Poly[oxy(methyl-1,2-ethanediyl)], .alpha.-[2-[bis(phosphonomethyl)amino]methylethyl]-.omega.-[2-[bis(phosphonomethyl)amino]methylethoxy]-, sodium salt (1:?)

Endpoint summary

Description of key information

Combined 28 -day Repeated Dose Toxicity and Reproduction/Developmental Screening (OECD 422):

No evidence of local or systemic toxicity in males or females was observed up to the highest dose level of 1000 mg/kg bw/day. The NOEL (No Observed Effect Level) for both males and females was considered to be 1000 mg/kg bw day, the highest dose level used.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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:

First test item administration: 1 September 2011. Termination (last necropsies): 15 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

as at 22nd March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test) as at July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC 440/2008 (laying down test methods pursuant to Regulation EC 1907/2006) as at 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
- Number of Animals: 40 males (10 per group), 40 females (10 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): Male (298 to 332 g), Females (179 to 210 g)
- Identification: Cage card and individual animal number (ear tattoo). Pups: On day 1 post partum, pups were individually tattooed with Indian ink.
- Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK and ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/lot nos. 02105110601, 02105110801, 6960C.CS-100099 and 72. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 28/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

DOSE FORMULATIONS
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

Poly[oxy(methyl-1,2-ethanediyl)], alpha-[2-[bis(phosphonomethyl)amino]methylethyl]-omega-[2-[bis(phosphonomethyl)amino]methylethoxy]-sodium salt was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Each formulation was divided in daily aliquots.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers.

Based upon the results of stability analyses performed dose formulations were stable for at least one week.

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.
- Frequency of Administration: Once daily
- Target Dose Levels*: Group 1: 0 mg/kg/day (control group), Group 2: 100 mg/kg/day, Group 3: 500 mg/kg/day, Group 4: 1000 mg/kg/day
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, using dose levels of 0, 100, 300 and 1000 mg/kg/day.
*target dose levels are given in terms of active ingredient.
- Dose Volume: 10 mL/kg body weight (daily adjusted to the actual body weight of individual animal)
- Duration of Acclimatization Period: 6 days
- Duration of Treatment Period: Males (29 days), females (approximately 7 weeks)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE FORMULATIONS
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (7 days) samples of about 2 g of each concentration were taken from the middle only of each aliquot used on the first treatment day. During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Harlan Laboratories Ltd., Itingen / Switzerland, and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC Chromatography coupled to an electric light scattering detector (ELSD) following an analytical procedure provided by the sponsor and adapted by Harlan Laboratories. The test item was used as the analytical standard.

In blank sample chromatograms no peak appeared at the retention time of the test item and, therefore, the absence of the test item in the vehicle control samples (highly purified water) was confirmed.

The application formulations investigated during the study were found to comprise the test item in the range of 93.2% to 118.6% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of the test item in the preparations was approved because single results found did not deviate more than 2.3% (<15%) from the corresponding mean.
In addition, the test item was found to be stable in application formulations when kept for seven days under storage conditions (room temperature) due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item and purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

- Males: 29 days
- Females: approximately 7 weeks

Frequency of treatment:

- Once daily

Remarks:

Doses / Concentrations:
100, 500 or 1000 mg/kg bw/day
Basis:
other: actual ingested (dose levels given in terms of active ingredient).

No. of animals per sex per dose:

10 per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

MALES
- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment End: On day before sacrifice
- Necropsy: After treatment for 29 days, when no longer needed for assessment of reproductive effects

FEMALES
- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment End: On day 4 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)

Positive control:

not required

Observations and examinations performed and frequency:

VIABILITY/MORTALITY
- Twice daily

CLINICAL SIGNS AND OBSERVATIONS
Daily cage-side clinical observation was performed once daily during acclimatization period. Thereafter, up to the necropsy, daily clinical observation was performed immediately before dosing, up to 30 minutes post-dosing and one hour after dosing. An additional observation was made five hours after dosing during the normal working week. Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION
- Males (weekly during pre-pairing and after pairing periods)
- Females (pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum). No food consumption was recorded during the pairing period.

WATER CONSUMPTION
- Monitored daily by visual inspection of water bottles. No changes indicated treatment related effects were noted and therefore no measurement was initiated.

