Phosphoric acid, dodecyl ester, potassium salt

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

After dosing rats (12 mels and 12 females per dose group) with 250, 500 or 1000 mg/kg bw/d of phosphoric acid, dodecyl ester, sodium salt in diet for a minimum of 42 days 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/d. No effects on reproductive parameters and pups were observed. Thereforem the NOEL (reproducitve/deveolpmental) is set at 1000 mg/kg bw/d.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated 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:

other: Well reported OECD 422 study with sufficient supporting.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan Inc., Atsugi Breeding Center
- Age at study initiation: 10 weeks
- Weight at study initiation: 338 - 395 g (males), 219 - 256 g (females)
- Housing: individually in a bracket type metal wire cage (250 mm (W) x 350 mm (D) x 200 mm (H), Japan Cage, Co., Ltd.); from gestation day 17 to day 5 of lactation, the females were housed individually in a plastic Econ cage (340 mm (W) x 400 mm (H) x 185 mm (H), CLEA Japan, Inc.)
- Diet (e.g. ad libitum): Solid chow NMF (Oriental Yeast Co., Japan), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 26°C
- Humidity (%): 37 - 77 %
- Air changes (per hr): 10 - 15 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

The test substance was administered by oral gavage . Each animal received 5 mL/kg bw of the test suspension. 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 weight of each animal.

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 female were judged to have copulated if the vaginal smear contained sperm or the presence of the vaginal plug was confirmed. Day to copulation was calculated from the day of mating, taken as day 0. The copulation rate, conception rate and fertility rate of each group were calculated according to the following:
- Copulation rate (%) = (number of pairs that copulated/number of pairs) x 100
- Conception rate (%) = (number of pregnant females/number of females that copulated) x 100
- Fertility rate (%) = number of pregnant females/number of males that copulated) x 100

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The suspensions of each concentration used for administration at week 1 and on the last week of administration were analysed by HPLC at 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% wit 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%).
The test sample (dosing suspension), 1 mL, was diluted wih 60 vol% of THF solution to 10 mL and centrifuged; then, 1 mL of the lower layer was diluted to 5 mL with the mobile phase of HPLC. The diluted solution was filtered and the filtrate was subjected to measurement by the HPLC system. Single samples were taken from the upper, middle and lower layers of the dosing suspension.

Duration of treatment / exposure:

- males of the main group and males/females of the recovery group:
14 days before mating, 14 days during mating and 14 days after mating => 42 days in total
- females of the main group:
14 days before mating and up to day 4 of lactation => 42 to 45 days in total

The recovery period of 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

Details on study schedule:

Pre-mating period: 14 days (males + females)
Mating period: 14 days (males + females)
Post-mating period: 14 days (males)
Gestation period: approx. 21 day (to delivery; females)
Lactation period: 4 days (females)

recovery period: 14 days (contol and high dose males/females)

Remarks:

Doses / Concentrations:
1000 mg/kg bw/d
Basis:
actual ingested

Remarks:

Doses / Concentrations:
500 mg/kg bw/d
Basis:
actual ingested

Remarks:

Doses / Concentrations:
250 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

main groups: 12
recovery groups (control and high doae): 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a previous 14 day repeated dose oral toxicity study of Sodium lauryl phosphate in rats (doses: 125, 250, 500 and 1000 mg/kg bw/d) administration of the test item did not produce any effect even at 1000 mg/kg bw/d. Therefore, 1000 mg/kg bw/d was set as the highest dose, and doses of 500 and 250 mg/kg bw/d were derived by dividing by a common factor of 2.

- Post-exposure recovery period in satellite groups:
The recovery period for the males and females of the recovery group (control and high dose group) was 14days after the end of administration, during which the substance was not given to the animals.

Positive control:

none

Parental animals: Observations and examinations:

- 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 h 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 groups, once every week during the pre-mating administration and mating period, and on predetermined days (gestation days 1, 7, 14 and 20, day 4 of lactation) during the gestation period and the lactation period for females of the main group, and once every wee during the administration and recvery period for the animals of the recovery groups.
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 groups, after F1 necropsy on day 4 of lactation (day 42-45 of administration) for the females of the main groups, 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 groups. These tests were performed on 5 animals each per group. In the above observation, tests, and measurements, obeservers were blinded to information such as the dose, and the animals were arranged in a random manner.
(1) Detailed observation of the general condition
Observation of animals in the home cage (posture convulsions, abnormal behavior), observation of the animals while being handled (ease of taking out from the home cage, respoonse to handling, conditions of the fur and skin, eyeballs, secretions from the eyes and nose, visible mucosa, automatic nerve functions), observation of animals in the open field (wakefulness, gait, posture, tremor, convulsion, standing frequency, excrement, stereotyped behavior, abnormal behavior.
(2) Function tests
Auditory response, approach reaction, contact reaction, pain reaction, pupillary reflex, air righting reflex, landing food splay, measurement of the grip strength, measurement of spontaneous motor activity

-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 necopsy for the males of the main groups
days 1, 4, 8, 11, and 14 of the recovery period and on the day of necropsy, in addition to the days of measurements for the males of the main groups, for the males and females of the recovery groups
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 gestation, and days 0 and 4 of lactation for the females of the main groups.

