Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The NOAEL (No Observed Adverse Effect Level) of Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts in this study for general toxicity and reproductive toxicity screening could be established at 1000 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name: Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts
CAS No.: 1893414-79-3
Physical state: white solid at 20 °C
Batch No.: PU61810016
Re-certification date of batch: 09 March 2018
Purity: 100 % (UVCB, lyophilized solid, water content 0.85 % (w/w))
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species/strain: healthy 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: approx. 14-15 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 326 - 368 g (mean: 352.48 g, ± 20 % = 281.98 - 422.97 g)
females: 210 - 268 g (mean: 236.33 g, ± 20 % = 189.06 – 283.59 g)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +- 3 °C
- Relative humidity: 55 +- 10 %
- Artificial light (< 60 lux in cage), sequence being 12 hours light (06:00 till 18:00), 12 hours dark
- Air change: 10 x / hour (open housing), 50 x / hour (IVC cages), HEPA filtered
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals). Water was supplied through bottles (exchanged at least once a week) with sipper tubes.
- Animals were housed in groups of 2 animals / sex / cage in IVC cages (type III H, polysulphone) during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period
males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Makrolon tunnels were provided for all males and for females until GD 18
- Nesting material was provided latest on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

aqua ad iniectabilia (sterile water)

Details on exposure:

The test item formulations were prepared with aqua ad iniectabilia. The vehicle was selected based on the test item’s characteristics and testing guideline. The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item and further vortexing and/or stirring it. The formulation was sonicated for approximately 10 minutes. Based on the results of stability testing, the test item formulations were prepared at least once every 10 days. The prepared formulation was stored protected from light and at room temperature. Homogeneity of the test item in the vehicle was maintained by keeping formulates under magnetic stirring during the daily administration. The vehicle was also used as control item.

Details on mating procedure:

Mating: Mating was performed using a ratio of 1:1 (male to female) (if possible). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimised.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration analysis of formulation samples was determined at three concentrations, 20 mg/mL, 60 mg/mL and 200 mg/mL in study weeks 1, 3, 5 and in the last week of the study. The mean weekly recoveries observed for the LD dose group was between 98.9% and 110.8% of the nominal value, between 94.1% and 112.8% for the MD dose group and between 96.7% and 118.9% of the nominal value for HD dose group. The mean overall recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 103.6%, 101.5%, and 105.2% of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15%. Homogeneity of formulation samples was determined at three concentrations, 20 mg/mL, 60 mg/mL and 200 mg/mL, in study weeks 1, 3, 5 and the last week of the study. The coefficients of variation of the different sampling locations (top, middle, bottom) was between 0.4% and 2.7% in LD dose group, between 0.7% and 3.2% in MD dose group and between 1.1% and 6.3% in HD dose group. All samples were homogenous, as COV was below or equal 15%.

Duration of treatment / exposure:

The test item is orally administered daily, i.e. 7 days per week in graduated doses to several groups of test animals (male and female), one dose level per group with a maximum exposure of 63 days in total in females (at least 14 days pre-mating, up to 14 days mating, approximately 22 days of gestation and up to post-natal day 12). Males are dosed for a minimum of four weeks until up to one day before the scheduled sacrifice.

Frequency of treatment:

The test item is orally administered daily, i.e. 7 days per week.

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

LD

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

MD

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

HD

No. of animals per sex per dose:

100 animals (40 males and 60 females) were included in the study. 60 females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/ group) showing regular estrous cycles were continued in the study. Remaining not selected 20 females were discarded without any observations or used for other appropriate studies/procedures.

Control animals:

yes, concurrent vehicle

Positive control:

not applicable

Parental animals: Observations and examinations:

Body Weight and Food Consumption: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter 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), on PND 4 and PND 13 along with pups. All animals were weighed directly before termination. Food consumption was measured 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.

Mating: Mating was performed using a ratio of 1:1 (male to female) (if possible). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement
were minimised.

