Reaction mass of orthophosphoric acid and potassium dihexadecyl phosphate and dipotassium hexadecyl phosphate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

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:

weight of evidence

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID 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 source

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, NOAEL for systemic effects for the test substance, is considered to be 1000 mg/kg bw/day.

Executive summary:

A screening study was conducted to determine the toxicity to reproduction of the read across substance, 'mono- C12 PSE, Na+' (purity: 100%, according to the OECD Guideline 422, in compliance with GLP. The read across 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 d before necropsy (42 d in total) and to female Sprague-Dawley SPF rats for 14-d before mating, through the mating period and the gestation period, up to Day 4 of lactation (42 to 45 d in total) to investigate the repeated-dose, reproductive and developmental toxicities. In the 0 and 1000 mg/kg bw groups, a 14-d recovery period was allowed after the 42-d administration period to investigate the reversibility of the toxic changes. Administration of the read across 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 haematological or blood chemistry tests. Administration of the read across 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 haematological 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 substance (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 read across 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 (BRC, 2005). Based on the results of the read across study, a similar systemic NOAEL can be expected for the test substance, ' mono- and di- C16 PSE, K+ and H3PO4'.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From April 10, 2008 to October 29, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals
- Source: Rat, Sprague-Dawley (Crl:CD(SD)), SPF (Supplier: ORIENTBIO INC., Korea)
- Age at study initiation: Male - 9 weeks old, Female - 8 weeks old
- Weight at study initiation: Male - 288.6–336.7 g, Female - 163.5–188.9 g
- Housing: Stainless wire cage, 260W×350D×210H (mm), Polycarbonate cage 260W×420L×180H (mm), 1 animal per cage (During the mating period: 1male and 1 female, During the lactation period: 1 female and neonate)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

Environmental conditions
- Temperature (°C): 19.2–23.4°C
- Humidity (%): 27.6–64.3 %
- Air changes (per h): 10–15 times/h
- Photoperiod (h dark / h light): 12–h light/dark cycle (lights on at 7 a.m., lights off at 7 p.m.)

Route of administration:

oral: gavage

Vehicle:

other: Water for injection

Details on oral exposure:

Preparation of dosing solutions:
At use, the test substance was diluted to necessary concentration using the Water for injection and prepared.

Vehicle
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): GBA8002, GBA8003

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

The males were administrated once daily during 2 weeks prior to mating, the 2 weeks of mating period, and 2 weeks after mating (total 6 weeks). The females of main group were administrated once daily from 2 weeks before mating to Day 4 post partum (approximately 54 d). The females of recovery group (without mating) were administrated approximately 54 d (applied equally main group females). Also, coitus was confirmed, but females did not show gestational signs, and were administrated until gestation Day 26.

Frequency of treatment:

Once a day

Remarks:

0, 125, 250 and 500 mg/kg bw
Basis: actual ingested

No. of animals per sex per dose:

Main group: 13 animals per sex per dose
Recovery group: 6 animals (control and high dose group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were determined based on the previous dose range finding study (Study No.: B08008P). In result, food consumption of males and females was decreased at 500 mg/kg bw, and females were observed in inhibition of weight gain. In addition, 1000 mg/kg bw dose level was observed in salivation and soft stool, and moribund in one male. As a result, 500 mg/kg bw was selected as the high dose level for this study and sequentially divided by a geometric ratio of 2 to produce two additional lower doses. 250 and 150 mg/kg bw were selected as the middle and low dose levels, respectively. In the control group, Water for injection was administered the same as dosing volume of the high dose.

- Rationale for animal assignment (if not random): lowing the period of quarantine and acclimation, 64 males and 64 females which were shown no clinical signs and satisfied body weight gains, were selected and allocated based on body weights to four groups. The control and high dose groups had 19 males and 19 females, and the low and mid dose groups had 13 males and 13 females, respectively.