- 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 groups, on days 1, 4, 8, 11 and 14of recovery period, in addtion to the days of measurement for the males of the main groups, for the males and females of the recovery groups, 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 groups.
Food consumption per animal was calculated from the data obtained.

- Vaginal smear
Vaginal smear was collected everyday from all females of the main groups, from day 1 of administration until copulation was confirmed, and subjected to microscopic examination. During the pre-mating period, the vaginal smear profile was classified into proestrus, estrus, metestrus, and anestrus, from which frequency of the estrus and the days from one estrus to the next estrus (estrus cycle) were calculated. During the mating period, vaginal smears were examined to confirm the absence/presence of sperms.

- Observation of mother animals
All females 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 from gestation day 21 to the morning of gestation day 25. The gestation period and delivery rate were calculated as follows:
Gestation period (days) = Day 0 of lactation - day 0 of gestation
Delivery rate (%) = (number of females giving birth to live pups/number of pregnant females) x 100
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 and observed for lactating behavior using pup gathering, nest building, and breastfeeding as incidences. In 5 females of each group the number of corpora lutea and number of implantation sites in these animals were calculated, and the implantation rate was calculated using the following equation:
Implantation rate (%) = (number of implantation sites/number of corpora lutea) x 100

- Urinalysis
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. Four-hour 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:pH, protein, Ketone bodies, Glucose, occult blood, Bilirubin, Urobilinogen, color, sediment, urine volume (4-hour and 20-hour), osmotic pressure and amount of water intake (24.hour volume).

- Haematology:
On the day after the last administration and on the last day of the recovery period, 5 males and 5 females of each group were, after having been denied access to food overnight from the previous day (approx. 16-20 h), laparotomised under ether anesthesia. Blood was colected from the abdominal aorta and the following parameters measured: Prothrombin time, activated partial Thromboplastin time, and Fibrinogen level were measured using the plasma obtained by centrifugation. Further parameters measured: red blood cell count, Haemoglobin, Haematocrit, maen corpuscular volume, mean corpuscular Haemoglobin, mean corpuscular Haemoglobin concentration, percentage of Reticulocytes, Platelet count, white blood cell cout, differential white blood cell count, Prothrombin time, activated patial Thromboplasin time, Fibrinogen.

- Clinical chemistry
At the same time as the blood sample collection for the haematological tests, blood samples were measured for the following parameters: AST, ALT, LDH, gamma-GTP, Albumin, total Cholesterol, Triglycerides, Phospholipids, total Bilirubin, GLucose, Urea nitrogen, Creatinine, Sodium, Potassium, Chloride, Calcium, inorganic Phosphorus, total protein.

Oestrous cyclicity (parental animals):

- Vaginal smear
Vaginal smear was collected everyday from all females of the main groups, from day 1 of administration until copulation was confirmed, and subjected to microscopic examination. During the pre-mating period, the vaginal smear profile was classified into proestrus, estrus, metestrus, and anestrus, from which frequency of the estrus and the days from one estrus to the next estrus (estrus cycle) were calculated. During the mating period, vaginal smears were examined to confirm the absence/presence of sperms.

Sperm parameters (parental animals):

no data

Litter observations:

- Observation of pups
The number of live and stillborn pups were counted on the day of birth. Live pups were observed for the presence/absence of any external abnormalities, checked for sex, their body weight was measured, and they were allowed to suckle the mother. Pups with external abnormalities were fixed in 10% Formalin solution and stored. The stillbirth rate, live birth rate, external anomaly rate and sex ratio were calculated as follows:
Stillbirth rate (%) = (number of stillborn pups/total number of pups) x 100
Live birth rate (%) = (number of live-born pups/total number of pups) x 100
External anomaly rate (%) = (number of pups with external anomalies/total number of pups) x 100
Sex ratio = (number of male pups)/(number of male + female pups)
Live pups were observed once everyday up to day 4 of lactation, and the viability rate was calculated:
Viability rate of live-born pups (%) = (number of pups alive on day 4 of lactation/number of pups alive on day 0 of lactation) x 100
Dead pups were discarded.
Body weight was measured for individual pups and the mean body weight was calculated seperately for each of the male and female littermates.

Postmortem examinations (parental animals):

- Macroscopy
All animals were subjected to detailed gross pathological examination of the body organs and tissues, including the external surface, head, chest and abdomen. In the females (mother animals), the number of corpora lutea and the number of implatation sites were counted on day 5 of lactation.

- Body weight
The absolute and relative organ weight of 5 males and 5 females of each group was determined: brain, thyroid gland (including parathyroid gland), thymus, heart, liver, spleen, kidneys, adrenals, testis and epididymis (weights from testes and epididymes was taken from all males).

- Histopathology
Specimens obtained from 5 males and 5 females of the control and high-dose group were subjected to microscopic examination. The following organs and tissues were examined: cerebrum, cerebellum, pituitary gland, spinal cord, sciatic nerve, thyroid gland, parathyroid gland, adrenals, thymus, spleen, submandibular lymph nodes, mesenteric lymph nodes, heart, lung, stomach, liver, kidneys, bladder, testis, epididymis, uterus, seminal vesicle, sternum (including bone marrow) and femur (including bone marrow).

