Phosphoric acid, mono- and di-C12-18-(even numbered)-alkyl esters, sodium salts

Endpoint summary

Description of key information

Based on the results of the read across studies, a NOAEL of 1000 mg/kg bw/day from the longer duration combined repeated dose and reproductive and development toxicity study (OECD 422) conducted with mono- and di- C12 PSE, Na+ has been taken forward for hazard assessment. This is further supported by a similar or higher NOAELs observed in a longer duration (90-day) repeated dose study available with mono- and di- C12 PSE, K+ in rats as well as in a OECD 422 screening study with dodecanol.

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

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

due to RA

Justification for type of information:

Refer to the section 13 for details on the read across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

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:
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: 8 - 9 weeks old
Body weight at the allocation of the animals to the experimental groups: males: 214 - 246 g; females: 143 - 175 g
The animals were derived from a controlled full-barrier maintained breeding system (SPF).
According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding conditions:
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/-10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1455)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological
controls at regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fiber bedding (lot no. 060411).
- Certificates of food, water and bedding are filed at BSL BIOSERVICE.
- Adequate acclimatisation period (at least five days)

Route of administration:

oral: gavage

Details on route of administration:

- Preparation of the test substance formulation:
The test substance was weighed into a tarred plastic vial on a precision balance and the vehicle (aqua ad injectionem) was added to give the appropriate final concentration of the test substance. The formulation vials were placed on a Vortex machine for short period to ensure homogenistation of the formulation. The vehicle was selected as suggested by the sponsor and on the basis of the test substance’s characteristics. The test substance formulations were prepared freshly on each administration day before the administration procedure. The vehicle was used as control substance.
- Preparation of the animals:
Prior to the start of the treatment period a detailed clinical observation was made to confirm that all animals were in good health condition. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.
- Administration of doses:
The test substance formulation and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 10 mL/kg bw. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Vehicle:

water

Details on oral exposure:

- Preparation of the test substance formulation:
The test substance was weighed into a tarred plastic vial on a precision balance and the vehicle (aqua ad injectionem) was added to give the appropriate final concentration of the test substance. The formulation vials were placed on a Vortex machine for short period to ensure homogenistation of the formulation. The vehicle was selected as suggested by the sponsor and on the basis of the test substance’s characteristics. The test substance formulations were prepared freshly on each administration day before the administration procedure. The vehicle was used as control substance.
- Preparation of the animals:
Prior to the start of the treatment period a detailed clinical observation was made to confirm that all animals were in good health condition. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.
- Administration of doses:
The test substance formulation and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 10 mL/kg bw. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the dosing formulations was tested once at the beginning of the treatment period. From all dose groups, samples of control or dosing formulations were frozen at 0 hours and 6 hours after the preparation and stored between -15° and -35°C. Stability was tested in samples of dosing formulations of the LD and HD groups only (4 samples in total). In the first week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared HD, MD and LD formulations and stored between -15° and -35°C. Homogeneity was tested in samples of dosing formulations of the LD and HD groups only (6 samples in total). Analysis of the dosing preparations of the test substance in the vehicle (nominal concentration) was performed once at the first week of treatment
and at the end of the treatment period and stored between -15 and -35°C. Concentrations were tested in all samples (8 samples in total).

Duration of treatment / exposure:

7 days per week for a period of 14 days

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control group

Dose / conc.:

60 mg/kg bw/day (actual dose received)

Remarks:

Low dose group (LD)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group (MD)

Dose / conc.:

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

Remarks:

How dose group (HD)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: random
- Section schedule rationale : all male animals together and all female animals together

Positive control:

No

Observations and examinations performed and frequency:

- Body weight and Food consumption
The body weight was recorded once before assignment to the experimental groups and on study days 1, 8 and 14 during the treatment period as well as on the day of necropsy. Food consumption was measured on study days 1, 8 and 14 for each animal.
- Clinical observations
Animals were observed for clinical signs during the entire treatment period of 14 days. General clinical observations were made at least once a day,
approximately at the same time each day and considering the peak period of anticipated effects after dosing.
The health condition of the animals was recorded. At least once daily all animals were observed for morbidity and mortality.
- Haematology
Haematological parameters were examined at the end of the treatment prior as part of the sacrifice of the animals. After overnight fasting, 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), red blood cell count (RBC), MCV, MCH, MCHC, reticulocytes (RE), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso).
- Clinical biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea),
total protein (TP), albumin (Alb), urea, total cholesterol (Chol), trigycerides (TG), sodium (Na), potassium (K).

Sacrifice and pathology:

- Pathology
On study day 15, all surviving animals of the study were sacrificed using anesthesia (Ketamin/Xylazine 3:1; Ketamine), and 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. All macroscopic findings were recorded. The following tissues from all animals were preserved
in 10% neutral buffered formalin except testes and epididymis that were fixed in Modified Davidson’s fixative for approx. 24 hours before they were transferred to 10% neutral buffered formalin.
Preserved and examined tissues: all gross lesions, uterus with cervix, oesophagus, ovaries, stomach, vagina, small and large intestines (including Peyer´s patches), testes, lungs (inflated with fixative), epididymides
- Organ Weight
The wet weight of the following organs was taken from all sacrificed animals as soon as possible. Paired organs were weighed separately. Organs weighed at Necropsy: liver, ovaries, kidneys, uterus with cervix (females), adrenals, thymus, testes, spleen, epididymides, heart, prostate with seminal vesicles and coagulating glands brain.

Other examinations:

- Blood sampling for possible determination of serum levels of inflammatory markers
Additionally, blood of each surviving animals was sampled at terminal sacrifice for a possible determination of serum levels of inflammatory markers. Blood from the abdominal aorta was collected in serum separator tubes. Thereafter, the samples were allowed to clot at room temperature and then were centrifuged for 10 min at 4°C at approx. 1000 g. After centrifugation, serum was transferred into
a tube labeled with “Serum”, study no., animal ID number and the time point of blood collection. All samples were stored at ≤ -70°C.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results. Due to the small size of groups a statistical evaluation of the results was not performed in this study.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Pilo erection, salivation:
During the first treatment week moderate (from study days 2 to 6) and then slight (study days 7 and 8) piloerection was observed in 1/3 female animals of the HD group. No clinical findings were noted in this animal during the second treatment week. Besides, only slight salivation was noted on a single day of the treatment period in 1/3 female animals of the MD group. Abnormal breathing in 1/3 male animals of the MD group and a wound, scratches and slight alopecia at the neck of 1/3 male animals of the LD group are not assumed to be test substance related but technically caused or incidental. One female in the control group bit off and swallowed a piece of gavaging cannula on study day 10 and subsequently (on study days 11 and 14) showed piloerection and body weight loss.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

After 14 days of treatment, no considerable differences were observed in the body weights when compared control animals. In all groups the mean body weight was in the normal range of variation for this strain. This slight effect in the mean values was mainly caused by one animal of the HD group that showed a body weight loss in the first treatment week. This effect is considered to be treatment related, but as the animals recovered in the second treatment week, it is unlikely to be a sign of systemic toxicity – but a local reaction. As a consequence, mean body weight gain of male– but not female – animals of the HD group was the slightly lower than in controls in the first treatment week and slightly higher than controls in the second treatment week.
A body weight loss of one female animal in the Control group in the second treatment week was associated with a reduced food intake. This was most likely related to a piece of gavaging cannula in stomach reaching into esophagus that was bitten off and swallowed on study day 10.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

A body weight decrease in one male animal in the first treatment week and in one female animal in the Control group in the second treatment week was associated with a reduced food intake. As a consequence, mean food consumption was slightly lower in the male animals of the HD group (first treatment week) than in the respective control group. Besides, there were no considerable differences between treated and control animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Increase in neutrophils, decrease in lymphocytes:
The treatment had no effect on the red blood cell system or on the platelets analyzed at the end of the treatment period. All parameters were in the normal range of variation for this strain. In the white blood cell system, however, slight alterations were observed. In the HD group and in female animals of the MD group, an increase in neutrophils (males: approx. 2-fold, females up to approx. 4-fold) with a concurrent decrease in lymphocytes (males: approx. 16% lower than controls, females: down to approx. 22% lower than controls) was observed in the differential white blood cell count at the end of the treatment period. Monocytes were slightly increased in the HD group, however, still in the normal range of variation. In one female animal of the Control group, also a slight increase in the rate of neutrophils and a decreased rate of lymphocytes was found. This was associated with a slight increase in blood monocytes. These findings in the differential white blood cell count are assumed to be related to a piece of gavaging cannula that had been swallowed and subsequently affect food consumption and body weight development.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Alkaline phospatase slightly increased:
At the end of the treatment period a slightly and dose dependently elevated serum level of AP was found in male and female animals of the HD group when compared to the control group (approx. 87% and approx. 86%, respectively). This was not associated with considerable differences in other serum markers for hepatotoxicity (ALT, ASAT, Chol). However, a relation to treatment cannot be excluded. Besides, all parameters of clinical chemistry were within the normal range of variation for this strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In male animals of the HD group prostate gland was slightly reduced in weight (25% below controls). A tendency was also observed in the MD group. The differences were less prominent when related to brain weight or body weight. A slight increase in absolute (15 % higher than controls) and relative epididymis weight (related to brain weight: 31 % above controls; related to body weight: 25 % above controls) was observed in the HD group. After 14 days, a slight increase in absolute and relative spleen weight was found in female animals of all dose groups (absolute weights between 20 and 36 % higher than controls). This effect is not assumed to be adverse and possibly not related to the test substance, as it was not dose-dependent and, at the same time, a slight decrease in absolute spleen weight of male animals was observed in the MD and HD groups.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on increase in neutrophils and alkaline phosphatase, slightly reduced prostate weight and increased epididymides weight

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: male reproductive system and alkaline phosphatase and neutrophils levels

Organ:

other: prostate and epididymides

Treatment related:

yes

Analytical data:

Stability of samples retained during and 6 hours after preparation was demonstrated with recovery rates of test substance between 84 to 92%. The recovery of test substance in samples retained for determination of homogeneity was between 79% and 89%. The recovery of test item in samples retained for determination of test substance concentration was between 79% and 96%.