Clinical Observations: General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily. Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Functional Observations: 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 during the last week of lactation of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests. Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Haematology: Haematological parameters were examined in 5 randomly selected males and females (only lactating females were evaluated) from each group at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. The following haematological parameters were examined:

haematocrit value (Hct) %
haemoglobin content (Hb) g/dL
red blood cell count (RBC) 10^12/L
mean corpuscular volume (MCV) fL
mean corpuscular haemoglobin (MCH) pg/erythrocyte
mean corpuscular haemoglobin concentration (MCHC) g/dL
reticulocytes (Re) %
platelet count (PLT) 10^9/L
white blood cells (WBC) 10^9/L
neutrophils (Neu) %
lymphocytes (Lym) %
monocytes (Mono) %
eosinophils (Eos) %
basophils (Baso) %
large unstained cells (Luc) %

Blood Coagulation: Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in citrate tubes.
prothrombin time (PT) sec
activated partial thromboplastin time (aPTT) sec

Clinical Biochemistry: Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examine:

alanine aminotransferase (ALAT) U/L
aspartate-aminotransferase (ASAT) U/L
alkaline phosphatase (AP) U/L
creatinine (Crea) µmol/L
total protein (TP) g/L
albumin (Alb) g/L
urea mmol/L
total bile acids (TBA) µmol/L
total cholesterol (Chol) mmol/L
glucose (Gluc) mmol/L
sodium (Na) mmol/L
potassium (K) mmol/L

From up to 2 female pups/litter on day 4 after birth from all dams and 2 pups/litter at termination on day 13 and from all adult males at termination, blood samples were collected from the defined site in serum separator tubes. All blood samples were stored
under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Pup blood was pooled by litter for thyroid hormone analysis. Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments. The two pups per litter were female pups to reserve male pups for nipple retention evaluations. No pups were eliminated if litter size was below 8 pups. At a litter size of 8, only one pup was sacrificed.

Urinalysis: An urinalysis was performed with samples from 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded. The following parameters were measured using qualitative indicators.
specific gravity
nitrite
ph-value (pH)
protein
glucose
ketone bodies (ketones)
urobilinogen (ubg)
bilirubin
blood
leukocytes

Oestrous cyclicity (parental animals):

Estrous Cycles: Estrous cycles were monitored before treatment starts to select for the study females with regular cyclicity (using vaginal smears). Further on, vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

A full histopathology was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Litter observations:

Litter Observations: The duration of gestation was recorded and is calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring were recorded. The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.

Postmortem examinations (parental animals):

Pathology: All males will be sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females will be sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine). All surviving pups were killed by cervical dislocation on PND 13. Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle. Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice. Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating. All animals were subjected to a detailed gross necropsy which included 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), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Organ Weights: The wet weight of the organs of of 5 randomly selected male and female animals (only lactating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals. Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.
Organs to be Weighed at Necropsy:

testes (paired weight)
uterus with cervix
epididymides (paired weight)
ovaries (paired weight)
prostate, seminal vesicles and coagulating glands (complete weight)
thymus
thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) - will be weighed after fixation (complete weight)
liver
kidneys (paired weight)
spleen
adrenal (paired weight)
brain
pituitary gland
heart

The following tissues from the five randomly selected male and female animals were preserved in 4 % neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they will be transferred to 70 % ethanol.

Tissue/Organ (Preserved at Necropsy / Histopathological Examination)

adrenal glands x / x
all gross lesions x / x
aorta x / --
brain (incl. medulla/pons, cerebellar and cerebral cortex) x / x
caecum x / x
colon x / x
duodenum x / x
epididymides x / x
eyes x / x
femur with knee joint x / --
Harderian glands x / --
heart x / x
ileum (including Peyer´s patches) x / x
jejunum x / x
kidneys x / x
liver x / x
lungs x / x
lymph nodes (axillary) x / --
lymph nodes (mandibular) x / x
lymph nodes (mesenteric) x / x
mammary gland area (male and female) x / --
oesophagus x / --
optic nerves x / --
ovaries x / x
oviducts x / --
pancreas x / --
pituitary x / --
prostate and seminal vesicles with coagulating glands as a whole x / x
rectum x / x
salivary glands (sublingual, submandibular) x / --
sciatic nerve x / x
skeletal muscle x / x
skin x / --
spinal cord (cervical, thoracic and lumbar segments) x / x
spleen x / x
sternum (with bone marrow) x / x
stomach x / x
testes x / x
thymus x / x
thyroid/parathyroid glands x / x
tongue x / --
trachea x / x
ureters x / --
urinary bladder x / x
uterus with cervix and vagina x / x