BODY WEIGHTS
- Recorded daily from treatment start to day of necropsy.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical observations were performed outside the home cage in all P generation animals. In all males of P generation it was performed once prior to the first administration of the test item and weekly thereafter. In females of P generation, it was prepared once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.

Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

FUNCTIONAL OBSERVATION BATTERY
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
- Cage-side observations: Faeces-balls, urine and posture as well as resistance to removal.
- Hand-held observations: Muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
- Open field observations: Level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
- Reflexes: Blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
- Measurements / Counts: Hind limb / fore limb grip strength, landing foot splay, rectal temperature.

Any abnormal findings were recorded and, where appropriate, graded in severity.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS
Blood samples were obtained on the day before or on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

HEMATOLOGY
The following hematology parameters were determined.
Complete Blood Cell Count:
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Leukocyte count, total
- Differential leukocyte count:
- Platelet count
Coagulation:
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined.
- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Bile acids
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

Sacrifice and pathology:

TERMINATION AND NECROPSY
Males were sacrificed after treatment for 29 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 5 post partum.

If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

All parent animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

At the scheduled sacrifice, all parent animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes.

For the parent animals, special attention was directed at the organs of the reproductive system.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS
At the scheduled sacrifice following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken of all parental males:
- Testes (as pairs)
- Epididymides (as pairs)
- Seminal vesicles (as pairs)
- Prostate
Of all parental females:
- Ovaries (as pairs)
- Uterus and Cervix
Of all parental males and females:
- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Pituitary
- Thymus
- Spleen
- Thyroid

TISSUE PRESERVATION
Tissues from the following reproductive organs from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution (or in Bouin’s fixative, if indicated):
- Prostate
- Seminal vesicles with coagulating gland
- Testes (in Bouin’s fixative)
- Epididymides (in Bouin’s fixative)
Tissues from the following reproductive organs from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries
- Uterus
- Vagina
- Cervix
In addition, from all males and females at scheduled necropsy and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Gross lesions
- Brain (including cerebrum, cerebellum and pons)
- Spinal cord (cervical, mid-thoracic and lumbar)
- Small and large intestines (incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids, and parathyroids if possible
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Aorta (thoratic)
- Bone and bone marrow (femur including stifle joint)
- Bone and bone marrow (sternum)
- Muscle (skeletal)
- Oesophagus
- Pancreas
- Pituitary
- Salivary glands (submaxillary / mandibular)
- Skin (abdominal region)
- Eyes ( fixed in Davidson's fluid)
- Mammary tissue

HISTOTECHNIQUE
All organ and tissue samples to be examined by the principal investigator for histopathology phase were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis were stained by PAS-hematoxylin.

HISTOPATHOLOGY
Slides of all organs and tissues collected at terminal sacrifice from the first five males and first five females which gave birth and slides of all reproductive organs from all males and females of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions, to all animals, which died spontaneously or had to be terminated in extremis and to all unfertile animals.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made.

A histopathology peer review was performed a principal investigator.

Other examinations:

MATING, GESTATION AND LACTATION
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

LITTER OBSERVATIONS
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible) and (with identification) on days 1 and 4 post partum. In addition a surface righting assessment was performed on day 1 post partum.

POSTMORTEM EXAMINATION (OFFSPRING)
Pups were sacrificed on day 4 post partum.

All pups sacrificed were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

At the scheduled sacrifice, all pups were sacrificed by an injection of sodium pentobarbital.

Dead pups, except those excessively cannibalized, were examined macroscopically.

All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

REPRODUCTIVE INDICES
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, pre- and post-implantation losses, mean litter size.

OFFSPRING VIABILITY INDICES
From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex rations and postnatal losses (up to day 4 post partum).

Statistics:

The following statistical methods were used to analyze functional observational battery, locomotor activity, food consumption, body weights, reproduction data, clinical laboratory investigations, organ weight and ratios and incidence and severity of histopathological findings:
- Means and standard deviations of various data were calculated and included in the report.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

Dose levels refered to belwo are given in terms of active ingredient.

IN-LIVE DATA - PARENTAL ANIMALS

VIABILITY / MORTALITY
All animals survived the scheduled study period.