Conclusions:

Based on the results of the read across study, the 14d NOAEL of the test substance was determined to be 600 mg/kg bw (rat, gavage).

Executive summary:

A study was conducted to determine the oral repreated dose toxicity of the read across substance according to OECD Guideline 407 and EU Method B.7, in compliance with GLP. Male and female Wistar rats were exposed to the test substance 7 days per week for a period of 14 days. They received by gavage 10 mL/kg bw test substance at concentrations of 0, 60, 600 and 1000 mg/kg bw (Control (water alone), Low dose, Mid dose and High Dose groups, respectively) During the treatment period, the animals were observed precisely each day for signs of toxicity. Body weight and food consumption were measured once weekly. At the conclusion of the treatment period, hematological and clinical biochemistry parameters were analyzed and all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved. The test substance formulations (test substance suspended in aqua ad injectionem) were prepared freshly on each day of administration. Dose volumes were adjusted individually based on weekly body weight measurement. No mortality occurred in the control or any of the dose groups during the treatment period of this study. Slight to moderate clinical signs (pilo erection and salivayion) were observed during the first treatment week in 1/3 female animals of the HD group. Besides, there were no considerable test substance related clinical signs in this study. In 1/3 male animals of the HD group, body weight and food consumption decreased in the first treatment week. As the animal recovered in the second treatment week it is not considered to be a sign of systemic toxicity but rather a local reaction. Besides, treatment with the test substance did not influence food consumption and body weight gain of male and female animals of this study. Test substance administration had a slight effect on haematological parameters analyzed at the end of the treatment period. In the HD group and in female animals of the MD group, a slight to moderate increase in neutrophils with a concurrent slight decrease in lymphocytes was observed in the differential blood cell count at the end of the treatment period. At the end of the treatment period a slightly and dose dependently elevated serum level of alkaline phosphatase was found in male and female animals of the MD and HD group when compared to the control group. No specific test substance related gross pathological changes were recorded for the male and female animals of this study. In male animals of the HD group prostate gland was slightly reduced and epididymides weight was slightly increased in weight, compared to controls. Based on the results of the read across study, the 14d NOAEL of the test substance was determined to be 600 mg/kg bw/day (2012).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined repeated dose toxicity and reproductive and developmental toxicity study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

due to RA

Justification for type of information:

Refer to the section 13 for details on the read across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Seventy each of 8-week-old male and female Sprague-Dawley SPF rats (Crj;CD (SD) IGS, Charles River Laboratories Japan, Inc., Atsugi Breeding Center) were purchased (number of animals received: 73 each of males and females)
- The animals were quarantined and acclimatized for 14 days. During this period, the general condition, body weight, and estrous cycle (for 9 days after the quarantine period) were examined, and 58 each of males and females without any abnormality of the general condition (males) (or without any abnormality of the general condition or estrous cycle and with good weight increase for females) were selected and administered the test substance at the age of 10 weeks.
- The body weight at the start of the test substance administration ranged from 338 to 395 g for the males and 219 to 256 g for the females.
- Animals were housed individually in a bracket type metal wire cage in an animal breeding room maintained at a temperature of 21°C to 26°C, relative humidity of 37% to 77%, ventilation frequency of 10 to 15 air changes per hour, and illuminated for 12 hours per day (07:00 to 19:00).
- The animals were allowed free access to Solid chow NMF (nonsterilized: Oriental Yeast Co., Ltd., batch numbers 040713, 040806, 040913) from a stainless steel feeder and to tap water (city water of Gotenba, water bottle was used).

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

The test substance was administered by oral gavage, a method commonly employed for oral administration to rodents. Each animal received 5 mL/kg body weight of the test suspension by forced oral administration via a gastric tube (08:20-14:24 h). Animals of the control group received the vehicle (olive oil) in a similar manner. The volume of the test suspension was calculated based on the latest body weight of each animal.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC

Details on analytical verification of doses or concentrations:

- An appropriate amount of the test substance for each concentration was weighed and suspended in olive oil in an agate mortar to prescribed concentrations (50, 100, and 200 mg/mL). The test suspensions were prepared at a frequency of at least once every 7 days and stored in a cold dark place (refrigerator, observed temperature: 3°C-5°C) in light-protected containers (brown glass bottles) until use.
- Stability of the test suspensions was confirmed at Bozo Research Center and showed that 50 and 200 mg/mL suspensions of the test substance (vehicle: olive oil) remained stable for 24 hours at room temperature after storage in light-protected containers in a cold dark place (refrigerator) for 7 days.
- Confirmation of the concentrations and uniformity of the test suspensions of each concentration used for administration at week 1 and on the last week of administration were analyzed by HPLC at the Bozo Research Center. The results showed that for all the suspensions tested, the percentage of the test substance relative to the nominal value was in the range of 96.5% to 105.0%, with a C.V. in the range of 1.0% to 5.3%, which were within the acceptable range (concentration, nominal value ± 10%; C.V., ≤ 10%).
- Analytical method:
The test sample (dosing suspension), 1 mL, was diluted with 60 vol% of THF solution to 10 mL and centrifuged (2000 rpm, 1000 × g, 20°C, 5 minutes); then, 1 mL of the lower layer was diluted to 5 mL with the mobile phase of HPLC. The diluted solution was filtered through a Milex HV filter and the filtrate was subjected to measurement by the HPLC system. Single test samples were taken from the upper, middle and lower layers of the dosing suspension.

Duration of treatment / exposure:

The duration of administration was 14 days before mating, 14 days during the mating period, and 14 days after the mating period, that is, 42 days in total, for the males of the main group and the males and females of the recovery group, and 14 days before mating and up to day 4 of lactation throughout the mating and gestation periods, that is, 42 to 45 days in total, for the females of the main group. The recovery period for the males and females of the recovery group was 14 days after the end of administration, during which period the test substance was not given to the animals.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

A total of 4 dose groups were set up: 250, 500, and 1000 mg/kg bw groups and the control group.
Each main group consisted of 12 male and female animals each, and each recovery group consisted of 5 each of males and female animals in the control and high- dose groups.

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for selection of doses. In a previous study “14-day repeated-dose oral toxicity study of the test substance in rats (a preliminary study)” (doses: 125, 250, 500, and 1000 mg/kg bw, Bozo Research Center study No.: C-R016), administration of the test substance did not produce any effect even at 1000 mg/kg bw, the level defined as the limiting dose by the OECD Guideline for Testing of Chemicals 422. Therefore, 1000 mg/kg was set as the highest dose, and doses of 500 and 250 mg/kg bw were derived by dividing by a common factor of 2. A total of 3 doses were thus set up.

Details on mating procedure
- After the end of the pre-mating administration period, each pair of male and female animals in the same dose group of the main group was housed in the same cage overnight, and the females were judged to have copulated if the vaginal smear contained sperm or the presence of the vaginal plug was confirmed. Days to copulation was calculated from the day of mating, taken as day 0. From gestation day 17 to day 5 of lactation, the animals were housed individually in a plastic Econ cage with
bedding.
- Observation of mother animals. All female animals confirmed to have copulated were allowed to undergo spontaneous delivery, and observed for the presence/absence of abnormalities in the delivery. Delivery completion was checked twice daily (morning, afternoon) from gestation day 21 to the morning of gestation day 25, from which the gestation period was calculated in units of 0.5 days. If delivery was complete by 5:00 h in the afternoon, that day was regarded as day 0 of lactation.
- Mother animals that completed the delivery were observed for the presence/absence of pup licking and ingestion of the placenta and amnion. They were allowed to suckle pups up to day 4 of lactation (the date of delivery completion was regarded as day 0 of lactation) and observed for lactating behavior, using pup gathering, nest building, and breastfeeding as indices.