Histopathology: A full histopathology was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals. Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Postmortem examinations (offspring):

Pathology: All males will be sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females will be sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine). All surviving pups were killed by cervical dislocation on PND 13. Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle. Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice. Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating. All animals were subjected to a detailed gross necropsy which included 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), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Organ Weights: The wet weight of the organs of of 5 randomly selected male and female animals (only lactating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals. Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.
Organs to be Weighed at Necropsy:

testes (paired weight)
uterus with cervix
epididymides (paired weight)
ovaries (paired weight)
prostate, seminal vesicles and coagulating glands (complete weight)
thymus
thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) - will be weighed after fixation (complete weight)
liver
kidneys (paired weight)
spleen
adrenal (paired weight)
brain
pituitary gland
heart

The following tissues from the five randomly selected male and female animals were preserved in 4 % neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they will be transferred to 70 % ethanol.

Tissue/Organ (Preserved at Necropsy / Histopathological Examination)

adrenal glands x / x
all gross lesions x / x
aorta x / --
brain (incl. medulla/pons, cerebellar and cerebral cortex) x / x
caecum x / x
colon x / x
duodenum x / x
epididymides x / x
eyes x / x
femur with knee joint x / --
Harderian glands x / --
heart x / x
ileum (including Peyer´s patches) x / x
jejunum x / x
kidneys x / x
liver x / x
lungs x / x
lymph nodes (axillary) x / --
lymph nodes (mandibular) x / x
lymph nodes (mesenteric) x / x
mammary gland area (male and female) x / --
oesophagus x / --
optic nerves x / --
ovaries x / x
oviducts x / --
pancreas x / --
pituitary x / --
prostate and seminal vesicles with coagulating glands as a whole x / x
rectum x / x
salivary glands (sublingual, submandibular) x / --
sciatic nerve x / x
skeletal muscle x / x
skin x / --
spinal cord (cervical, thoracic and lumbar segments) x / x
spleen x / x
sternum (with bone marrow) x / x
stomach x / x
testes x / x
thymus x / x
thyroid/parathyroid glands x / x
tongue x / --
trachea x / x
ureters x / --
urinary bladder x / x
uterus with cervix and vagina x / x

Histopathology: A full histopathology was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals. Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry will be statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Reproductive indices:

Reproductive Indices - Copulation Index (%), Fertility Index (%), Delivery Index (%),Viability Index (%) (PND 0-4), Viability Index (%) (PND 4-13) was assessed for test item-related effects.

Offspring viability indices:

Pup Survival Data - Mean Pup Survival Data was assessed for test item-related effects.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Low incidences of clinical signs like alopecia in few animals of treatment groups and scratches in one HD female were seen without dose dependency and are considered as incidental. Moving the bedding was observed transiently in few males (6/10) and females (5/10) of the HD group. Furthermore, salivation was noted transiently in 1/10 females of the HD group. The slight clinical signs of moving the bedding and salivation were mainly seen at the end of the treatment period in males and during gestation and lactation period in females. Both signs were observed in short timely relation to dose administration or in anticipation thereof and thus were considered to be a sign of discomfort or a local reaction to the test item. These slight signs were not considered as adverse systemic effects. One LD female transiently showed clinical signs of abnormal breathing and slight piloerection. They were most probably caused by regurgitation of the test item and are a subsequent sign of discomfort. Thus, these signs were not considered as adverse systemic effects.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the treatment period with Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts in any of the dose groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

During the entire study period, there was no statistically significant effect on body weight of male and female animals when comparing the dose groups with the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

During the entire study period, there was no considerable and statistically significant difference in food consumption of male and female animals between the dose groups and the control group.