CLINICAL OBSERVATIONS (DAILY)
No test item-related clinical signs were found in males or females at any dose level.

Incidentally, scabs were noted in one male in the control group (no. 5), one male at the dose level of 1000 mg/kg bw/day (no. 39) and one female at the dose level of 1000 mg/kg bw/day (no. 75), exophthalmos was noted in one female at the dose level of 1000 mg/kg bw/day (no. 77) and hair loss was noted in one female in the control group (no. 47).

No further observations were noted in males or females at any dose level.

DETAILED CLINICAL OBSERVATIONS (WEEKLY)
No test item-related effects were noted during detailed weekly clinical observations in males or females at any dose level.

The two findings recorded during the examination: a crust in one female in the control group (no. 47) and exophthalmos noted in one female at the dose level of 1000 mg/kg bw/day (no. 77), were considered to be incidental.

FUNCTIONAL OBSERVATIONAL BATTERY
No test item-related effects were noted during functional observational battery in males or females at any dose level.

At the dose level of 500 mg/kg bw/day in females, statistically significantly lower body temperature was noted. Mean body temperature at mid dose level was 38.5 °C compared to 39.0 °C in the control group. The difference was only minor and no dose dependent change of body temperature was noted across all dose groups. For these reasons the difference in body temperature at mid dose level was considered not to be test item-related.

Incidentally, decreased rearings were noted in two males in the control group (nos. 2 and 3) and one male at the dose level of 100 mg/kg bw/day (no. 14), increased rearings were noted in two males at the dose level of 1000 mg/kg bw/day (nos. 32 and 35) and one female in the control group (no. 41), exophthalmos and increased pupil size were noted in one female at the dose level of 1000 mg/kg bw/day (no. 77) and increased incidence of urine puddles was noted in one female at the dose level of 1000 mg/kg bw/day (no. 76).

No further findings were noted during functional observational battery in males or females at any dose level.

LOCOMOTOR ACTIVITY
No effects were noted during measurement of locomotor activity in males or females at any dose level.

In general, mean total beam crossing counts within 30 minutes of measurement were 1386, 1300, 1370 and 1419 in males and 911, 902, 1011 and 996 in females, given in the order of ascending dose levels.

FOOD CONSUMPTION OF MALES
Pre-pairing and After Pairing Periods:
No effects on food consumption of males were observed at any dose level.

At the dose levels of 100, 500 and 1000 mg/kg bw/day, differences in mean food consumption were respectively: +4%, ±0% and +4% over the pre-pairing period and +8%, +4% and +8% over the after pairing period (percentages refer to the respective values in the control group).

FOOD CONSUMPTION OF FEMALES
Pre-pairing, Gestation and Lactation Periods:
No effects on food consumption of females were observed at any dose level.

At the dose levels of 100, 500 and 1000 mg/kg bw/day, differences in mean food consumption were respectively: ±0%, ±0% and ±0% over the pre-pairing period, ±0%, ±0% and +4% over the gestation period and -7%, -7% and -3% over the lactation period (percentages refer to the respective values in the control group).

BODY WEIGHTS OF MALES
Pre-pairing, Pairing and After Pairing Periods:
No test item-related effects on body weights or body weight gain of males were observed at any dose level.

Overall differences in mean body weight gain at the dose levels of 0, 100, 500 and 1000 mg/kg bw/day were respectively: +13.3%, +13.6%, +12.0% and +14.4% during the pre-pairing period, +6.8%, +7.1%, +6.8% and +6.1% during the pairing period and +1.2%, +1.5%, +1.5% and +1.3% during the after pairing period (percentages refer to the body weight gain within the period).

At the dose level of 1000 mg/kg bw/day, statistically significantly higher body weights on two days and statistically significantly higher body weight gain on one day were noted. Statistically significantly higher were: mean body weights recorded on days 3 and 4 of the pairing period; 367 g and 370 g at the high dose level and 352 g and 354 g in the control group, respectively, and mean body weight gain recorded on day 12 of the pre-pairing period; 13.0% at the high dose level and 11.0% in the control group. Body weights and body weight gain of males at the high dose level were slightly higher than the respective control values during the entire study period. However, the differences were only minor and they did not develop in correlation to the length of the treatment. For these reasons they were considered not to be related to the treatment.