Postmortem examinations (offspring):

- Macroscopy
All animals were subjected to detailed gross pathological examination of the body organs and tissues, including the external surface, head, chest and abdomen.

Statistics:

Body weight, food consumption, water intake, number of occurrences of the estrus profile, estrous cycle, days to copulation, gestation period, number of corpora lutea, number of implantation sites, number of live-born pups, open field observations, function tests, grip strength and spontaneous motor activity, quantitative urinaliysis parameters, haematological tests, blood chemistry tests, and organ weight were subjected to the Bartlett test for homoscedasticity.
Data were then subjected to Dunnett's test if homoscedastic, else to a Dunnett-type test.

The implantation rate, stillbirth rate, live birth rate, external anomaly rate, and viability rate of the pups were calculated for each mother animal, and subjected to the Bartlett test for homoscedasticity anad then to the Dunnett test if homoscedastic, else to Dunnett-type test.

As for the copulation rate, conception rate, delivery rate, sex ratio of the pups, auditory response, approach reaction, contact reaction, pain reaction, pupillary reflex, and air righting reflex, the number of animals that copulated, the number of pregnant animals, the number of females that gave birth to live pups, the number of live male pups, the number of live female pups, and the number of animals that showed normal reflexes were calculated for each group and subjected to x(2) test with Yates' continuity correction (2-sided significance level, 0.05 and 0.01). If there were cells with an expected frequency of 5 or less, the data were subjected to Fisher's exact test (2-sided significance level, 0.05 and 0.01).

Reproductive indices:

Copulation rate (%) = (number of pairs that copulated/number of pairs) x 100
Conception rate (%) = (number of pregnant females/number of females that copulated) x 100
Fertility rate (%) = (number of pregnant females/number of males that copulated) x 100
Implantation rate (%) = (number of implantation sites/number of corpora lutea) x 100

Offspring viability indices:

Stillbirth rate (%) = (number of stillborn pups/total number of pups) x 100
Live birth rate (%) = (number of live-born pups/total number of pups) x 100
External anomaly rate (%) = (number of pups with external anomalies/total number of pups) x 100
Sex ratio = (number of male pups)/(number of male + female pups)
Viability rate of live-born pups (%) = (number of pups alive on day 4 of lactation/number pf pups alive on day 0 of lactation) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

effects on stomach

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

1. General condition:
In the main group, one female in the 500 mg/kg bw/d 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/d group showed decreased sponaeous 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.

2. Detailed observation of the general condition, function tests, and measurement of the grip strength and spontaneous motor activity:
- 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/d 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 maingroup or the recovery group. No significant differences were observed in the standing frequency between the control group and any of the treatment groups.

3. Function tests:
Function tests were performed at the end of the administration (week 6 of administration) in the males of the main group, on day 4 of lactation in the females of the main group, and in the last week of administration (week 6 of administration and last week of the recovery period in the males and females of the recovery group.
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.

4. Measurement of the grip strength:
Subsequent to the function tests, the grip strength of the front and hind limbs was measured.
No significant differences were observed between the control group and any of the treatment groups in either the main group or the recovery group.

5. Measurement of spontaneous motor activity:
Subsequent to the measuement of the grip strength, the spontaneous motot activity was 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.

6. Body weight:
After the administration of the test substance, there was no significant difference in the body weight between males and females of the main group. A significantly greater increase in the body weight was observed in the females of the 250 and 1000 mg/kg group during lactation period, but the increase was not dose-related.
In the recovery group, males in the 1000 mg/kg bw/d showed decreased body weight gain during the administration period and decreased body weight during the recovery period. This was caused by the abnormality in 1 out of 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 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.

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

8. Urinalysis:
No abnormailties were observed in the qualitative parameters in any of the animlas in either the main group or in the recovery group.
No significant difference was observed in any of the quantitative parameters between the control group and any of the treatment groups.

9. Hematological tests:
- 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/d 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/d group. However, none of these changes were observed in the 1000 mg/kg bw/d group, suggesting that they were within the range of physiological variations.
Nio significant differences were observed in the male animals between the control group and any of the treamtent groups.
- Tests at the end of the 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 lymphocyes, and significant decrease in the percentage of segmented neutrophils were observed in females of the 1000 mg/kg bw/d 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.

10. Blood chemistry tests:
- Tests at the end of the administation period:
A significant increase in the serum level of ALT was observed in the males of the 1000 mg/kg bw/d group. A significant decrease of the serum level of inorganic phosphorus was observed in the males of the 250 mg/kg bw/d group. However, since the decrease was not dose-related, it was considered 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 femlaes of the 1000 mg/kg bw/d group. However, since no change was observed at the end of the administration period, the increae was considered to be within the range of physiological variations.

11. Organ weights:
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.
- Measurement at the end of the administration period:
Thymus gland: a significant decrease in both the absolute and relative weight eas observed in the males of the 500 mg/kg bw/d group.
Testis: A significant increase in the relative weight was observed in the males of the 500 mg/kg bw/d group.
Heart: A significant increase in the absolute weight was observed in the femlaes of the 500 mg/kg bw/d group.
- Measurements at the end of the recovery period:
- Thyriod gland: A significant decrease in both the absolute and relative weights was observed inthe females of the 1000 mg/kg bw/d group.