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

Description (incidence and severity):

No toxicologically relevant effects on clinical biochemistry parameters were found at the end of the treatment period of this study. Single deviating values in individual animals compared to controls were considered as incidental. Statistically significant higher mean value for glucose were observed in the male MD and male HD group as well as in the female MD group, when compared to corresponding controls. As the differences were small and values were within the normal range of variation, they are not considered as toxicological relevant but incidental. A high serum level of AP (503 U/L) associated with an increase in TBA (46.31 µmol/L) in female no. 52 of the LD group indicates possible cholestasis. However, this did not correlate with histopathological findings or elevated urinary bilirubin and is not considered toxicologically relevant. Occasionally, further dosed female animals (i.e. no. 56, 64, 70, 71) in all dose groups showed isolated slightly elevated serum AP values. No high ALP values were found in male animals.

Urinalysis findings:

no effects observed

Description (incidence and severity):

All parameters of urinalysis of the dose groups at the end of the treatment period were not considerably different compared to the corresponding control group and were within the normal range of variation.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

In males, no relevant effects were observed in any of the parameters of the functional observation battery at the end of the treatment period. The parameter rearing (supported) showed a dose-dependently decreased count with HD females revealing statistically significant lower rearing in the last week of treatment when compared to control females. However, this effect is considered to be within the normal range of variation and without biological relevance. In the week before treatment females of the MD and HD group were observed with a significantly higher count of rearings (not supported) compared to the control group. The difference is based on the inter-individual variation and biologically not relevant. There were no biologically or statistically significant differences in body temperature in both males and females when comparing the dose groups with the control animals.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Males and females: No test-item related changes were noted.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In stomach, minimal to slight squamous cell hyperplasia located at the limiting ridge was observed in some animals form the high dose and control group. This finding was considered to be most likely incidental, although a test item involvement cannot be fully excluded. In kidneys, hyaline droplets were observed in the cytoplasm of tubular epithelial cells in male rats from the high dose and control group. The hyaline droplets represent an accumulation of secondary lysosomes within the cytoplasm and contain alpha-2u-globulin. This is a male rat specific event, and it is of no toxicological relevance in humans. In the adrenal gland the cortical vacuolization was noticed in some cells of the Zona fasciculata. This change occurred most likely secondary to stress and it was considered incidental and not test item related. The test item did not produced histological evidence of toxicity in reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus, cervix, and vagina. The sperm staging did not revealed any treatment-related effects on the testicular
histomorphology including spermatogenesis and interstitial cell structure. The treatment with the test item did not induced histomorphological effects in the reproductive organs of the non-pregnant low dose group female (no. 58) and its pairing partner (no. 18) and the non-pregnant high dose group female 73 and their pairing partner 33.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Males and females: No test-item related changes were noted.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

One female of the HD group followed normal cycle. However, in the smear following the proestrous stage a metestrus stage (instead of an estrous stage) was determined. Although not common, it is assumed that this animal had passed a seemingly short estrous stage between these 2 time points. This is supported by the observation that a regular estrous stage was present in the previous and the following cycle of this animal. Thus, it was not regarded as an abnormal cycle. Beside one HD female displaying a seemingly abnormal cycle the other observed parameters including the length and sequence of cycle stages were comparable between the test item-treated groups and the control group.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The sperm staging did not revealed any treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test item related effects on the reproductive indices (copulation, viability and delivery indices) in the dose groups when compared to the control group. Fertility in the HD group was slightly reduced due to a single non-pregannt female which is assumed to be an incidental finding.

Precoital Interval and Duration of Gestation: Precoital interval was slightly longer in the HD group when compared to the control group (3 days compared to 1.7 days in controls). However, the duration of gestation was unaffected.

Litter Data: There were no test item treatment related or statistically significant effects observed on litter data parameters like number of male pups, number of female pups, sex ratio, number of live pups, still birth, runt on PND 0 as well as number of male pups, number of female pups, number of live pups and sex ratio on PND 4 and PND 13 when compared with the controls.