Further statistically significant differences were observed in body weight gain. Value at the dose level of 500 mg/kg bw/day recorded on day 7 of the pre-pairing period was statistically significantly lower then the control value; 5.8% at the mid dose level and 7.0% in the control group were noted. Value at the dose level of 100 mg/kg bw/day recorded on day 2 of the after pairing period was statistically significantly higher than the control value; 1.6% at the low dose level and 1.0% in the control group were noted. The differences were only minor, occurred on one day only and did not follow a dose dependency. For these reasons they were considered not to be related to the treatment.

BODY WEIGHTS OF FEMALES
Pre-pairing, Pairing, Gestation and Lactation Periods:
No effects on body weights or body weight gain of females were observed at any dose level.

Overall differences in mean body weight gain at the dose levels of 0, 100, 500 and 1000 mg/kg bw/day were respectively: +9.4%, +9.4%, +8.3% and +8.5% during the pre-pairing period, +63.6%, +56.7%, +59.0% and +63.0% during the gestation period and +5.6%, +5.0%, +3.0% and +1.7% during the lactation period (percentages refer to the body weight gain within the period).


CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY
Males:
No effects on hematology parameters, which were considered to be test item-related, were noted at any dose level.

Incidentally, statistically significantly higher value of red cell volume distribution width (RDW) was noted at the dose level of 1000 mg/kg bw/day; mean value at the high dose level was 0.182 compared to 0.143 in the control group. The value at the high dose level was within the 95% of a tolerance limit for the historical controls (a range from 0.115 to 0.234) and therefore the difference was considered not to be test item-related.

No significant differences were noted among remaining values.

Females:
No effects on hematology parameters, which were considered to be test item-related, were noted at any dose level.

Incidentally, statistically significantly lower value eosinophils (EOS) was noted at the dose level of 1000 mg/kg bw/day; mean value at the high dose level was 0.02 g/l compared to 0.10 g/l in the control group. The value at the high dose level was within the 95% of a tolerance limit for the historical controls (a range from 0.02 g/l to 0.13 g/l) and therefore the difference was considered not to be test item-related.

BIOCHEMISTRY
Males:
No effects on clinical biochemistry parameters were noted at any dose level.
Females:
No effects on clinical biochemistry parameters were noted at any dose level.

MATING PERFORMANCE AND FERTILITY
No test item-related effect on mating performance or fertility was observed at any dose level.

All females were mated within twelve days after initiation of pairing. Mean (median) precoital times were 2.4 (3), 3.3 (3), 2.2 (2) and 2.9 (2) days at the dose level of 0, 100, 500 and 1000 mg/kg bw/day, respectively.

Four females were not pregnant; two at the low dose level (female nos. 53 and 55 mated with male nos. 13 and 15, respectively), one at the mid dose level (female no. 64 mated with male no. 24) and one at the high dose level (female no. 71 mated with male no. 31).
Consequently, fertility indexes (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 100%, 80%, 90% and 90% at the dose levels of 0, 100, 500 and 1000 mg/kg bw/day, respectively.

The incidence of infertility did not follow a dose dependency and the number of non-pregnant females per group was in the range of the historical background, up to four non-pregnant females were noted in historical control groups. For these reasons lack of pregnancy was considered not to be related to the treatment with the test item.

All pregnant females gave birth to living pups. Consequently, the gestation index (number of females with living pups as a percentage of females pregnant) was 100% in all groups.

CORPORA LUTEA COUNT
No effects on corpora lutea count were observed at any dose level.

Mean number of corpora lutea per dam was 15.5, 17.1, 17.1 and 17.2 in order of ascending dose levels.

PRE-IMPLANTATION LOSS
No test item-related effects on pre-implantation loss were observed at any dose level.

Mean pre-implantation loss per dam was 2.0, 4.0, 6.0 and 4.4, in order of ascending dose level.

At the dose level of 500 mg/kg bw/day, mean pre-implantation loss was statistically significantly higher than in the control group. No statistically significant difference in pre-implantation loss was noted at the high dose level and therefore the difference at the mid dose level was considered not to be test item-related.

DURATION OF GESTATION
No effects on duration of gestation were observed at any dose level.

Mean duration of gestation was 21.5, 21.6, 21.6 and 21.3 days, in order of ascending dose level.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS
No effects on implantation rate or post implantation loss were observed at any dose level.