12. Necropsy findings:
Adminstration of the test substance had effects on the stomach of animals in the 250 mg/kg bw/d and higher dose groups.
- 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/d groups, respectively. White foci were observed in 1 male of the 500 mg/kg bw/d 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/d groups, respectively. Indentation of the glandular stomach was observed in 1 female of the 500 mg/kg bw/d group.
All the other findings observed in the organs and tissues, as follows, were considered to be incidental, as judged from the frequency of their occurrence and the pathological findings.
Stomach: 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/d groups, respectively.
Kidney: Pyelectasis was observed in 2 and 1 males of the 250 mg/kg bw/d groups, respectively.
Eyeballs: Corneal opacity (unilateral) was observed in 1 female of the 500 mg/kg bw/d 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/d 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 folowing 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/d group.

13. Histopathological findings:
Adminstration of the test substance had effects on the stomach of the animals in the 250 mg/kg bw/d 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/d groups, respectively. Very mild to moderate thickening of the anterior stomach mucosa was observed in 1, 4 and 5 males and 1, 4 and 4 females of the 250, 500 and 1000 mg/kg bw/d 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/d 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/d group.
Kidney: Very mild basophilic tubules were observed in 3 males of the control and 1 male and 1 female in the 1000 mg/kg bw/d group.
Liver: Very mild, minute granulomas were observed in 3 males of the control group and 1 male of the 1000 mg/kg bw/d group.
Lung (including bronchi): Very mild mineral deposits in the arterial walls were observed in the 1 male of the control group and 1 female of the 1000 mg/kg bw/d 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/d group.
Spleen: Very mild to mild extramedullary hematopoesis was observed in 5 females each in the control group and 1000 mg/kg bw/d group.
Stomach: Inclusion cysts were observed in 1 male of the 500 mg/kg bw/d group. Very mild to mild erosions in the glandular stomach were observed in 0, 0, 0, and 1 females on the control group, 250, 500 and 1000 mg/kg bw/d group.
- 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/d group.

14. Estrus cycle:
Therw were no animals that showed abnormal estrus cycles. No significant differences were observed in the mean length of the estrus cycle between the control group and any of the treatment groups.

15. Mating results:
One pair inthe 500 mg7kg bw/d group did not copulate, wheras copulation occurred in all of the other pairs by day 4 after the start of mating, resulting in pregnancy in all of the females that copulated. Thus, there were no significant differences in the days to copulation, copulation time, copulation rate, fertility rate, or conception rate between the control group and any of the treatment groups.

16. Delivery results and delivery/lactating state:
All pregnant females delivered normally from day 21.5 to 23.0 of gestation. After administration of the test substance, there were no significant differences in the delivery rate, gestation period, number of corpora lutea, number of implantation rate, stillbirth rate, number of live-born pups, and live birth rate. Significant increases in the number of corpora lutea and in the live-born pups were observed in the 250 mg/kg bw/d group, but they were not dose-related.
In regard to nursing, no abnormalities were observed in nest building, pup gathering, or lactating behavior in any of the mother animals.

Key result

Dose descriptor:

NOAEL

Effect level:

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

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

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

1. Observation of pups:
No significant differences were observed in the sex ratio, body weight at birth, or external anomaly rate between the control group and any of the treatment groups. Observation for external anomlaies showed kinking of the tail in 1 animal of the 500 mg/kg bw/d group, which was considered to be a spontaneous occurrence as judged from the frequency of occurrence and the type of the anomaly.

2. Viability of the pups:
During the lactation period, death occurred in only 4 pups in the control group and 2 pups in the 1000 mg/kg bw/d group. Thus, there were no significant differences in the viability rate on day 4 of lactation between the control group and any of the treatment groups.

3. Body weight of the pups:
No significant differences were observed in the body weight of the males or females at birth or on day 4 of lactation between the control and any of the treatment groups.

4. Necropsy findings of pups on day 4 of lactation:
Thymic cervical residue was observed in the 1, 1, 0 and 2 male and 2, 1, 0, and 3 femalaes of the control group, 250, 500, and 1000 mg/kg bw/d groups, respectively, however, the occurrences werenot dose-related.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

other: no effects observed

Key result

Reproductive effects observed:

no

Conclusions:

After dosing rats (12 males and 12 females per dose group) with 250, 500 or 1000 mg/kg bw/d in diet for a minimum of 42 days 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/d. No effects on reproductive parameters and pups were obserserved. Therefore, the NOEL (reproductive/developmental) is set at 1000 mg/kg bw/d.