Observations and examinations performed and frequency:

Cage side observations: Yes
- Time schedule: The parent’s animals were observed for clinical signs including abortion, dystocia, premature birth, parturition, and lactation condition once daily. The moribund and mortality were observed twice daily. The recovery groups were observed during 2 weeks after the end of administration.
- Cage side observations checked and were included.

Detailed clinical observations: Yes
- Time schedule: once a week
- Detailed clinical signs were performed the day before the first administration and once a week during the treatment and recovery period for general appearance, posture, activity, response to handling, nervous system, function of autonomic nervous system, and abnormal behavior.

Body weight: Yes
- Time schedule for examinations: once a week
Body weights of males were measured just before administration and once a week during the treatment and recovery period. Body weights of females were measured just before administration, on Day 0, 7, 14, and 20 of gestation and on Day 0 and 4 of lactation. During the administration and recovery period, body weights were measured once a week. Only, body weights were not measured during the mating period.

Haematology: Yes
- Time schedule for collection of blood: on the day necropsy
- Anaesthetic used for blood collection: Yes (with isoflurane)
- Animals fasted: Yes (fasted from approximately over 18 hours prior to necropsy)
- How many animals: 6 males and 6 females selected from each group
- Parameters checked and were examined.

Clinical chemistry: Yes
- Time schedule for collection of blood: on the day necropsy
- Animals fasted: Yes (fasted from approximately over 18 h prior to necropsy)
- How many animals: 6 males and 6 females selected from each group
- Parameters checked and were examined.

Urinanalysis: Yes
- Time schedule for collection of urine: Urine samples were collected for approximately 3 hours as fresh urine and approximately 24 h as cumulative urines
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked and were examined.

Neurobehavioural examination: Yes
- Time schedule for examinations: on the day before necropsy
- Dose groups that were examined: Six males and six females selected from each group in the main group and all animals of the recovery group
- Battery of functions tested:
- Pinna reflex: Checked the reflex at the stimulation of animal’s auricle.
- Auditory reflex: Checked the reflex to sound at the sound generation.
- Corneal reflex: Checked the reflex at the stimulation of animal’s cornea.
- Papillary reflex: Checked the reflex of pupil by the light.
- Traction test: Measured the times to grasp the metal horizontal bar by forepaws.
- Rotarod test: Measured the times to endure on the rotating rod.
- Open field test: Animals were allocated to the open filed test boxes. The frequency for movement of each animal was recorded.

Sacrifice and pathology:

Necrospy findings: Yes
After completion of observation period of main and recovery groups, all live and dead animals were performed to detailed macroscopic examination about body surface and organ and tissue of the whole body.

Histopathology: Yes (see table 18-1, 18-2, appendix 18-1 and 18-2)
Histopathological examinations for 6 males and 6 females selected from each group in the main group such as the control group and the high dose group, and the recovery group, the number of dead animals, and organ of macroscopic lesions were performed.

Other examinations:

Organ weights
The following organs which were selected from six males and six females each group in the main group and all animals of the recovery group were weighed individually at necropsy. Organ-to-body weights (terminal body weights) percentages were calculated. Only, testis and epididymides were weighed from all male animals. Paired organs (*) were weighed together. [Brain · Heart · Liver · Thymus · Spleen · Kidney* · Adrenal* · Testis* · Epididymis* · Ovary* . Uterus]

Statistics:

Statistical analysis of this study was performed by the SAS program (SAS 9.1.3, SAS Institute Inc., U.S.A.). For the data including body weights, food consumption, urine, hematology, blood biochemistry parameters, organ weights, sensory reflex, and motor function test, the Bartlett test was conducted to test for variance homogeneity at the 5.0% one-tailed probability level. One-way analysis of variance (ANOVA) test was employed on homogeneity, if significant, followed by Dunnett’s t- test for multiple comparisons. Kruskal-wallis was employed on heterogeneity, if significant, followed by Mann-whitney u-test for multiple comparisons. Group comparison was performed at the 5.0 and 1.0% two-tailed probability level. For the data of recovery, Folded-F test was conducted to test for variance homogeneity at the 5.0% two-tailed probability level. Student t-test was employed on homogeneity, if overrule, Aspin-Welch t-test was performed at the 5.0 and 1.0% two-tailed probability level. Non pregnancy animals were excluded.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