Positive control:

No

Observations and examinations performed and frequency:

- Observation of the general condition. All animals were observed for the presence of any abnormality of the general condition, such as in the external appearance, nutritional condition, posture, behavior, and excrements, 3 times everyday (before, immediately after, and 2 hours after the administration) during the administration period and once every morning during the recovery period.
Detailed observation of the general condition, function tests, measurement of the grip strength and spontaneous motor activity. Detailed observation of the general condition was performed once before the start of administration for all animals, once every week during the administration period for the males of the main group, once every week during the pre-mating administration and mating periods, and on predetermined days (gestation days 1, 7, 14 and 20, day 4 of lactation) during the gestation period and the lactation period for the females of the main group, and once every week during the administration and recovery periods for the animals of the recovery group. Function tests, measurement of the grip strength, and measurement of the spontaneous motor activity were performed on the last week of administration (day 39 of administration) for the males of the main group, after F1 necropsy on day 4 of lactation (day 42-45 of administration) for the females of the main group, and on the last week of administration (day 39 of administration) and last week of the recovery period (day 11 of recovery) for the males and females of the recovery group. These tests were performed on 5 animals each per group. - Measurement of body weight. Body weight was measured on days 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39 and 42 of administration, and on the day of necropsy for the males of the main group, on days 1, 4, 8, 11 and 14 of the recovery period and on the day of necropsy, in addition to the days of measurement for the males of the main group, for the males and females of the recovery group, and on days 1, 4, 8, 11 and 15 of administration (and days 18, 22 and 25 of the administration period as well as in non-copulated animals), days 0, 4, 7, 11, 14, 17 and 20 of gesta tion, and days 0 and 4 of lactation for the females of the main group. Body weight was measured from 08:06 to 10:45 h, except for the measurement on day 0 of lactation for females whose end of delivery was confirmed during the observation in the afternoon. On the day of necropsy, the body weight was measured after the animals had been denied access to food for approximately 16 hours from the previous day, in order to calculate the relative organ weight.
- Measurement of food consumption. The amount of food remaining relative to that supplied on the previous day was measured on days 1, 4, 8, 11, 15, 32, 36, 39 and 42 of administration for the males of the main group, on days 1, 4, 8, 11 and 14, in addition to the days of measurements for the males of the main group, for the males and females of the recovery group, on days 1, 4, 8, 11 and 15 of administration, days 1, 4, 7, 11, 14, 17 and 20 of gestation, and days 2 and 4 of lactation for the females of the main group. Food consumption per animal was calculated from the data thus obtained. The amount of food supplied and the amount of food remaining were measured from 08:26 to 11:25 h.
- Urinalysis (including measurement of water intake). On the last week of administration (day 36 to 37 of administration) and on the last week of the recovery period (day 8 to 9 of recovery), each of the male animals was individually housed in a cage equipped with a urine collector. Four-hour urine specimens were collected under fasting conditions of the animals with free access to water, followed by 20-hour urine specimen collection under free access to food and water. The parameters tested are
as shown below. The first 4-week urine specimens were subjected to tests from pH up to sediments, as well as measurement of the urine volume, and the subsequent 20-hour urine specimens were subjected to measurement of the osmotic pressure and urine volume. Urine volume was calculated as the sum of the volumes of 4-hour and 20-hour urine specimens. The amount of water intake from the previous day was measured using a water bottle while the animals were housed in the cage
equipped with the urine collector.

Sacrifice and pathology:

After delivery, the mother animals were exsanguinated to death by dissecting the abdominal aorta on Day 5 of lactation, after the animals had been denied access to food overnight (approx. 16-20 h) from Day 4 of lactation: 5 in each group after blood collection for hematological tests and blood chemistry tests, and the remaining animals under ether anesthesia.

- Necropsy and organ weight measurement. All of the 5 male and female animals in each group from which blood samples were collected for the hematology and blood chemistry tests on the day after the last day of administration and on the last day of the recovery period were exsanguinated to death after the blood collection, and all the other animals were exsanguinated to death by dissecting the abdominal aorta under ether anesthesia. They were then subjected to detailed gross pathological e
xamination of the body organs and tissues, including the external surface, head, chest and abdomen, and the results were recorded. In the females (mother animals), the number of corpora lutea and number of implantation sites were counted on day 5 of lactation. Then, in 5 each of the male and female animals from which blood samples were collected for the hematology and blood chemistry tests, the weight (absolute) of the following organs (testes and epididymes of all the animals) was measured
and the relative weight of each organ per 100 g body weight was calculated from the absolute organ weight and the body weight at necropsy. For bilateral organs marked with an asterisk, the weight of each side was measured separately and the sum of the weights was calculated. Brain, thyroid gland* (including parathyroid gland), thymus gland, heart, liver, spleen, kidney*, adrenal*, testis*, and epididymis*.

- Histopathological examination. The following organs and tissues of all the animals were fixed and st ored in 10 vol% formalin solution in phosphate buffer (the testes and epididymes were fixed in Bouin's fluid, followed by storage in 10 vol% formalin solution in phosphate buffer). Then, organs and tissues (see below) were embedded in paraffin, and sections were stained with hematoxylin and eosin (H-E). Specimens obtained from 5 each of the male and female animals of the control and high-dose groups
from which blood specimens were collected for the hematology and blood chemistry tests were subjected to microscopic examination (for bilateral organs, both sides were isolated and one side was subjected to the microscopic examination). The results revealed the effect of the test substance on the stomach. Therefore, specimens from 5 each of the male and female animals of the low- and medium-dose groups were also subjected to microscopic examination. Representative cases of normal
and abnormal findings were photographed.

(Cerebrum, cerebellum, pituitary gland, spinal cord (thoracic), sciatic nerve, thyroid gland, parathyroid gland, adrenal, thymus gland, spleen, submandibular lymph nodes, mesenteric lymph nodes, heart, lung (including bronchus), stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, kidney, bladder, testis, epididymis, ovary, uterus, seminal vesicle, sternum (including bone marrow), femur (including bone marrow), and the animal identification site (auricle))

Statistics:

- Bartlett test
- Dunnett’s test
- χ2 test with Yates’ continuity correction
- Fisher’s exact test

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- In the main group, one female in the 500 mg/kg bw group showed opacity of an eyeball (unilateral) from gestation day 5, which was not related to the dose and was therefore considered to be an incidental change.
- In the recovery group, one male in the 1000 mg/kg bw group showed decreased spontaneous motor activity from day 37 of administration up to day 7 of the recovery period, and wheezing from day 37 of administration until the end of the recovery period.
- No abnormality was observed in the other animals, either in the main or in the recovery groups.

Mortality:

no mortality observed

Description (incidence):

- There were no significant difference in the body weight between males and females of the main group. A significantly greater increase in body weight was observed in the females of the 250 and 1000 mg/kg bw groups during the lactation period, but the increase was not dose-related.
- In the recovery group, males in the 1000 mg/kg bw group showed decreased body weight gain during the administration period and decreased body weight (-21g) during the recovery period. This was caused by the abnormality in 1 out of the 5 animals. This animal showed continued body weight decrease (and also decreased spontaneous activity and wheezing in the observation of the general condition; the body weight was 466g before manifestation of the symptom and 261g on day 14 of the recovery period). The body weights of the other 4 males and 5 females in the same group were similar to those of the animals in the control group, showing no statistically significant differences.

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Administration of the test substance did not have any effect on the food consumption in the males or females of either the main group or the recovery group. A significant increase was observed on day s 2 and 4 of lactation in the females of the 250 mg/kg dose group in the main group, but this was not dose-related.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