Executive summary:

The substance tested in this OECD 422 study, Phosphoric acid, dodecyl ester, sodium salt, and the substance to be registered, Phosphoric acid, dodecyl ester, potassium salt, differ only in the cation. Both Potassium and Sodium are Alkali metal cations which are comparable in their physico-chemical properties. Furthermore, both cationic species are endogenous. A daily uptake of 2400 mg Sodium (adult) is considered to be safe (please refer to BfR (Federal Institute for Risk Assessment) statement "Gesundheitliche Bewertung des Salzgehalts industriell vorgefertigter Gerichte", August 2011). For Potassium a safe daily uptake of 1000 mg (humans 4 years and older; BfR) was proposed. Based on the before mentioned facts very similar toxicological profiles of Sodium and Postassium are assumed. Therefore, the toxic properties of the test substance, Phosphoric acid, dodecyl ester, sodium salt and the substance to be registered, Phosphoric acid, dodecyl ester, potassium salt are supposed to be driven by the anionic phosphoric acid ester component solely and a read across between both substances is justified.

Sodium lauryl phosphate (Phosphoric acid, dodecyl ester, sodium salt) was administered at 0 (control group), 250, 500 or 1000 mg/kg bw/d 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 and gestation periods, and up to day 4 of lactation (42 to 45 days in total), to investigate repeated-dose, reproductive and developmental toxicity. In the 0 and 1000 mg/kg bw/d group, a 14-day recovery period was allowed after the 42-day administration period to investigate the reversibility of the toxic changes.

- Repeated-dose toxicity:

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.

Histopathological examination at the end of the administration period showed, upon gross observation, indentation of the anterior stomach in the 250 mg/kg bw/d and higher dose groups, and rough mucosa or white foci in the anterior stomach in the 500 mg/kg bw/d and higher dose groups, and upon histological examination, erosions/ulcers of the anterior stomach, thickening of the anterior stomach mucosa, and edema of the submucosal tissue in the 250 mg/kg bw/d and higher dose groups.

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.

- Reproductive and developmental toxicity:

Administration of the test substance had no effect on any of the estrous cycle, days to mating, copulation rate, fertility rate, or conception rate. Similarly, administration of the test substance had no effect on gestation period, delivery rate, number of corpora lutea, number of implatation sites, implantation rate, delivery/nursing behavior, stillbirth rate, number of live pups, or the live birth rate of the mother animals, or on the sex ratio of the littermates. It had also no effect on the findings of observation at birth, necropsy findings postpartum day 4, body weight or viability of the pups.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable with restrictions

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There is strong evidence from structural considerations and a metabolism study performed with Phosphoric acid 2-ethylhexyl ester in rats that Phosphoric acid dodecyl ester is metabolised to 1 -Dodecanol and Phosphate (please refer to chapter 7.1). In urine and feces of rats which were dosed with Phosphoric acid 2 -ethylhexyl ester only inorganic Phosphate was detected. No ester was found (Dekant, W. et al; 2001; Clariant Study Report No. 000942). It was concluded, that Phosphoric acid 2-ethylhexyl ester is completely metabolised into Phosphate and 2 -Ethylhexanol by endogenous enzymes (e.g. esterases) after ingestion. This metabolic pathway is very likely to be also effective in case of Phosphoric acid Dodecyl ester. Phosphoric acid dodecyl ester is supposed to be metabolised to Phosphate and 1 -Dodecanol after entering the organism. Phosphate is known to be an endogenous compound and food additive without reported toxic properties. Therefore, a read across to the second metabolite, 1 -Dodecanol, was conducted in order to estimate the toxicological properties in a combined repeated dose/reproductive toxicity study.
24 rats (12 males and 12 females) were dosed with 1 -Dodecanol in diet in concentrations of 0, 1500, 7500 or 30000 ppm (corresponding to 0, 100, 500 or 2000 mg/kg bw/d) for a period of a minimum of 41 days (males). This study was performed according to OECD 422. No effect was seen on the reproductive and developmental parameters as well as in the macroscopic and histological examination.
A NOEL (reproductive/developmental) was set at 30000 ppm (2000 mg/kg bw/d).

The substance tested in the OECD 422 study described below, Phosphoric acid, dodecyl ester, sodium salt, and the substance to be registered, Phosphoric acid, dodecyl ester, potassium salt, differ only in the cation. Both Potassium and Sodium are Alkali metal cations which are comparable in their physico-chemical properties. Furthermore, both cationic species are endogenous. A daily uptake of 2400 mg Sodium (adult) is considered to be safe (please refer to BfR (Federal Institute for Risk Assessment) statement "Gesundheitliche Bewertung des Salzgehalts industriell vorgefertigter Gerichte", August 2011). For Potassium a safe daily uptake of 1000 mg (humans 4 years and older; BfR) was proposed. Based on the before mentioned facts very similar toxicological profiles of Sodium and Postassium are assumed. Therefore, the toxic properties of the test substance, Phosphoric acid, dodecyl ester, sodium salt and the substance to be registered, Phosphoric acid, dodecyl ester, potassium salt are supposed to be driven by the anionic phosphoric acid ester component solely and a read across between both substances is justified.
Sodium lauryl phosphate (Phosphoric acid, dodecyl ester, sodium salt) was administered at 0 (control group), 250, 500 or 1000 mg/kg bw/d 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 and gestation periods, and up to day 4 of lactation (42 to 45 days in total), to investigate repeated-dose, reproductive and developmental toxicity. In the 0 and 1000 mg/kg bw/d group, a 14-day recovery period was allowed after the 42-day administration period to investigate the reversibility of the toxic changes.