Clinical signs and mortality
In the main group, death was observed in one female (2403) of the 500 mg/kg bw treatment group on Day 16 of administration. And in the recovery group, death was observed in one female (2416) of the 500 mg/kg treatment group on Day 52 of administration. In the main group, no clinical signs were observed in males and females of 125 mg/kg bw treatment group and in females of the 250 mg/kg bw treatment group. But, transient salivations which were appeared immediately after administrations were sporadically observed in four males of the 250 mg/kg bw treatment group during from Day 22 of administration to completion of administration. Also, transient salivations were observed in all males of the 500 mg/kg bw treatment group from Day 20 of administration to completion of administration and in four females of the 500 mg/kg bw treatment group from Day 2 of gestation to completion of administration, sporadically or persistently. In addition, mucous stool, soft stool, and dirty nose were observed in one male (1407) of the 500 mg/kg bw treatment group on Day 22 and 23. In the recovery group, transient salivations were observed in almost all males of the 500 mg/kg bw treatment group from Day 16 of administration to completion of administration and in almost all females of the 500 mg/kg treatment group from Day 20 of administration to completion of administration, sporadically or persistently. Detailed clinical signs were performed once a week during the study period, and no clinical signs were observed in all animals of the control and treatment groups.

Body weight and weight gain
In the main group, no treatment-related abnormalities in body weight gain were noted in all animals of the control and treatment groups. In females of the recovery group, body weights on Day 55 of administration were significantly decreased (p<0.05) compared with the control group in the 500 mg/kg bw treatment group. Besides, no treatment-related abnormalities in body weight gain were noted in all males.

Food consumption
In the main group, no treatment-related abnormalities in food consumption were noted in all animals of the control and treatment groups. In females of the recovery group, food consumption was significantly decreased (p<0.05) compared with the control group on Day 13 and 34 of administration in the 500 mg/kg bw treatment group. Besides, no treatment-related abnormalities in food consumption were noted in all males.

Haematology
In the main group, no treatment-related abnormalities in haematology were noted in all animals of the control and treated groups. In the recovery group, eosinophils (EOS) of males and prothrombin time (PT) of females were significantly increased (p<0.05) compared with the control group in the 500 mg/kg bw treatment group.

Clinical chemistry
In blood biochemistry of the main group, phosphorus (P) was significantly decreased (P<0.05) compared with the control group in females of the 125 mg/kg bw treatment group. In female of the recovery group, triglycerides (TG, p<0.05), total proteins (TP, p<0.05), and albumin (Alb, p<0.01) were significantly decreased compared with the control group in the 500 mg/kg bw treatment group.

Urinanalysis
In the main and recovery groups, no treatment-related abnormalities were noted in the urinalysis parameters.

Neurobehaviour
No treatment-related abnormalities in the pinna reflex, auditory reflex, corneal reflex, papillary reflex, traction, rota rod, and open field test were observed in all animals of the control and treatment groups.

Organ weights
In the main group, no treatment-related abnormalities in absolute and relative organ weights of the males were noted compared with the control group. However, in females, absolute organ weights of kidney were significantly increased (p<0.05, p<0.01) compared with the control group in all treatment groups; however, dose-dependent was not observed. Relative organ weights of uterus were significantly decreased (p<0.05) compared with the control group in the 500 mg/kg bw treatment group; however, the difference was slight. In the recovery group, no treatment-related abnormalities in absolute and relative organ weights of the males were noted. However, in females, fasted body weights (p<0.01) and absolute organ weights of heat (p<0.05) were significantly decreased compared with the control group in the 500 mg/kg bw treatment group.