-Tests at the end of the administration period. A significant increase in the serum level of fibrinogen was observed in the females of the 250 mg/kg bw group and a significant decrease in the percentage of lymphocytes and significant increase in the percentage of segmented neutrophils were observed in the females of the 500 mg/kg bw group. However, none of these changes were observed in the 1000 mg/kg bw group, suggesting that they were within the range of physiological variations. No significant differences were observed in the male animals between the control group and any of the treatment groups.
- Tests at the end of recovery period. A significant increase in the mean corpuscular volume of the red blood cells, significant decrease of the platelet count, significant increase in the percentage of lymphocytes, and significant decrease in the percentage of segmented neutrophils were observed in females of the 1000 mg/kg bw group. However, these changes were not observed at the end of the administration period, which suggested that they were within the range of physiological variations. No significant differences were observed in the male animals between the control group and any of the treatment groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Test at the end of the administration period. A significant increase in the serum level of ALT was observed in the males of the 1000 mg/kg bw group. A significant decrease of the serum level of inorganic phosphorus was observed in the males of the 250 mg/kg bw group. However, since the decrease was not dose-related, it was considered to be within the range of physiological variations.
- Tests at the end of recovery period. A significant increase in the serum level of total protein was observed in the females of the 1000 mg/kg bw group. However, since no such change was observed at the end of the administration period, the increase was considered to be within the range of physiological variations.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis (including measurement of water intake) showed no abnormalities in the qualitative parameter values in any of animals in either the main group or in the recovery group. No significant difference was observed in any of the quantitative parameter values between the control group and any of the treatment groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- Observation of animals in the home cage. No abnormalities were observed in any of the animals in either the main group or the recovery group.
- Observation of the animals while being handled. No abnormality was observed in any of animals in either the main group or the recovery group.
- Observation of animals in the open field. Males of the 1000 mg/kg bw group in the main group showed a significant increase of the defecation frequency during weeks 1 and 2 of administration, which was a very mild transient change and considered to be within normal range. No abnormalities were observed in the other parameters in any of the animals in either the main group or the recovery group. No significant differences were observed in the standing frequency between the control group and any of the treatment groups.
- Function tests. No abnormalities were observed in any of the animals in either the main group or the recovery group. No significant differences were observed in the air righting reflex or landing foot splay between the control group and any of the treatment groups.
- Measurement of the grip strength. No significant differences were observed between the control group and any of the treatment groups in either the main group or the recovery group.
- Measurement of spontaneous motor activity (measured for 10-minute periods and a total of 60 minutes). No significant difference was observed between the control group and any of the treatment groups in either the main group or the recovery group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No dose-related changes in either direction (increase or decrease) were observed in either the absolute or the relative weight. Although significant differences in the weights of the following organs were observed, they were considered to be within the range of normal variations, because they were neither dose-related nor were observed at the end of the administration period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Findings at the end of the administration period. Stomach: Indentation of the anterior stomach was observed in 0, 5 and 7 males, and 1, 1 and 3 females of the 250, 500 and 1000 mg/kg bw groups, respectively. White foci were observed in 1 male of the 500 mg/kg bw group. Rough mucosa in the anterior stomach was observed in 5 and 9 males, and 5 and 6 females of the 500 and 1000 mg/kg bw groups, respectively. Indentation of the glandular stomach was observed in 1 female of the 500 mg/kg bw group. All the other findings observed in the organs and tissues were considered to be incidental, as judged from the frequency of their occurrence and the pathological findings (Dark red foci in the glandular stomach were observed in 0, 1, 2 and 1 males and 4, 2, 1 and 1 females of the control
group, 250, 500 and 1000 mg/kg bw groups, respectively. Kidney: Pyelectasis was observed in 2 and 1 males of the 250 and 1000 mg/kg bw groups, respectively. Eyeballs: Corneal opacity (unilateral) was observed in 1 female of the 500 mg/kg bw group).
- Findings at the end of the recovery period. Stomach: Rough mucosa in the anterior stomach was observed in 1 male of the 1000 mg/kg bw group. This animal also showed expansion of the digestive tract from the stomach to the colon due to gas accumulation, and a mild decrease in the size of the testis. Other findings observed in the following organs were considered to be incidental as judged from the frequency of their occurrence and the pathological findings (Lung: Dark red foci were observed in 1 female of the 1000 mg/kg bw group).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Administration of the test substance had effects on the stomach of the animals in the 250 mg/kg bw and higher dose groups.
- Findings at the end of the administration period.
Stomach: Mild to moderate erosions or ulcers of the anterior stomach were observed in 0, 4 and 4 males and 1, 1 and 1 females of the 250, 500, and 1000 mg/kg bw groups, respectively. Very mild to moderate thickening of the anterior stomach mucosa was observed in 1, 4 and 5 males and 1, 4 and 3 females of the 250, 500, and 1000 mg/kg bw groups, respectively. Very mild to mild edema of the submucosal tissue in the anterior stomach was observed in 1, 5 and 5 males and 0, 4 and 3 females of the 250, 500, and 1000 mg/kg bw groups, respectively. Most of these changes in the anterior stomach were localized findings. All of the other findings observed, as follows, were considered to be incidental changes as judged from the frequency of their occurrence and the histopathological findings
Epididymis: Very mild infiltration by stromal cells was observed in 1 male of the control group. Heart: Very mild localized myocarditis was observed in 4 males of the control group and 1 male of the 1000 mg/kg bw group.
Kidney: Very mild basophilic tubules were observed in 3 males of the control group and 1 male and 1 female in the 1000 mg/kg bw group.
Liver: Very mild, minute granulomas were observed in 3 males of the control group and 1 male of the 1000 mg/kg bw group.
Lung (including bronchi): Very mild mineral deposits in the arterial walls were observed in 1 male of the control group and 1 female of the 1000 mg/kg bw group. Very mild accumulation of foam cells was observed in 2 males and 1 female of the control group, and 1 male and 3 females of the 1000 mg/kg bw group.
Spleen: Very mild to mild extramedullary hematopoiesis was observed in 5 females each in the control group and 1000 mg/kg bwgroup.
Stomach: Inclusion cysts were observed in 1 male of the 500 mg/kg bw group. Very mild to mild erosions in the glandular stomach were observed in 0, 0, 0 and 1 male and 3, 1, 1, and 0 females in the control group, 250, 500, and 1000 mg/kg bw group, respectively).

- Findings at the end of the recovery period. Stomach: Moderate thickening of the anterior stomach mucosa was observed in 1 male of the 1000 mg/kg bw group.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Refer to the other related RSS for details on reproductive and development toxicity.

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic effects observed up to 1000 mg/kg bw/d; the changes in forestomach are of local nature due to the irritant properties of the substance

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects on forestomach

Key result

Critical effects observed:

no

Conclusions:

Based on the results of the read across study, the NOAEL for systemic effects is considered to be 1000 mg/kg bw/day.

Executive summary:

A study was conducted to determine the toxicity to reproduction of the read across substance, phosphoric acid, dodecyl ester, sodium salt (mono- C12 PSE, Na+), according to OECD Guideline 422, in compliance with GLP. The test substance was administered at 0 (control group), 250, 500 or 1000 mg/kg bw to male Sprague-Dawley SPF rats for 14 days before mating, through the mating period, and up to 1 day before necropsy (42 days in total) and to female Sprague-Dawley SPF rats for 14 days before mating, through the mating period and the gestation period, up to day 4 of lactation (42 to 45 days in total) to investigate the repeated-dose, reproductive and developmental toxicities. In the 0 and 1000 mg/kg bw groups, a 14-day recovery period was allowed after the 42-day administration period to investigate the reversibility of the toxic changes. Administration of the test substance had no effect on any of the following: general condition, findings on detailed observation of the general condition, results of function tests, grip strength, spontaneous motor activity, body weight, food consumption, results of urinalysis (including water intake), or in the results of hematological or blood chemistry tests. Administration of the test substance had no effect on any of the following: general condition, findings on detailed observation of the general condition, results of function tests, grip strength, spontaneous motor activity, body weight, food consumption, results of urinalysis (including water intake), or in the results of hematological or blood chemistry tests. Changes in the forestomach may be considered to be adverse, however, they are also considered to be a result of local irritation of the (irritant) test item (which is brought directly in contact to the mucosa in a massive amount by gavage application) than a true effect of systemic toxicity. Furthermore, in the light of the absence of a forestomach in humans, observed effects on this tissue are of questionable relevance with reference to the extrapolation of the toxic properties of a test substance in humans. Therefore, based on the results of the read across study, the NOAEL for systemic effects is considered to be 1000 mg/kg bw/day (METI, 2005).

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Appearance: semi-solid (amorphous): gel

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animals:
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: 8 - 9 weeks old
Body weight at the allocation of the animals to the experimental groups: males: 214 - 246 g; females: 143 - 175 g
The animals were derived from a controlled full-barrier maintained breeding system (SPF).
According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding conditions:
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/-10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1455)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological
controls at regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fiber bedding (lot no. 060411).
- Certificates of food, water and bedding are filed at BSL BIOSERVICE.
- Adequate acclimatisation period (at least five days)

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- Preparation of the test substance formulation:
The test substance was weighed into a tarred plastic vial on a precision balance and the vehicle (aqua ad injectionem) was added to give the appropriate final concentration of the test substance. The formulation vials were placed on a Vortex machine for short period to ensure homogenistation of the formulation. The vehicle was selected as suggested by the sponsor and on the basis of the test substance’s characteristics. The test substance formulations were prepared freshly on each administration day before the administration procedure. The vehicle was used as control substance.
- Preparation of the animals:
Prior to the start of the treatment period a detailed clinical observation was made to confirm that all animals were in good health condition. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.
- Administration of doses:
The test substance formulation and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 10 mL/kg bw. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the dosing formulations was tested once at the beginning of the treatment period. From all dose groups, samples of control or dosing formulations were frozen at 0 hours and 6 hours after the preparation and stored between -15° and -35°C. Stability was tested in samples of dosing formulations of the LD and HD groups only (4 samples in total). In the first week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared HD, MD and LD formulations and stored between -15° and -35°C. Homogeneity was tested in samples of dosing formulations of the LD and HD groups only (6 samples in total). Analysis of the dosing preparations of the test substance in the vehicle (nominal concentration) was performed once at the first week of treatment
and at the end of the treatment period and stored between -15 and -35°C. Concentrations were tested in all samples (8 samples in total).