Mean number of implantations per dam was 13.5, 13.1, 12.2 and 12.8 whereas mean number of post-implantation loss per dam was 1.2, 1.4, 0.9 and 1.7; both cited in order of ascending dose levels.

LITTER SIZE AT FIRST LITTER CHECK
No effects on litter size were observed at any dose level.

Mean numbers of living pups per dam at first litter check were 12.3, 11.8, 11.3 and 11.1, whereas birth indexes (number of pups borne alive as a percentage of implantations) were 91.1%, 89.5%, 92.7% and 87.0% at the dose levels of 0, 100, 500 and 1000 mg/kg bw/day, respectively.

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
No effects on postnatal loss were observed at any dose level.

In the control group, one pup (from litter no. 49) was found dead at first litter check and further one pup from the same litter was missing on day 2 post partum.
At the dose level of 100 mg/kg bw/day, one pup (from litter no. 57) was missing on day 3 post partum.
At the dose level of 500 mg/kg bw/day, one pup (from litter no. 65) was missing on day 2 post partum.
At the dose level of 1000 mg/kg bw/day, two pups (both from litter no. 74) were found dead at first litter check.
No further mortality of pups was observed at any dose level. Because the incidence of pups mortality did not correlate with the dose levels, the postnatal loss was considered not to be test item-related.


TERMINAL FINDINGS - PARENT ANIMALS

ORGAN WEIGHTS
Males:
No test item-related effects on absolute organ weights or organ weights relative to body or brain weights were noted at any dose level.

At the dose level of 1000 mg/kg bw/day, statistically significantly lower absolute brain weight and brain weight to body weight ratio were noted. Mean brain weights were 1.94 g compared to 2.02 in the control group whereas mean brain weight relative to body weight were 0.53 at the high dose level and 0.57 in the control group. Historical controls contained values of brain weights from 1.93 g to 2.10 g and values of brain weights to body weights ratios from 0.50 to 0.65. Both values at the high dose level were in the range of the historical controls and therefore these differences were considered not to be related to the treatment.
Males receiving 1000 mg/kg bw/day showed statistically significantly higher values of heart weight to brain weight and liver weight to brain weight ratios but in the absence of statistically significant differences in the absolute weights of these organs. For this reason these differences were considered to be a result of the lower brain weight and not to be a primary effect on the organ weights.
Further statistically significant difference at the high dose level was lower spleen weight to body weight ratio; mean value at the high dose level was 0.18 compared to 0.22 in the control group. No significant differences in absolute spleen weight or spleen weight to brain weight ratio were noted. Mean spleen weight to body weight ratio at the high dose level was in the range of the historical controls (containing values from 0.18 to 0.24). For these reasons this difference was considered not to be related to the treatment.

No further significant differences in absolute or relative organ weights were noted in males at any dose level.

Females:
No test item-related effects on absolute organ weights or organ weights relative to body or brain weights were noted at any dose level.

At the dose level of 500 mg/kg bw/day, statistically significantly lower ovarian weights were noted but in the absence of statistically significant differences in ovarian weights to body weights or to brain weights ratios. Mean ovarian weights at the mid dose level was 0.115 g compared to 0.142 g in the control group. No significant differences in absolute or relative ovarian weights were noted at the high dose level and therefore the difference at the mid dose level was considered not to be test item-related.
No further significant differences in absolute or relative organ weights were noted in females at any dose level.

MACROSCOPICAL FINDINGS
Males:
No test item-related findings were noted during necropsy of males at any dose level.
Following findings were considered to be within the range of normal background alterations:
- foci on thymus found in one male at the low dose level (no. 14),
- urinary bladder distended with urine found in one male at the low dose level (no. 18),
- foci on lung found in one male at the high dose level (no. 33).
No further findings were noted in males at any dose level.

