Registration Dossier

Administrative data

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 changes 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 the test item, a dose of 50 mg/kg/day was considered to be the NOAEL (No Observed Adverse Effect Level) for pregnant females and 450 mg/kg bw/d for males.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-08-01 to 2013-04-??
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
Vehicle:
water
Details on oral 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each 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:
daily
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
Positive control:
none
Observations and examinations performed and frequency:
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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes (ketamine/xylazin, 3:1)
- Animals fasted: No
- How many animals: five randomly selected males and females of each group
- Parameters examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT),
white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), prothrombin time (PT),
activated partial thromboplastin time (aPTT)



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Animals fasted: No
- How many animals: five randomly selected males and females of each group
- Parameters examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP),
creatinine (Crea), total protein (TP), albumin (Alb), urea, total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na),
potassium (K), Calcium (Ca), Phosphorus (PHS)


URINALYSIS: Yes
- Time schedule for collection of urine: 5 randomly selected males at necropsy.
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters were examined: specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg),
bilirubin, blood, leukocytes


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in the week before the first treatment and during the last week of the treatment
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

OTHER:
Sperm analysis
At necropsy (one day after the last administration) one epididymis and one testis were separated and used for evaluation of sperm parameters.
Epididymal sperm motility and testicular sperm count were evaluated in all male animals using Hamilton Thorn Sperm Analyser
(TOX IVOS Version 13.0C).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
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.
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.

The wet weight of the organs (liver, kidneys, adrenals, testes, epididymides, prostate, seminal vesicles and coagulating glands,
ovaries, uterus with cervix, thymus, thyroid/parathyroid glands, spleen, brain, pituitary gland, 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 (all gross lesions, lung, brain, urinary bladder, stomach lymph nodes (mesenteric and axillary), small and large intestines
(including Peyer´s patches), trachea, liver, kidneys, thymus adrenal glands, spleen, heart) 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. 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.


HISTOPATHOLOGY: Yes

All organs and tissues listed 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.
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 animals 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).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality:
mortality observed, treatment-related
Description (incidence):
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
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
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
Details on results:
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.

Haematology and Coagulation
At the end of the treatment period WBC and Basophil values were slightly increased in both male and female treated groups,
without attaining the statistical significance. Statistically significant increase was noted PLT values in female MD and HD groups,
but without dose response. No dose response patterns were noted for basophil values (males and females) and WBC value (females).
Besides, all haematological parameters were within the normal range of variation and the changes were not related to treatment.
There were no test item related changes noted for blood coagulation parameters in males and females.

Clinical Biochemistry
At the end of the treatment period ALAT (male HD group) and CREA (LD, MD and HD groups) values were slightly higher,
but without attaining the statistical significance.
In females, no treatment related changes were recorded. However, there were decrease in mean values noted for ASAT, ALAT, ALP and
TBA values in MD or HD groups without attaining the statistical significance.
Besides, all parameters of clinical chemistry were within the normal range of variation and the changes were not related to treatment.

Urinalysis
The urinalysis performed in male animals revealed no test- item related effect in any of the treatment groups compared to the control group.

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 mall spleen and red discoloured axillary lymph node and thymus at necropsy, and it was non-pregnant. Based on the results of histopathological valuation, 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,
statistically 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
other: pregnant females
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
Critical 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 changes 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 the test item, a dose of 50 mg/kg/day was considered to be the NOAEL (No Observed Adverse Effect Level) for pregnant females and 450 mg/kg bw/d for males.
Executive summary:
The aim of this study was to assess the possible effects of Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate on male and female fertility and embryofetal development after repeated dose administration in Wistar 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 femaleWistarrats.

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 fromfive 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 of kidney, 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) 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 these animals were 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 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 the survival 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.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable without restriction

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Endpoint summary “Repeated dose toxicity”

 

Oral:

Relevant NOAEL (OECD 422, gavage, rat): 50 mg/kg bw/d (pregnant females); 150 mg/kg bw/d (males)

 

The aim of this OECD 422 study was to assess the possible effects of Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate on male and female fertility and embryofetal development after repeated dose administration in Wistar 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 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. 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 the survival 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 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 changes 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 the test item, a dose of 50 mg/kg/day was considered to be the NOAEL (No Observed Adverse Effect Level) for pregnant females and 450 mg/kg bw/d for males.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The OECD 422 study was selected as relevant available repeated dose toxicity study due to its reliability and since it provides a sensitive NOAEL.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
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 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 repeated dose toxicity inhalation - local effects endpoint:
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 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 repeated dose toxicity dermal - systemic effects endpoint:
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.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
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.

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: kidneys

Justification for classification or non-classification

Due to the NOAEL of 50 mg/kg bw/day in an OECD 422 repeated dose/reproduction/developmental toxicity screening test in rats with Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate, this substance has to be classified with H373 – May cause damage to organs through prolonged or repeated exposure regarding systemic and target organ toxicity after repeated exposure according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC) and does not have to be classified according to the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC).
Since the substance has to be classified with reference to the repeated dose toxicity further testing is considered to be unnecessary.