Duration of treatment / exposure:

7 days per week for a period of 14 days

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control group

Dose / conc.:

60 mg/kg bw/day (actual dose received)

Remarks:

Low dose group (LD)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group (MD)

Dose / conc.:

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

Remarks:

How dose group (HD)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: random
- Section schedule rationale : all male animals together and all female animals together

Positive control:

No

Observations and examinations performed and frequency:

- Body weight and Food consumption
The body weight was recorded once before assignment to the experimental groups and on study days 1, 8 and 14 during the treatment period as well as on the day of necropsy. Food consumption was measured on study days 1, 8 and 14 for each animal.
- Clinical observations
Animals were observed for clinical signs during the entire treatment period of 14 days. General clinical observations were made at least once a day,
approximately at the same time each day and considering the peak period of anticipated effects after dosing.
The health condition of the animals was recorded. At least once daily all animals were observed for morbidity and mortality.
- Haematology
Haematological parameters were examined at the end of the treatment prior as part of the sacrifice of the animals. After overnight fasting, 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), red blood cell count (RBC), MCV, MCH, MCHC, reticulocytes (RE), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso).
- Clinical biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea),
total protein (TP), albumin (Alb), urea, total cholesterol (Chol), trigycerides (TG), sodium (Na), potassium (K).

Sacrifice and pathology:

- Pathology
On study day 15, all surviving animals of the study were sacrificed using anesthesia (Ketamin/Xylazine 3:1; Ketamine), and 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. All macroscopic findings were recorded. The following tissues from all animals were preserved
in 10% neutral buffered formalin except testes and epididymis that were fixed in Modified Davidson’s fixative for approx. 24 hours before they were transferred to 10% neutral buffered formalin.
Preserved and examined tissues: all gross lesions, uterus with cervix, oesophagus, ovaries, stomach, vagina, small and large intestines (including Peyer´s patches), testes, lungs (inflated with fixative), epididymides
- Organ Weight
The wet weight of the following organs was taken from all sacrificed animals as soon as possible. Paired organs were weighed separately. Organs weighed at Necropsy: liver, ovaries, kidneys, uterus with cervix (females), adrenals, thymus, testes, spleen, epididymides, heart, prostate with seminal vesicles and coagulating glands brain.

Other examinations:

- Blood sampling for possible determination of serum levels of inflammatory markers
Additionally, blood of each surviving animals was sampled at terminal sacrifice for a possible determination of serum levels of inflammatory markers. Blood from the abdominal aorta was collected in serum separator tubes. Thereafter, the samples were allowed to clot at room temperature and then were centrifuged for 10 min at 4°C at approx. 1000 g. After centrifugation, serum was transferred into
a tube labeled with “Serum”, study no., animal ID number and the time point of blood collection. All samples were stored at ≤ -70°C.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results. Due to the small size of groups a statistical evaluation of the results was not performed in this study.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Pilo erection, salivation:
During the first treatment week moderate (from study days 2 to 6) and then slight (study days 7 and 8) piloerection was observed in 1/3 female animals of the HD group. No clinical findings were noted in this animal during the second treatment week. Besides, only slight salivation was noted on a single day of the treatment period in 1/3 female animals of the MD group. Abnormal breathing in 1/3 male animals of the MD group and a wound, scratches and slight alopecia at the neck of 1/3 male animals of the LD group are not assumed to be test substance related but technically caused or incidental. One female in the control group bit off and swallowed a piece of gavaging cannula on study day 10 and subsequently (on study days 11 and 14) showed piloerection and body weight loss.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

After 14 days of treatment, no considerable differences were observed in the body weights when compared control animals. In all groups the mean body weight was in the normal range of variation for this strain. This slight effect in the mean values was mainly caused by one animal of the HD group that showed a body weight loss in the first treatment week. This effect is considered to be treatment related, but as the animals recovered in the second treatment week, it is unlikely to be a sign of systemic toxicity – but a local reaction. As a consequence, mean body weight gain of male– but not female – animals of the HD group was the slightly lower than in controls in the first treatment week and slightly higher than controls in the second treatment week.
A body weight loss of one female animal in the Control group in the second treatment week was associated with a reduced food intake. This was most likely related to a piece of gavaging cannula in stomach reaching into esophagus that was bitten off and swallowed on study day 10.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

A body weight decrease in one male animal in the first treatment week and in one female animal in the Control group in the second treatment week was associated with a reduced food intake. As a consequence, mean food consumption was slightly lower in the male animals of the HD group (first treatment week) than in the respective control group. Besides, there were no considerable differences between treated and control animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Increase in neutrophils, decrease in lymphocytes:
The treatment had no effect on the red blood cell system or on the platelets analyzed at the end of the treatment period. All parameters were in the normal range of variation for this strain. In the white blood cell system, however, slight alterations were observed. In the HD group and in female animals of the MD group, an increase in neutrophils (males: approx. 2-fold, females up to approx. 4-fold) with a concurrent decrease in lymphocytes (males: approx. 16% lower than controls, females: down to approx. 22% lower than controls) was observed in the differential white blood cell count at the end of the treatment period. Monocytes were slightly increased in the HD group, however, still in the normal range of variation. In one female animal of the Control group, also a slight increase in the rate of neutrophils and a decreased rate of lymphocytes was found. This was associated with a slight increase in blood monocytes. These findings in the differential white blood cell count are assumed to be related to a piece of gavaging cannula that had been swallowed and subsequently affect food consumption and body weight development.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Alkaline phospatase slightly increased:
At the end of the treatment period a slightly and dose dependently elevated serum level of AP was found in male and female animals of the HD group when compared to the control group (approx. 87% and approx. 86%, respectively). This was not associated with considerable differences in other serum markers for hepatotoxicity (ALT, ASAT, Chol). However, a relation to treatment cannot be excluded. Besides, all parameters of clinical chemistry were within the normal range of variation for this strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In male animals of the HD group prostate gland was slightly reduced in weight (25% below controls). A tendency was also observed in the MD group. The differences were less prominent when related to brain weight or body weight. A slight increase in absolute (15 % higher than controls) and relative epididymis weight (related to brain weight: 31 % above controls; related to body weight: 25 % above controls) was observed in the HD group. After 14 days, a slight increase in absolute and relative spleen weight was found in female animals of all dose groups (absolute weights between 20 and 36 % higher than controls). This effect is not assumed to be adverse and possibly not related to the test substance, as it was not dose-dependent and, at the same time, a slight decrease in absolute spleen weight of male animals was observed in the MD and HD groups.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on increase in neutrophils and alkaline phosphatase, slightly reduced prostate weight and increased epididymides weight

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: male reproductive system and alkaline phosphatase and neutrophils levels

Organ:

other: prostate and epididymides

Treatment related:

yes

Analytical data:

Stability of samples retained during and 6 hours after preparation was demonstrated with recovery rates of test substance between 84 to 92%. The recovery of test substance in samples retained for determination of homogeneity was between 79% and 89%. The recovery of test item in samples retained for determination of test substance concentration was between 79% and 96%.

Conclusions:

Under the study conditions, the 14d NOAEL of the test substance was determined to be 600 mg/kg bw (rat, gavage).

Executive summary:

A study was conducted to determine the oral repreated dose toxicity of the test substance according to OECD Guideline 407 and EU Method B.7, in compliance with GLP. Male and female Wistar rats were exposed to the test substance 7 days per week for a period of 14 days. They received by gavage 10 mL/kg bw test substance at concentrations of 0, 60, 600 and 1000 mg/kg bw (Control (water alone), Low dose, Mid dose and High Dose groups, respectively) During the treatment period, the animals were observed precisely each day for signs of toxicity. Body weight and food consumption were measured once weekly. At the conclusion of the treatment period, hematological and clinical biochemistry parameters were analyzed and all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved. The test substance formulations (test substance suspended in aqua ad injectionem) were prepared freshly on each day of administration. Dose volumes were adjusted individually based on weekly body weight measurement. No mortality occurred in the control or any of the dose groups during the treatment period of this study. Slight to moderate clinical signs (pilo erection and salivayion) were observed during the first treatment week in 1/3 female animals of the HD group. Besides, there were no considerable test substance related clinical signs in this study. In 1/3 male animals of the HD group, body weight and food consumption decreased in the first treatment week. As the animal recovered in the second treatment week it is not considered to be a sign of systemic toxicity but rather a local reaction. Besides, treatment with the test substance did not influence food consumption and body weight gain of male and female animals of this study. Test substance administration had a slight effect on haematological parameters analyzed at the end of the treatment period. In the HD group and in female animals of the MD group, a slight to moderate increase in neutrophils with a concurrent slight decrease in lymphocytes was observed in the differential blood cell count at the end of the treatment period. At the end of the treatment period a slightly and dose dependently elevated serum level of alkaline phosphatase was found in male and female animals of the MD and HD group when compared to the control group. No specific test substance related gross pathological changes were recorded for the male and female animals of this study. In male animals of the HD group prostate gland was slightly reduced and epididymides weight was slightly increased in weight, compared to controls. Under the study conditions, the 14d NOAEL of the test substance was determined to be 600 mg/kg bw/day (2012).