Pre- and Post-Natal Data: There were no test item treatment related effects observed on the number of corpora lutea, implantation sites, percent preimplantation loss and post implantation loss in treatment groups when compared with the control group.

Reproductive Indices: There were no test item related effects on the reproductive indices (copulation, viability and delivery indices) in the dose groups when compared to the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item related external findings in the pups of this study. Single clinical findings were a dark snout, small appearance, no indication of suckling or a dark tag noted in a single pups in this study. These are considered as incidental findings. Absent hair coat on the back, neck or head was seen in occasional litters of all dose groups at death.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No test item-related effect on mortality of pups occurred from PND 0 to PND 4 and from PND 4 to PND 13 in the dose groups when compared to the control group. A marginally higher mean mortality of pups was observed in the MD group (1.48 %)
between PND 0 and PND 4 when compared to the control group (0.00 %). The mortality was attributed to one missing (possible cannibalism) pup of dam no. 64 (pup no. 14 on PND 1) and one pup of dam no. 68 (pup no. 6 on PND 1). This is considered incidental. No mortality occurred in the LD and the HD groups. Between PND 4 and PND 13 no mortality was observed in any of the dose groups and the control group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup weight was slightly and dose-dependently – but not statistically significantly lower in the dose groups compared to controls (4, 6 and 8 % in LD, MD and HD, respectively, higher than in controls. On day 13 mean pup weight in the HD group was approx. 10 % lower than in controls. This was reflected in male litter weight (27 % below controls on day 13) – but not in female litter weight, which was slightly higher in the HD group compared to the respective controls. No effect was seen, when comparing total litter weights.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The test item had no effect on serum T4 levels of males of the parental generation or on 13 day old pups. No considerable differences were found between dose groups and control group of this study.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No adverse effects of test item were found on male and female clinical observations, functional observations, body weight development, food consumption, estrous cyclicity, litter data, litter weight data, precoital interval and duration of gestation, pre and post-natal data, reproductive indices, pup survival data, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis in parental males and pups sacrificed on PND 13, pup external findings, haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treatment groups.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Pup Survival Data: No test item-related effect on mortality of pups occurred from PND 0 to PND 4 and from PND 4 to PND 13 in the dose groups when compared to the control group. Between PND 4 and PND 13 no mortality was observed in any of the dose groups and the control group.

Anogenital Distance and Nipple Retention: The mean absolute anogenital distance of male pups was marginally but statistically significantly lower in the HD group (2.42 mm) when compared to the control group (2.59 mm, p<0.05). As no statistical significance was achieved for the relative (to cube root of pup weight) anogenital distance of male pups, this is not assumed to be toxicologically relevant. No toxicologically relevant effect was observed for nipple retention in the male pups of
any dose group. The mean absolute anogenital distance of female pups was slightly but statistically significantly higher in the LD (0.77 mm, p<0.05) and the MD (0.80 mm p<0.01) group when compared to the control group (0.63 mm). Also the relative (to cube root of pup weight) anogenital distance of females was shown to be slightly but significantly higher in the LD (0.41 p<0.05), the MD (0.44, p<0.001), and also the HD (0.40, p<0.05) group when compared to the control group (0.34).
The alterations in the anogenital distance of dosed females are not considered to be test item-related as they do not follow a dose-dependency and are within the normal range of variation.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

Key result

Reproductive effects observed:

no

Conclusions:

The NOAEL (No Observed Adverse Effect Level) of Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts in this study for general toxicity and reproductive toxicity screening could be established at 1000 mg/kg bw/day.

Executive summary:

On the basis of this combined repeated dose oral toxicity and reproduction/developmental toxicity screening test with Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made: No mortality occurred in this study. No adverse effects of test item were found on male

and female clinical observations, functional observations, body weight development, food consumption, estrous cyclicity, litter data, litter weight data, precoital interval and duration of gestation, pre and post-natal data, reproductive indices, pup survival data, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis in parental males and pups sacrificed on PND 13, pup external findings,

haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treatment groups.

The NOAEL (No Observed Adverse Effect Level) of Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts in this study for general toxicity and reproductive toxicity screening could be established at 1000 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available