- Reproductive and developmental toxicity:

Administration of the test substance had no effect on any of the estrous cycle, days to mating, copulation rate, fertility rate, or conception rate. Similarly, administration of the test substance had no effect on gestation period, delivery rate, number of corpora lutea, number of implatation sites, implantation rate, delivery/nursing behavior, stillbirth rate, number of live pups, or the live birth rate of the mother animals, or on the sex ratio of the littermates. It had also no effect on the findings of observation at birth, necropsy findings postpartum day 4, body weight or viability of the pups.
Therefore the NOAEL (reproductive/developmental) was considered to be 1000 mg/kg bw/d.

Short description of key information:
Sodium lauryl phosphate (Phosphoric acid, dodecyl ester, sodium salt) was administered at 0 (control group), 250, 500 or 1000 mg/kg bw/d 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 and gestation periods, and up to day 4 of lactation (42 to 45 days in total), to investigate repeated-dose, reproductive and developmental toxicity. In the 0 and 1000 mg/kg bw/d group, a 14-day recovery period was allowed after the 42-day administration period to investigate the reversibility of the toxic changes.
- Reproductive and developmental toxicity:
Administration of the test substance had no effect on any of the estrous cycle, days to mating, copulation rate, fertility rate, or conception rate. Similarly, administration of the test substance had no effect on gestation period, delivery rate, number of corpora lutea, number of implatation sites, implantation rate, delivery/nursing behavior, stillbirth rate, number of live pups, or the live birth rate of the mother animals, or on the sex ratio of the littermates. It had also no effect on the findings of observation at birth, necropsy findings postpartum day 4, body weight or viability of the pups.
Therefore the NOAEL (reproductive/developmental) was considered to be 1000 mg/kg bw/d.

Justification for selection of Effect on fertility via oral route:
The OECD 422 study performed with Phosphoric acid, dodecyl ester, sodium salt was selected as the most relevant available reproductive toxicity screening study due to its reliability and since it provides the most sensitive NOEL.
The substance tested in this OECD 422 study, Phosphoric acid, dodecyl ester, sodium salt, and the substance to be registered, Phosphoric acid, dodecyl ester, potassium salt, differ only in the cation. Both Potassium and Sodium are Alkali metal cations which are comparable in their physico-chemical properties. Furthermore, both cationic species are endogenous. A daily uptake of 2400 mg Sodium (adult) is considered to be safe (please refer to BfR (Federal Institute for Risk Assessment) statement "Gesundheitliche Bewertung des Salzgehalts industriell vorgefertigter Gerichte", August 2011). For Potassium a safe daily uptake of 1000 mg (humans 4 years and older; BfR) was proposed. Based on the before mentioned facts very similar toxicological profiles of Sodium and Postassium are assumed. Therefore, the toxic properties of the test substance, Phosphoric acid, dodecyl ester, sodium salt and the substance to be registered, Phosphoric acid, dodecyl ester, potassium salt are supposed to be driven by the anionic phosphoric acid ester component solely and a read across between both substances is justified.

Justification for selection of Effect on fertility via inhalation route:
In accordance with column 2 of REACH Annexes VIII and IX, the reproductive toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the inhalation route because exposure of human via inhalation, especially in a higher extent than via oral application as performed in the animal studies, is considered unlikely taking into account the vapour pressure of the substance and the physical form.

Justification for selection of Effect on fertility via dermal route:
In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the dermal route because
- no systemic effects or other evidence of absorption were observed in skin and eye irritation studies in rabbits as well as in the skin sensitization test in guinea pigs
- due its irritant properties only local skin effects are expected to occur; however, since the edema/erythema were reversible these are considered to have no impact on the absorption of the test substance via skin. Due to the combination of its polar (ionic) character and the long extent of the alcoholic chain it is unlikely that higher amounts than tested in a oral reproductive toxicity study will be systemically available via the skin barrier.

Effects on developmental toxicity

Description of key information

Based on the findings of an OECD 414 study it is concluded that NOAEL for

- maternal toxicity is 50 mg/kg bw/d due to treatment-related significant reduction in maternal body weight and food consumption at 250

and 1000 mg/kg bw/d along with significant reduction in corrected weight gain at 1000 mg/kg bw/d

- fetal development is 250 mg/kg bw/d due to treamtment-related significant reduction in fetal weights at 1000 mg/kg bw/d

- teratogenicity is 1000 mg/kg bw/d as there were no signs of teratogenicity in any of the tested dose levels up to the highest dose of 1000 mg/kg bw/d.

• Maternal toxicity is 50 mg/kg/day due to treatment-related significant reduction in maternal body weight and food consumption at 250 and 1000 mg/kg/day along with significant reduction in corrected body weight gain at 1000 mg/kg/day,  

• Fetal developmental toxicity is 250 mg/kg/day due to treatment –related significant reduction in fetal weights at 1000 mg/kg/day,

• Teratogenicity is 1000 mg/kg/day as there were no signs of teratogenicity in any of the tested dose levels up to the high dose of 1000 mg/kg/day

in Wistar rats when Leomin PN pa was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Species: Rat (Rattus norvegicus)

Strain: Wistar rats – Han Tac: WH rats

Source: Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311,Gajwel,Mandal, Medak District, Andhra Pradesh(A.P)

Justification for selection of species: Rat is a standard laboratory rodent species used for prenatal developmental toxicity assessment and also preferred by various regulatory authorities for toxicity assessment. The Wistar rat was selected due to the large amount of background data accumulated for this strain.