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined repeated dose toxicity and reproductive and developmental toxicity study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Appearance: white powder with a characteristic faint odor
- Structural formula or rational formula: C12H26NaO4P
- Molecular weight: 288.30
- Description (25°C): White powder with a characteristic faint odor
- Acid value: 110-150 (observed value: 131.7) (method 2, 2 g, the solvent used was a mixture of ethanol, benzene, and water at a ratio of 5 : 5 : 1)
- Loss on drying: not more than 2.5% (observed value: 0.7%) (1 g, 105°C, 1 hour)
- Heavy metals: not more than 20 ppm; Arsenic: not more than 2 ppm
- Stability: The test substance remaining after the animal study was returned to the manufacturer for analysis. The analysis results showed that no significant changes had occurred in the test substance during the administration period (attached document 2).
- Storage method: Room temperature (observed temperature, 15°C-28°C) under hermetically sealed conditions.
- Disposal of the remaining test substance: 5 g of the test substance was stored separately in the Test Substance Storage Room at Gotenba Laboratories, and the test substance remaining after the animal study was returned to the manufacturer.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Seventy each of 8-week-old male and female Sprague-Dawley SPF rats (Crj;CD (SD) IGS, Charles River Laboratories Japan, Inc., Atsugi Breeding Center) were purchased (number of animals received: 73 each of males and females)
- The animals were quarantined and acclimatized for 14 days. During this period, the general condition, body weight, and estrous cycle (for 9 days after the quarantine period) were examined, and 58 each of males and females without any abnormality of the general condition (males) (or without any abnormality of the general condition or estrous cycle and with good weight increase for females) were selected and administered the test substance at the age of 10 weeks.
- The body weight at the start of the test substance administration ranged from 338 to 395 g for the males and 219 to 256 g for the females.
- Animals were housed individually in a bracket type metal wire cage in an animal breeding room maintained at a temperature of 21°C to 26°C, relative humidity of 37% to 77%, ventilation frequency of 10 to 15 air changes per hour, and illuminated for 12 hours per day (07:00 to 19:00).
- The animals were allowed free access to Solid chow NMF (nonsterilized: Oriental Yeast Co., Ltd., batch numbers 040713, 040806, 040913) from a stainless steel feeder and to tap water (city water of Gotenba, water bottle was used).

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

The test substance was administered by oral gavage, a method commonly employed for oral administration to rodents. Each animal received 5 mL/kg body weight of the test suspension by forced oral administration via a gastric tube (08:20-14:24 h). Animals of the control group received the vehicle (olive oil) in a similar manner. The volume of the test suspension was calculated based on the latest body weight of each animal.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC

Details on analytical verification of doses or concentrations:

- An appropriate amount of the test substance for each concentration was weighed and suspended in olive oil in an agate mortar to prescribed concentrations (50, 100, and 200 mg/mL). The test suspensions were prepared at a frequency of at least once every 7 days and stored in a cold dark place (refrigerator, observed temperature: 3°C-5°C) in light-protected containers (brown glass bottles) until use.
- Stability of the test suspensions was confirmed at Bozo Research Center and showed that 50 and 200 mg/mL suspensions of the test substance (vehicle: olive oil) remained stable for 24 hours at room temperature after storage in light-protected containers in a cold dark place (refrigerator) for 7 days.
- Confirmation of the concentrations and uniformity of the test suspensions of each concentration used for administration at week 1 and on the last week of administration were analyzed by HPLC at the Bozo Research Center. The results showed that for all the suspensions tested, the percentage of the test substance relative to the nominal value was in the range of 96.5% to 105.0%, with a C.V. in the range of 1.0% to 5.3%, which were within the acceptable range (concentration, nominal value ± 10%; C.V., ≤ 10%).
- Analytical method:
The test sample (dosing suspension), 1 mL, was diluted with 60 vol% of THF solution to 10 mL and centrifuged (2000 rpm, 1000 × g, 20°C, 5 minutes); then, 1 mL of the lower layer was diluted to 5 mL with the mobile phase of HPLC. The diluted solution was filtered through a Milex HV filter and the filtrate was subjected to measurement by the HPLC system. Single test samples were taken from the upper, middle and lower layers of the dosing suspension.

Duration of treatment / exposure:

The duration of administration was 14 days before mating, 14 days during the mating period, and 14 days after the mating period, that is, 42 days in total, for the males of the main group and the males and females of the recovery group, and 14 days before mating and up to day 4 of lactation throughout the mating and gestation periods, that is, 42 to 45 days in total, for the females of the main group. The recovery period for the males and females of the recovery group was 14 days after the end of administration, during which period the test substance was not given to the animals.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

A total of 4 dose groups were set up: 250, 500, and 1000 mg/kg bw groups and the control group.
Each main group consisted of 12 male and female animals each, and each recovery group consisted of 5 each of males and female animals in the control and high- dose groups.

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for selection of doses. In a previous study “14-day repeated-dose oral toxicity study of the test substance in rats (a preliminary study)” (doses: 125, 250, 500, and 1000 mg/kg bw, Bozo Research Center study No.: C-R016), administration of the test substance did not produce any effect even at 1000 mg/kg bw, the level defined as the limiting dose by the OECD Guideline for Testing of Chemicals 422. Therefore, 1000 mg/kg was set as the highest dose, and doses of 500 and 250 mg/kg bw were derived by dividing by a common factor of 2. A total of 3 doses were thus set up.

Details on mating procedure
- After the end of the pre-mating administration period, each pair of male and female animals in the same dose group of the main group was housed in the same cage overnight, and the females were judged to have copulated if the vaginal smear contained sperm or the presence of the vaginal plug was confirmed. Days to copulation was calculated from the day of mating, taken as day 0. From gestation day 17 to day 5 of lactation, the animals were housed individually in a plastic Econ cage with
bedding.
- Observation of mother animals. All female animals confirmed to have copulated were allowed to undergo spontaneous delivery, and observed for the presence/absence of abnormalities in the delivery. Delivery completion was checked twice daily (morning, afternoon) from gestation day 21 to the morning of gestation day 25, from which the gestation period was calculated in units of 0.5 days. If delivery was complete by 5:00 h in the afternoon, that day was regarded as day 0 of lactation.
- Mother animals that completed the delivery were observed for the presence/absence of pup licking and ingestion of the placenta and amnion. They were allowed to suckle pups up to day 4 of lactation (the date of delivery completion was regarded as day 0 of lactation) and observed for lactating behavior, using pup gathering, nest building, and breastfeeding as indices.

Positive control:

No

Observations and examinations performed and frequency:

- Observation of the general condition. All animals were observed for the presence of any abnormality of the general condition, such as in the external appearance, nutritional condition, posture, behavior, and excrements, 3 times everyday (before, immediately after, and 2 hours after the administration) during the administration period and once every morning during the recovery period.
Detailed observation of the general condition, function tests, measurement of the grip strength and spontaneous motor activity. Detailed observation of the general condition was performed once before the start of administration for all animals, once every week during the administration period for the males of the main group, once every week during the pre-mating administration and mating periods, and on predetermined days (gestation days 1, 7, 14 and 20, day 4 of lactation) during the gestation period and the lactation period for the females of the main group, and once every week during the administration and recovery periods for the animals of the recovery group. Function tests, measurement of the grip strength, and measurement of the spontaneous motor activity were performed on the last week of administration (day 39 of administration) for the males of the main group, after F1 necropsy on day 4 of lactation (day 42-45 of administration) for the females of the main group, and on the last week of administration (day 39 of administration) and last week of the recovery period (day 11 of recovery) for the males and females of the recovery group. These tests were performed on 5 animals each per group. - Measurement of body weight. Body weight was measured on days 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39 and 42 of administration, and on the day of necropsy for the males of the main group, on days 1, 4, 8, 11 and 14 of the recovery period and on the day of necropsy, in addition to the days of measurement for the males of the main group, for the males and females of the recovery group, and on days 1, 4, 8, 11 and 15 of administration (and days 18, 22 and 25 of the administration period as well as in non-copulated animals), days 0, 4, 7, 11, 14, 17 and 20 of gesta tion, and days 0 and 4 of lactation for the females of the main group. Body weight was measured from 08:06 to 10:45 h, except for the measurement on day 0 of lactation for females whose end of delivery was confirmed during the observation in the afternoon. On the day of necropsy, the body weight was measured after the animals had been denied access to food for approximately 16 hours from the previous day, in order to calculate the relative organ weight.
- Measurement of food consumption. The amount of food remaining relative to that supplied on the previous day was measured on days 1, 4, 8, 11, 15, 32, 36, 39 and 42 of administration for the males of the main group, on days 1, 4, 8, 11 and 14, in addition to the days of measurements for the males of the main group, for the males and females of the recovery group, on days 1, 4, 8, 11 and 15 of administration, days 1, 4, 7, 11, 14, 17 and 20 of gestation, and days 2 and 4 of lactation for the females of the main group. Food consumption per animal was calculated from the data thus obtained. The amount of food supplied and the amount of food remaining were measured from 08:26 to 11:25 h.
- Urinalysis (including measurement of water intake). On the last week of administration (day 36 to 37 of administration) and on the last week of the recovery period (day 8 to 9 of recovery), each of the male animals was individually housed in a cage equipped with a urine collector. Four-hour urine specimens were collected under fasting conditions of the animals with free access to water, followed by 20-hour urine specimen collection under free access to food and water. The parameters tested are
as shown below. The first 4-week urine specimens were subjected to tests from pH up to sediments, as well as measurement of the urine volume, and the subsequent 20-hour urine specimens were subjected to measurement of the osmotic pressure and urine volume. Urine volume was calculated as the sum of the volumes of 4-hour and 20-hour urine specimens. The amount of water intake from the previous day was measured using a water bottle while the animals were housed in the cage
equipped with the urine collector.