Females:
No test item-related findings were noted during necropsy of females at any dose level.
Following findings were considered to be within the range of normal background alterations:
- foci on lung found in one female in the control group (no. 44),
- foci in stomach found in one female at the mid dose level (no. 67) and one female at the high dose level (no. 76),
- cyst in stomach and black-brown ileum contents found in one female at the mid dose level (no. 61),
- thickened and dark red discolored liver lobe found in one female in the control group (no. 44),
- watery cyst on ovary found in one female in the control group (no. 47),
- dark red discoloration of ovaries found in one female in the control group (no. 49),
- enlarged lymph nodes found in one female at the high dose level (no. 72),
- dark red discoloration of mandibular lymph node found in one female in the control group (no. 43),
- In addition, alopecia in one female in the control group (no. 47) and enlarged left eye in one female at the high dose level (no. 77) which were noted during the in live phase, were also recorded during the necropsy.
No further findings were noted in females at any dose level.

HISTOPATHOLOGY FINDINGS
No histological evidence of toxicological properties of the test item Poly[oxy(methyl-1,2-ethanediyl)], alpha-[2-[bis(phosphonomethyl)amino]methylethyl]-omega-[2-[bis(phosphono methyl) amino]methyl ethoxy]-sodium salt in the reproductive organs and tissues examined was detected.

Special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure did not reveal any differences between control (group 1) and high dose (group 4) males in the completeness of cell populations, completeness of stages, and potential degenerative changes.

In organs other than reproductive, the test item did also not induce any test item-related findings.

Key result

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Remarks:

No signs of general toxicity in males or females was observed up to 1000 mg/kg bw day, the highest dose level used.

Key result

Critical effects observed:

no

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

Group (mg/kg/day)	1 (0)	2 (100)	3 (500)	4 (1000)
Female numbers	41-50	51-60	61-70	71-80
Number of females paired	10	10	10	10
Number of females mated	10	10	10	10
Numbers of females, which which did not deliver any pups (A)	0	2	1	1
Number of females which reared their pups until day 4 post partum	10	8	9	9

(A)Female nos. 53, 55, 64 and 71.

 

LITTER DATA - F1 PUPS

 

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

No test item-related findings were noted in pups at any dose level.

 

Incidentally, no milk in the stomach in one pup in the control group (from litter no. 49) and 2 pups at the high dose level (both from litter no. 74), no milk in the stomach together with pup cold to touch in all pups from one litter (no. 51) at the low dose level as well as swelling together with a wound on hind limb in one pup at the high dose level (from litter 72) were noted at first litter check. The wound was noted still on days 2 and 3 post partum whereas the swelling was noted up to the termination of the pup

 

No further findings were noted at first litter check or during the first 4 days post partum in pups at any dose level.

 

SEX RATIOS

Pups sex ratio was not affected by exposure to the test item at any dose level.

 

At first litter check, percentages of male pups were 57%, 57%, 54% and 52%, in order of ascending dose level.

 

RIGHTING REFLEX

No effects on righting reflex of pups were observed at any dose level.

 

Percentages of pups for which righting reflex was observed within the first one minute of the test were 89%, 97%, 90% and 93% at the dose levels of 0, 100, 500 and 1000 mg/kg bw/day, respectively.

 

BODY WEIGHTS TO DAY 4 POST PARTUM

No effects on pups body weights or body weight gain were noted at any dose level.

 

Overall mean body weights of pups on day 1 post partum were: 6.0 g, 6.2 g, 6.4 g and 6.5 g, at the dose levels of 0, 100, 500 and 1000 mg/kg/day respectively. Overall, body weight gain of pups during the first four days of lactation period was +48.2%, +48.6%, +50.8% and +51.6%, respectively.

 

MACROSCOPICAL FINDINGS

No findings were noted during the necropsy of pups at any dose level.

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats over approximately 29 days. The test substance was administered in highly purified water as vehicle at dosages of 100, 500, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test substance was administered to male rats for 29 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

No evidence of general toxicity in males or females was found up to the highest dose level of 1000 mg/kg bw/day in terms of all of the endpoints examined within this study.

All animals survived the scheduled study period. During the live phase of the study no test item-related effects were noted during daily or detailed weekly clinical observations or functional observational battery tests. Food consumption, body weights and body weight gain were not affected in males or in females at any dose level.

Also terminal examinations did not indicate any toxicologically relevant effect. No test itemrelated effects on hematology or clinical biochemistry parameters, no effects on organ weights or findings during necropsy and histopathological examination of males or females were noted at any dose level.

Based on the results of this study, the NOEL (No Observed Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity was considered to be 1000 mg/kg bw day, the highest dose level used.

