification or non-classification

There is no evidence to suggest that a classification for reproductive toxicity is appropriate.

With reference to the OECD 422 sand OECD 414 study performed with Reaction mass of potassium ethyl octyl phosphonate and diethyl octylphosphonateand the lack of reproductive/developmental effects, it is concluded that Phosphoric acid, dodecyl ester, potassium salt is not subject to classification and labelling according to Directive 67/548/EEC and Regulation 1272/2008/EC regarding reproductive and developmetal toxicity.