Sacrifice and pathology:

After delivery, the mother animals were exsanguinated to death by dissecting the abdominal aorta on Day 5 of lactation, after the animals had been denied access to food overnight (approx. 16-20 h) from Day 4 of lactation: 5 in each group after blood collection for hematological tests and blood chemistry tests, and the remaining animals under ether anesthesia.

- Necropsy and organ weight measurement. All of the 5 male and female animals in each group from which blood samples were collected for the hematology and blood chemistry tests on the day after the last day of administration and on the last day of the recovery period were exsanguinated to death after the blood collection, and all the other animals were exsanguinated to death by dissecting the abdominal aorta under ether anesthesia. They were then subjected to detailed gross pathological e
xamination of the body organs and tissues, including the external surface, head, chest and abdomen, and the results were recorded. In the females (mother animals), the number of corpora lutea and number of implantation sites were counted on day 5 of lactation. Then, in 5 each of the male and female animals from which blood samples were collected for the hematology and blood chemistry tests, the weight (absolute) of the following organs (testes and epididymes of all the animals) was measured
and the relative weight of each organ per 100 g body weight was calculated from the absolute organ weight and the body weight at necropsy. For bilateral organs marked with an asterisk, the weight of each side was measured separately and the sum of the weights was calculated. Brain, thyroid gland* (including parathyroid gland), thymus gland, heart, liver, spleen, kidney*, adrenal*, testis*, and epididymis*.

- Histopathological examination. The following organs and tissues of all the animals were fixed and st ored in 10 vol% formalin solution in phosphate buffer (the testes and epididymes were fixed in Bouin's fluid, followed by storage in 10 vol% formalin solution in phosphate buffer). Then, organs and tissues (see below) were embedded in paraffin, and sections were stained with hematoxylin and eosin (H-E). Specimens obtained from 5 each of the male and female animals of the control and high-dose groups
from which blood specimens were collected for the hematology and blood chemistry tests were subjected to microscopic examination (for bilateral organs, both sides were isolated and one side was subjected to the microscopic examination). The results revealed the effect of the test substance on the stomach. Therefore, specimens from 5 each of the male and female animals of the low- and medium-dose groups were also subjected to microscopic examination. Representative cases of normal
and abnormal findings were photographed.

(Cerebrum, cerebellum, pituitary gland, spinal cord (thoracic), sciatic nerve, thyroid gland, parathyroid gland, adrenal, thymus gland, spleen, submandibular lymph nodes, mesenteric lymph nodes, heart, lung (including bronchus), stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, kidney, bladder, testis, epididymis, ovary, uterus, seminal vesicle, sternum (including bone marrow), femur (including bone marrow), and the animal identification site (auricle))

Statistics:

- Bartlett test
- Dunnett’s test
- χ2 test with Yates’ continuity correction
- Fisher’s exact test

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- In the main group, one female in the 500 mg/kg bw group showed opacity of an eyeball (unilateral) from gestation day 5, which was not related to the dose and was therefore considered to be an incidental change.
- In the recovery group, one male in the 1000 mg/kg bw group showed decreased spontaneous motor activity from day 37 of administration up to day 7 of the recovery period, and wheezing from day 37 of administration until the end of the recovery period.
- No abnormality was observed in the other animals, either in the main or in the recovery groups.

Mortality:

no mortality observed

Description (incidence):

- There were no significant difference in the body weight between males and females of the main group. A significantly greater increase in body weight was observed in the females of the 250 and 1000 mg/kg bw groups during the lactation period, but the increase was not dose-related.
- In the recovery group, males in the 1000 mg/kg bw group showed decreased body weight gain during the administration period and decreased body weight (-21g) during the recovery period. This was caused by the abnormality in 1 out of the 5 animals. This animal showed continued body weight decrease (and also decreased spontaneous activity and wheezing in the observation of the general condition; the body weight was 466g before manifestation of the symptom and 261g on day 14 of the recovery period). The body weights of the other 4 males and 5 females in the same group were similar to those of the animals in the control group, showing no statistically significant differences.

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Administration of the test substance did not have any effect on the food consumption in the males or females of either the main group or the recovery group. A significant increase was observed on day s 2 and 4 of lactation in the females of the 250 mg/kg dose group in the main group, but this was not dose-related.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

-Tests at the end of the administration period. A significant increase in the serum level of fibrinogen was observed in the females of the 250 mg/kg bw group and a significant decrease in the percentage of lymphocytes and significant increase in the percentage of segmented neutrophils were observed in the females of the 500 mg/kg bw group. However, none of these changes were observed in the 1000 mg/kg bw group, suggesting that they were within the range of physiological variations. No significant differences were observed in the male animals between the control group and any of the treatment groups.
- Tests at the end of recovery period. A significant increase in the mean corpuscular volume of the red blood cells, significant decrease of the platelet count, significant increase in the percentage of lymphocytes, and significant decrease in the percentage of segmented neutrophils were observed in females of the 1000 mg/kg bw group. However, these changes were not observed at the end of the administration period, which suggested that they were within the range of physiological variations. No significant differences were observed in the male animals between the control group and any of the treatment groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Test at the end of the administration period. A significant increase in the serum level of ALT was observed in the males of the 1000 mg/kg bw group. A significant decrease of the serum level of inorganic phosphorus was observed in the males of the 250 mg/kg bw group. However, since the decrease was not dose-related, it was considered to be within the range of physiological variations.
- Tests at the end of recovery period. A significant increase in the serum level of total protein was observed in the females of the 1000 mg/kg bw group. However, since no such change was observed at the end of the administration period, the increase was considered to be within the range of physiological variations.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis (including measurement of water intake) showed no abnormalities in the qualitative parameter values in any of animals in either the main group or in the recovery group. No significant difference was observed in any of the quantitative parameter values between the control group and any of the treatment groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- Observation of animals in the home cage. No abnormalities were observed in any of the animals in either the main group or the recovery group.
- Observation of the animals while being handled. No abnormality was observed in any of animals in either the main group or the recovery group.
- Observation of animals in the open field. Males of the 1000 mg/kg bw group in the main group showed a significant increase of the defecation frequency during weeks 1 and 2 of administration, which was a very mild transient change and considered to be within normal range. No abnormalities were observed in the other parameters in any of the animals in either the main group or the recovery group. No significant differences were observed in the standing frequency between the control group and any of the treatment groups.
- Function tests. No abnormalities were observed in any of the animals in either the main group or the recovery group. No significant differences were observed in the air righting reflex or landing foot splay between the control group and any of the treatment groups.
- Measurement of the grip strength. No significant differences were observed between the control group and any of the treatment groups in either the main group or the recovery group.
- Measurement of spontaneous motor activity (measured for 10-minute periods and a total of 60 minutes). No significant difference was observed between the control group and any of the treatment groups in either the main group or the recovery group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No dose-related changes in either direction (increase or decrease) were observed in either the absolute or the relative weight. Although significant differences in the weights of the following organs were observed, they were considered to be within the range of normal variations, because they were neither dose-related nor were observed at the end of the administration period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Findings at the end of the administration period. Stomach: Indentation of the anterior stomach was observed in 0, 5 and 7 males, and 1, 1 and 3 females of the 250, 500 and 1000 mg/kg bw groups, respectively. White foci were observed in 1 male of the 500 mg/kg bw group. Rough mucosa in the anterior stomach was observed in 5 and 9 males, and 5 and 6 females of the 500 and 1000 mg/kg bw groups, respectively. Indentation of the glandular stomach was observed in 1 female of the 500 mg/kg bw group. All the other findings observed in the organs and tissues were considered to be incidental, as judged from the frequency of their occurrence and the pathological findings (Dark red foci in the glandular stomach were observed in 0, 1, 2 and 1 males and 4, 2, 1 and 1 females of the control
group, 250, 500 and 1000 mg/kg bw groups, respectively. Kidney: Pyelectasis was observed in 2 and 1 males of the 250 and 1000 mg/kg bw groups, respectively. Eyeballs: Corneal opacity (unilateral) was observed in 1 female of the 500 mg/kg bw group).
- Findings at the end of the recovery period. Stomach: Rough mucosa in the anterior stomach was observed in 1 male of the 1000 mg/kg bw group. This animal also showed expansion of the digestive tract from the stomach to the colon due to gas accumulation, and a mild decrease in the size of the testis. Other findings observed in the following organs were considered to be incidental as judged from the frequency of their occurrence and the pathological findings (Lung: Dark red foci were observed in 1 female of the 1000 mg/kg bw group).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Administration of the test substance had effects on the stomach of the animals in the 250 mg/kg bw and higher dose groups.
- Findings at the end of the administration period.
Stomach: Mild to moderate erosions or ulcers of the anterior stomach were observed in 0, 4 and 4 males and 1, 1 and 1 females of the 250, 500, and 1000 mg/kg bw groups, respectively. Very mild to moderate thickening of the anterior stomach mucosa was observed in 1, 4 and 5 males and 1, 4 and 3 females of the 250, 500, and 1000 mg/kg bw groups, respectively. Very mild to mild edema of the submucosal tissue in the anterior stomach was observed in 1, 5 and 5 males and 0, 4 and 3 females of the 250, 500, and 1000 mg/kg bw groups, respectively. Most of these changes in the anterior stomach were localized findings. All of the other findings observed, as follows, were considered to be incidental changes as judged from the frequency of their occurrence and the histopathological findings
Epididymis: Very mild infiltration by stromal cells was observed in 1 male of the control group. Heart: Very mild localized myocarditis was observed in 4 males of the control group and 1 male of the 1000 mg/kg bw group.
Kidney: Very mild basophilic tubules were observed in 3 males of the control group and 1 male and 1 female in the 1000 mg/kg bw group.
Liver: Very mild, minute granulomas were observed in 3 males of the control group and 1 male of the 1000 mg/kg bw group.
Lung (including bronchi): Very mild mineral deposits in the arterial walls were observed in 1 male of the control group and 1 female of the 1000 mg/kg bw group. Very mild accumulation of foam cells was observed in 2 males and 1 female of the control group, and 1 male and 3 females of the 1000 mg/kg bw group.
Spleen: Very mild to mild extramedullary hematopoiesis was observed in 5 females each in the control group and 1000 mg/kg bwgroup.
Stomach: Inclusion cysts were observed in 1 male of the 500 mg/kg bw group. Very mild to mild erosions in the glandular stomach were observed in 0, 0, 0 and 1 male and 3, 1, 1, and 0 females in the control group, 250, 500, and 1000 mg/kg bw group, respectively).