Executive summary:

The potential effects of the test item on repeated dose, reproductive and developmental toxicity were assessed in accordance with OECD 422 (combined 28-day repeated dose toxicity study with reproductive and developmental screening). The test item was administered to male rats for 29 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following dose levels were applied:

Group 1:                          0 mg/kg body weight/day (control group)

Group 2:                      100 mg/kg body weight/day

Group 3:                      500 mg/kg body weight/day

Group 4:                    1000 mg/kg body weight/day

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

 

The following results were obtained:

 

PARENT ANIMALS

Mortality and General Tolerability

 

All animals survived the scheduled study period.

 

No test item-related findings were noted during daily clinical or detailed weekly clinical observations in males or females at any dose level.

 

Functional Observational Battery

 

No test item-related effects were noted during functional observational battery in males or females at any dose level.

 

Food Consumption

 

No effects on food consumption of males or females were observed at any dose level.

 

Body Weights

 

No effects on body weights or body weight gain of males or females were observed at any dose level.

 

Clinical Laboratory Investigations

 

No test item-related effects on hematology or clinical biochemistry parameters were noted in males or females at any dose level.

 

Reproduction and Breeding Data

 

No test item-related effect on mating performance, fertility, duration of gestation, corpora lutea count, pre-implantation loss, implantation rate, post implantation and postnatal loss or litter size were noted at any dose level.

 

Organ Weights

 

No test item-related effects on absolute organ weights or organ weights relative to body or brain weights were noted in males or females at any dose level.

 

Macroscopical Findings and Histopathological Examinations

 

No test item-related findings were noted during necropsy of males or females at any dose level.

 

No histological evidence of toxicological properties of the test item in the reproductive and other than reproductive organs and tissues examined were detected.

 

 

LITTER DATA - F1 PUPS

Findings at First Litter Check and during Lactation

 

No test item-related findings were noted at first litter check or during the first 4 days post partum in pups at any dose level.

 

No effects on righting reflex were observed in pups at any dose level.

 

Pups sex ratio was not affected by the exposure to the test item at any dose level.

 

Pup Weights to Day 4 Post Partum

 

No effects on pups body weights or body weight gain were noted at any dose level.

 

Macroscopical Findings

 

No findings were noted during the necropsy of pups at any dose level.

Conclusion

Based on the results of this study, the NOEL (No Observed Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity was considered to be 1000 mg/kg bw day, the highest dose level used.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with Annex XI, Section 1.2, a sub-chronic 90-day repeated dose toxicity study is not considered necessary for this substance.

In all available toxicology studies, the substance shows no indication of systemic toxicity, nor does the substance present any structural alerts for systemic toxicity. The available information suggests that absorption of the test substance from the gastrointestinal tract can take place. Some absorption may also take place via the skin. Once absorbed, the substance would be distributed in the serum and urine is the significant route of excretion. There is limited evidence suggesting that the test substance may be metabolised.

There was no indication of local or systemic toxicity in acute toxicity studies conducted via the oral and dermal routes of exposure, with both studies concluding an LD50 = > 2000 mg/kg bw. The substance is not classified for skin and eye irritation/corrosion, although some mild irritation was observed in the studies that were conducted.

The substance was non-mutagenic in bacteria (Ames test), non-clastogenic in mammalian cells in vitro and non-mutagenic in mammalian (CHO) cells in vitro in both the presence and absence of metabolic activation. The substance is not a skin sensitiser, suggesting that the substance does not bind to circulatory proteins.

In a short-term 28-day repeated dose toxicity study, combined with reproduction and developmental screening, no signs of local or systemic toxicity were observed for the repeated dose toxicity or reproductive and developmental endpoints. A NOEL of 1000 mg/kg bw/d was concluded for all endpoints associated with this study, i.e. the regulatory limit concentration.