No. of groups: 4 groups
• Vehicle control : 0 mg/kg/day (G1)
• Low dose: 50 mg/kg/day (G2)
• Mid dose: 250 mg/kg/day (G3)
• High dose: 1000 mg/kg/day (G4)

No. of Day 0 mated females (sperm positive in vaginal smears)/ group: 24 /group, Total = 96 females

Age at the start of treatment: 14 to 15 weeks

Mean Body weight (g) and body weight range of Day 0 mated females: The weight variation did not exceed ± 20 % of the mean body weight in each group.

Mean body weight(g) / Body weight range(g)
G1 : 227.336 ± 16.88 / 202.16 to 257.40
G2 : 227.491 ± 16.67 / 202.87 to 259.32
G3 : 227.466 ± 16.78 / 203.12 to 256.62
G4 : 227.158 ± 16.66 / 202.69 to 256.17

Identification: Temporary Identification; Before mating, the rats were allotted temporary identification numbers and were identified by crystal violet body marking.

Permanent Identification; After mating, the rats were identified by the last three digits of the permanent accession numbers written on the tail. The permanent accession number was included on the cage cards.

Acclimatization: After physical examination for good health and suitability for the study, the rats were acclimatized for 12 days. During the acclimatization period, all rats were observed once daily. Females used in this study were nulliparous and non-pregnant.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analyzed for active ingredient concentration (a.i.) and homogeneity at the initiation of treatment and at termination of treatment period.

The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another was kept as back up set which was stored in the experimental room depending upon the obtained stability results. For each set, two replicate samples were drawn from top, middle and bottom layers of each dose formulation. In case of control, two replicate samples from middle layer were drawn. Dose formulations were sent to Analytical R&D Department of Advinus Therapeutics Limited for formulation analysis to determine the concentration and homogeneity of the test item in dose formulations.

The dose formulations collected at initiation of treatment (23 May 2016) and at termination of treatment (06 June 2016) were initially analyzed by adopting an in-house developed and validated method under Advinus
Study No. G11296.

As per sponsor’s suggestion, in addition, on each day of treatment, a back-up sample (a composite sample of approximately 50 mL) from each dose formulation was stored at frozen conditions at -10 to -20°C for sampling at a later date. Later another analytical method was provided by sponsor and the revised analytical method was also validated in-house, after suitable modifications. The stored formulation samples corresponding to those collected on 23 May 2016 and 06 June 2016 are being analyzed using the revised analytical method.

Formulations were considered acceptable when the overall mean result (calculated using all the 6 replicate values) of all the layers and mean of each layers was within ± 15.0 % of the claimed concentration and the relative standard deviation (% RSD, calculated using all the 6 replicate values) of assay of top, middle and bottom layers was equal to or less than 10.0 %.

The unused back up samples (analyzed initially) were discarded as the results of first set of analysis were within the acceptable limits.

Details on mating procedure:

- Impregnation procedure: [cohoused]

- If cohoused:
- M/F ratio per cage: [1:1]
- Length of cohabitation: During the mating period, female rats were cohabited with males in a 1:1 ratio. When sperm was detected in a vaginal smear or vaginal plug was observed in the morning, the animal was considered to be mated. This day was considered as day 0 of gestation.

The mated female rats obtained each day were assigned to the treatment groups and vehicle control groups by body weight stratification. This procedure was continued till the required numbers of Day 0 mated females were obtained (24 per group).

- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy

Duration of treatment / exposure:

The dose formulations of Leomin PN pa was administered orally by gavage using disposable plastic syringe attached with a metal feeding/intubation cannula to rats of low dose (G2), mid dose (G3) and high dose (G4) groups once daily from GD 5 to GD 19 of presumed gestation, at approximately the same time each day (varying by± 2 hours).

Frequency of treatment:

Gestation Day 5 to 19

Duration of test:

15 Days

Dose / conc.:

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

Remarks:

Doses / Concentrations:
Group 1
Basis:
nominal conc.
0 mg/mL

Dose / conc.:

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

Remarks:

Doses / Concentrations:
Group 2
Basis:
nominal conc.
5 mg/mL

Dose / conc.:

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

Remarks:

Doses / Concentrations:
Group 3
Basis:
nominal conc.
25 mg/mL

Dose / conc.:

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

Remarks:

Doses / Concentrations:
Group 4
Basis:
nominal conc.
100 mg/mL

No. of animals per sex per dose:

No. of Day 0 mated females (sperm positive in vaginal smears)/ group: 24 /group, Total = 96 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: As per the Sponsor’s suggestion, based on the results of a dose range finding study the following doses were selected for main embryo-fetal developmental toxicity study with Leomin PN pa in Wistar rats by oral route:

• G1 - Vehicle control - 0 mg/kg/day
• G2 - Low dose - 50 mg/kg/day
• G3 - Mid dose - 250 mg/kg/day
• G4 - High dose - 1000 mg/kg/day

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The rats were subjected to physical examination after mating i.e., on day ‘0’, of gestation and at weekly interval during the presumed gestation period and findings were recorded.