- Findings at the end of the recovery period. Stomach: Moderate thickening of the anterior stomach mucosa was observed in 1 male of the 1000 mg/kg bw group.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Refer to the other related RSS for details on reproductive and development toxicity.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

ca. 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic effects observed up to 1000 mg/kg bw/d; the changes in forestomach are of local nature due to the irritant properties of the substance

Key result

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

ca. 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects on forestomach

Key result

Critical effects observed:

no

Conclusions:

Under the study conditions, only (local) changes of the forestomach mucosa were observed which are due to the irritant properties of the substance. Therefore, the NOAEL (systemic) is considered to be 1000 mg/kg bw/day.

Executive summary:

A study was conducted to determine the toxicity to reproduction of the test substance according to OECD Guideline 422, in compliance with GLP. The test substance was administered at 0 (control group), 250, 500 or 1000 mg/kg bw to male Sprague-Dawley SPF rats for 14 days before mating, through the mating period, and up to 1 day before necropsy (42 days in total) and to female Sprague-Dawley SPF rats for 14 days before mating, through the mating period and the gestation period, up to day 4 of lactation (42 to 45 days in total) to investigate the repeated-dose, reproductive and developmental toxicities. In the 0 and 1000 mg/kg bw groups, a 14-day recovery period was allowed after the 42-day administration period to investigate the reversibility of the toxic changes. Administration of the test substance had no effect on any of the following: general condition, findings on detailed observation of the general condition, results of function tests, grip strength, spontaneous motor activity, body weight, food consumption, results of urinalysis (including water intake), or in the results of hematological or blood chemistry tests. Administration of the test substance had no effect on any of the following: general condition, findings on detailed observation of the general condition, results of function tests, grip strength, spontaneous motor activity, body weight, food consumption, results of urinalysis (including water intake), or in the results of hematological or blood chemistry tests. Changes in the forestomach may be considered to be adverse, however, they are also considered to be a result of local irritation of the (irritant) test item (which is brought directly in contact to the mucosa in a massive amount by gavage application) than a true effect of systemic toxicity. Furthermore, in the light of the absence of a forestomach in humans, observed effects on this tissue are of questionable relevance with reference to the extrapolation of the toxic properties of a test substance in humans. Under the study conditions, only (local) changes of the forestomach mucosa were observed at 250 mg/kg bw/day and above, which are due to the irritant properties of the substance. Therefore, the NOAEL (systemic) is considered to be 1000 mg/kg bw/day (METI, 2005).

Endpoint conclusion

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 compliant read across study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Study 1:

A dose range finding study was conducted to determine the oral repeated dose toxicity of the read across substance, mono- and di- C12 PSE, K+, according to OECD Guideline 407 and EU Method B.7, in compliance with GLP. In this study, 3 male and female Wistar rats each received by gavage 10 mL/kg bw test substance at concentrations of 0 (water), 60, 600 and 1000 mg/kg bw, 7 days per week for a period of 14 days. During the treatment period, the animals were observed precisely each day for signs of toxicity. Body weight and food consumption were measured once weekly. At the conclusion of the treatment period, hematological and clinical biochemistry parameters were analyzed and all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved. The test substance formulations (test substance suspended in aqua ad injectionem) were prepared freshly on each day of administration. Dose volumes were adjusted individually based on weekly body weight measurement. No mortality occurred in the control or any of the dose groups during the treatment period of this study. Slight to moderate clinical signs (pilo erection and salivation) were observed during the first treatment week in 1/3 female animals of the high dose (HD) group. Besides, there were no considerable test substance-related clinical signs in this study. In 1/3 male animals of the HD group, body weight and food consumption decreased in the first treatment week. As the animal recovered in the second treatment week, it is not considered to be a sign of systemic toxicity but rather a local reaction. Besides, treatment with the test substance did not influence food consumption and body weight gain of male and female animals of this study. Test substance administration had a slight effect on haematological parameters analyzed at the end of the treatment period. In the HD group and in female animals of the mid dose (MD) group, a slight to moderate increase in neutrophils with a concurrent slight decrease in lymphocytes was observed in the differential blood cell count at the end of the treatment period. These findings in the differential white blood cell count were assumed to be related to a piece of gavaging cannula that had been swallowed and had subsequently affected food consumption and body weight development. At the end of the treatment period, a slightly and dose dependent elevated serum level of alkaline phosphatase was found in male and female animals of the MD and HD group when compared to the control group. This was not associated with considerable differences in other serum markers for hepatotoxicity (ALT, ASAT, Chol). No specific test substance-related gross pathological changes were recorded for the male and female animals of this study. In male animals of the HD group prostate gland was slightly reduced and epididymis weight was slightly increased in weight, compared to controls. Based on the results of the read across study, the 14 d NOAEL of the test substance was determined to be 600 mg/kg bw/day (Allingham, 2012).

Study 2:

A study was conducted to determine the toxicity to reproduction of the read across substance, mono- C12 PSE, Na+, according to OECD Guideline 422, in compliance with GLP. The test substance was administered at 0 (control group), 250, 500 or 1000 mg/kg bw to male Sprague-Dawley SPF rats for 14 days before mating, through the mating period, and up to 1 day before necropsy (42 days in total) and to female Sprague-Dawley SPF rats for 14 days before mating, through the mating period and the gestation period, up to day 4 of lactation (42 to 45 days in total) to investigate the repeated-dose, reproductive and developmental toxicities. In the 0 and 1000 mg/kg bw groups, a 14-day recovery period was allowed after the 42-day administration period to investigate the reversibility of the toxic changes. Administration of the test substance had no effect on any of the following: general condition, findings on detailed observation of the general condition, results of function tests, grip strength, spontaneous motor activity, body weight, food consumption, results of urinalysis (including water intake), or in the results of hematological or blood chemistry tests. Administration of the test substance had no effect on any of the following: general condition, findings on detailed observation of the general condition, results of function tests, grip strength, spontaneous motor activity, body weight, food consumption, results of urinalysis (including water intake), or in the results of hematological or blood chemistry tests. Changes in the forestomach may be considered to be adverse; however, they are also considered to be a result of local irritation of the (irritant) test item (which is brought directly in contact to the mucosa in a massive amount by gavage application) than a true effect of systemic toxicity. Furthermore, in the light of the absence of a forestomach in humans, observed effects on this tissue are of questionable relevance with reference to the extrapolation of the toxic properties of a test substance in humans. Therefore, based on the results of the read across study, the NOAEL for systemic effects is considered to be 1000 mg/kg bw/day (METI, 2005). This is further supported by a NOAEL of 1000 mg/kg bw/day determined in a longer duration (90-day) repeated dose study available with mono- and di- C12 PSE, K+ in rats.

Based on the available data, a NOAEL of 1000 mg/kg bw/day from the longer duration OECD 422 study can be considered for the test substance, indicating a low systemic toxicity potential. Further, the higher fatty alcohol content in the test substance is not expected to have a differential impact on the repeated dose toxicity potential, as available studies with dodecanol resulted in a systemic NOAEL at 2000 mg/kg bw/day (highest dose) due to the absence of toxicologically significant effects at any dose level in a OECD 422 screening study in rats (OECD, 2006).

Justification for classification or non-classification

Based on the results of the read across studies, the test substance is assessed not to meet the criteria for STOT RE classification according to CLP criteria (Regulation 1272/2008/EC).​​