In conclusion, the substance displays no suggestion of any toxic properties that may result in hazardous properties being identified for human health hazard assessment. The registrant proposes that a 90-day sub-chronic toxicity study should not be conducted on animal welfare grounds.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

The conclusion is based on a reliable (Klimisch 1) OECD 422 study assessing the 28-day repeated dose toxicity of the substance. This is considered suitable fot hazard assessment.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to REACH Annex VIII, 8.6.1 (short-term repeat dose toxicity). Column 2, testing by the inhalation route is appropriate if exposure of humans via inhalation is likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size. The substance has a low measured vapour pressure of 7.9 x 10-6 Pa at 25°C and is not considered to be volatile (predicted negative for explosive and oxidising properties). It is considered that the substance has low potential to become airborne from the aqueous preparations in which the substance is used. The potential for exposure to humans to aerosols, particles or droplets of an inhalable size is considered unlikely based on the vapour pressure and use of the substance. Therefore, inhalation is not considered a significant route of exposure as the substance is not available as a vapour. A study has therefore not been conducted via this route. Repeat dose toxicity testing has been instead conducted via the oral route.

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:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to REACH Annex VIII, 8.6.1 (short-term repeat dose toxicity). Column 2, testing by the inhalation route is appropriate if exposure of humans via inhalation is likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size. The substance has a low measured vapour pressure of 7.9 x 10-6 Pa at 25°C and is not considered to be volatile (predicted negative for explosive and oxidising properties). It is considered that the substance has low potential to become airborne from the aqueous preparations in which the substance is used. The potential for exposure to humans to aerosols, particles or droplets of an inhalable size is considered unlikely based on the vapour pressure and use of the substance. Therefore, inhalation is not considered a significant route of exposure as the substance is not available as a vapour. A study has therefore not been conducted via this route. Repeat dose toxicity testing has been instead conducted via the oral route.

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:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In this instance, the default oral route of exposure was considered most appropriate for the repeated dose toxicity endpoint.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In this instance, the default oral route of exposure was considered most appropriate for the repeated dose toxicity endpoint.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable, no evidence of local or systemic toxicity was observed in the available studies.

Additional information

28 -Day Repeated Dose Toxicity (OECD 422)

The potential effects of the test item on repeated dose, reproductive and developmental toxicity were assessed in accordance with OECD 422 (combined 28-day repeated dose toxicity study with reproductive and developmental screening). The test item was administered to male rats for 29 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following dose levels were applied:

Group 1:                          0 mg/kg body weight/day (control group)

Group 2:                      100 mg/kg body weight/day

Group 3:                      500 mg/kg body weight/day

Group 4:                    1000 mg/kg body weight/day

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

 

The following results were obtained:

 

PARENT ANIMALS

Mortality and General Tolerability

All animals survived the scheduled study period.

No test item-related findings were noted during daily clinical or detailed weekly clinical observations in males or females at any dose level.

 

Functional Observational Battery

No test item-related effects were noted during functional observational battery in males or females at any dose level.

 

Food Consumption

No effects on food consumption of males or females were observed at any dose level.

 

Body Weights

No effects on body weights or body weight gain of males or females were observed at any dose level.

 

Clinical Laboratory Investigations

No test item-related effects on hematology or clinical biochemistry parameters were noted in males or females at any dose level.

 

Reproduction and Breeding Data

No test item-related effect on mating performance, fertility, duration of gestation, corpora lutea count, pre-implantation loss, implantation rate, post implantation and postnatal loss or litter size were noted at any dose level.

 

Organ Weights

No test item-related effects on absolute organ weights or organ weights relative to body or brain weights were noted in males or females at any dose level.

 

Macroscopical Findings and Histopathological Examinations

No test item-related findings were noted during necropsy of males or females at any dose level.

No histological evidence of toxicological properties of the test item in the reproductive and other than reproductive organs and tissues examined were detected.

 

 

LITTER DATA - F1 PUPS

Findings at First Litter Check and during Lactation

No test item-related findings were noted at first litter check or during the first 4 days post partum in pups at any dose level.

No effects on righting reflex were observed in pups at any dose level.

Pups sex ratio was not affected by the exposure to the test item at any dose level.

 

Pup Weights to Day 4 Post Partum

No effects on pups body weights or body weight gain were noted at any dose level.

 

Macroscopical Findings

No findings were noted during the necropsy of pups at any dose level.

Conclusion

Based on the results of this study, the NOEL (No Observed Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity was considered to be 1000 mg/kg bw day, the highest dose level used.

Justification for classification or non-classification

The substance does not meet the criteria for classification in accordance with Regulation (EC) 1272/2008 (CLP).