Gross pathology
In the necropsy findings of the main group, agenesis of spleen was observed in one male (1302) of the 250 mg/kg bw treatment group, and yellow spot of epididymis was observed in one male (1408) of the 500 mg/kg bw treatment group. The other abdominal signs were not observed, and the necropsy finding of recovery group was not noted. In the necropsy findings for dead animals, gaseous distension was observed in one female (2403) of the 500 mg/kg bw treatment group in the main group on Day 16 of administration. In the recovery group, gaseous distension, severe hydronephrosis of left kidney and dilation of left uterine horn were observed in one female (2416) of the 500 mg/kg bw treatment group in the recovery group on Day 52 of administration.

Histopathology: Non-neoplastic
In the main group, cast of kidney was observed in one male (1104) of the control group, and sperm granuloma of epididymis was observed in one male (1408) of the 500 mg/kg bw treatment group. The other abnormal findings were not observed. In the recovery group, basophilic tubules of kidney was observed in one female (2119) of the control group. The other abnormal findings were not observed. In the 500 mg/kg bw treatment group of the main group female, no histopathological change in one dead animal (2403) was observed. In the 500 mg/kg bw treatment group of the recovery group female, renal tissue in one dead animal (2416) was not observed by severe hydronephrosis of gross findings, and left uterine horn was dilated; however, no histopathological change was observed.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Two dead females in the 500 mg/kg bw treatment group were occurred, and findings of gaseous distension of gastrointestinal tract were observed. Also, mucous stool, soft stool, and dirty nose were observed in one male of the 500 mg/kg bw treatment group.

Remarks on result:

other: no dose dependence observed

Key result

Critical effects observed:

no

Conclusions:

Under the study conditions, NOAEL for repeated dose toxicity was determined to be 250 mg/kg bw/day.

Executive summary:

A screening study was conducted to determine the repeated dose toxicity with the reproduction/developmental toxicity of the test substance, phosphoric acid (85% in water), according to OECD Guideline 422, in compliance with GLP. The test substance dose levels of 0, 125, 250 and 500 mg/kg bw/day was orally administered once a day to male rats from 2 weeks prior to mating to 2 weeks after mating (42 days) and to female rats from 2 weeks prior to mating to on Day 4 post-partum (40–52 days) in 13 male and 13 female rats per group. Additional recovery groups of 6 males and 6 females were included in the control and high dose groups. In the recovery group, males were administered during 42 days, and females were administered during 53 days, and then were kept during the 2 weeks of recovery period. It was performed to confirm the presence or absence of reversibility of toxicity. One female in the 500 mg/kg bw/day treatment group died on Day 16 of administration, and gaseous distension was observed in gastrointestinal tract. In the recovery group, one female in the 500 mg/kg/day treatment group died on Day 52 of administration, and gaseous distension of gastrointestinal tract, severe hydronephrosis of left kidney, and dilation of left uterine horn were observed. However, severe hydronephrosis of left kidney and dilation of left uterine horn were considered as change that occurred spontaneously by individual difference without treatment-related effects of the test substance. In the clinical signs, mucous stool, soft stool, and dirty nose were observed in one male of the 500 mg/kg/day treatment group on Day 22 and 23 of administration. However, the detailed clinical signs were performed once a week, and no abnormality was observed in the main and recovery groups. The partial items of body weights, food consumption, urinalysis, haematology, and biochemistry have shown slight differences; however, no change by administration of the test substance was observed. Also in organ weights, difference by administration of the test substance was not observed. Spontaneous changes in the necropsy and histopathological examinations were adventitiously observed; however, change of pathology by test substance was not shown. However, two dead females in the 500 mg/kg bw/day treatment group occurred, and findings of gaseous distension of gastrointestinal tract were observed. Mucous stool, soft stool, and dirty nose were observed in one male of the 500 mg/kg treatment group. Based on these results, as effects of the test substance for all males and females were not observed at 250 mg/kg bw/day and below, the NOAEL was concluded by the study authors at 250 mg/kg bw/day (OECD SIDS, 2009).