- Cage side observations checked in table 2 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations for clinical signs were performed twice a day - pre dose and post dose (within 1-2 hours of administration) during treatment days and once on non-treatment days.

BODY WEIGHT: Yes /
- Time schedule for examinations: All females included in the study were weighed on gestation days 0, 3, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
About 200 g (food input) was provided on Day 0. The food output was recorded and replenished to about 200 g on Days 3, 5, 8, 11, 14 and 17 and food output on Day 20 of presumed gestation was recorded. There was no spillage of food.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:

OTHER:

- Dam examination
The following maternal data were recorded.
a. Pregnancy status
b. Gravid uterine weight
c. No. of corpora lutea
d. No. of implantation sites
e. No. of early resorptions
f. No. of late resorptions

- Fetal examination
The following litter data was recorded:
a. Total number of fetuses
b. Number of live fetuses
c. Number of dead fetuses
d. Individual fetal body weight (g)
e. Fetus sex

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: Yes: [half per litter]

Statistics:

The following statistical tests were used:

The data on maternal body weight, body weight change, gravid uterine weight, corrected body weight gain, maternal food consumption, number of corpora lutea , number of implantations, total number of fetuses, male and female fetus number and weight were analyzed using ANOVA model, after testing for homogeneity for intra group variance using Levene’s test.

Incidence of pre-implantation loss, post implantation loss, Number of early, late and total resorptions were analyzed using Kruskal Wallis test.

Overall percentage of normal and minor external, visceral and skeletal malformations, Sex ratio and number of dams with any resorptions were analyzed using 2 X 2 Contingency Table.

Statistically significant differences (p < 0.05), indicated by the aforementioned tests were designated by symbol ‘*’ throughout the report.

Historical control data:

Enclosed in Annexure 7

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There was treatment-related significant reduction in maternal body weights and food consumption at 250 and 1000 mg/kg/day as compared to vehicle control group along with significant reduction in corrected body weight gain at 1000 mg/kg/day indicating maternal toxicity.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

ca. 250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs of teratogenicity in any of the tested doses.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Developmental effects observed:

not specified

Conclusions:

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

• Maternal toxicity is 50 mg/kg/day due to treatment-related significant reduction in maternal body weight and food consumption at 250 and 1000 mg/kg/day along with significant reduction in corrected body weight gain at 1000 mg/kg/day,

• Fetal developmental toxicity is 250 mg/kg/day due to treatment –related significant reduction in fetal weights at 1000 mg/kg/day,

• Teratogenicity is 1000 mg/kg/day as there were no signs of teratogenicity in any of the tested dose levels up to the high dose of 1000 mg/kg/day

in Wistar rats when Leomin PN pa was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.

Executive summary:

The objective of this study was to evaluate theembryo-fetal developmentaltoxicity of test item Leomin PN pawhen administered to pregnant Wistar rats by oral route during gestation days (GD) 5 to GD19.The results of this study helped to establish the No Observed Adverse Effect Level (NOAEL) of the test item.

 

In this study, each group (G1, G2, G3 and G4) consisted of 24 presumed pregnant Wistar rats (gestation day 0). Day `0' of gestation for each individual female rat in the study was considered as the day on which vaginal smear was found sperm positive.

 

The test item,Leomin PN pawas dissolved in vehicle [Milli-Q water] and administered orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 50, 250 and 1000 mg/kg/day for low (G2), mid (G3) and high(G4) dose group rats, respectively. The rats in the vehicle control (G1) group received the vehicle alone. A constant dose volume of 10 mL/kg body weight was administered to all groups.

 

The dose formulation solutions were analyzed for active ingredient concentrations at the initiation and termination of treatment. The results of analysis of formulations revealed that the analyzed concentrations were within the acceptable limits.

 

The mated females were observed twice daily for clinical signs, mortality and morbidity. Body weights were recorded on GD0, 3, 5, 8, 11, 14, 17 and 20. About 200 g (food input) was provided on Day ‘0’. The food left over was recorded and replenished to about 200 g on GD 3, 5, 8, 11, 14 and 17. The food left over was also recorded on Day 20 of presumed gestation. The intermittent body weight gain and food intake was calculated and presented for rats found pregnant at caesarean section.

 

Caesarean section was performed for all the rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the dams were removed (by laparotomy) and the contents were examined.The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. The number of corpora lutea was counted on each ovary. All the fetuses were sexed, weighed and examined for external malformations.Approximately half the number of fetuses from each dam was examined for visceral malformations and the remaining half was evaluated for skeletal malformations.

 

Results of the study are presented below:

 

•        There were no mortalities and clinical signs at all the doses tested.
•        There was treatment-related significant reduction in maternal body weights and food consumption at 250 and 1000 mg/kg/day as compared to vehicle control group along with significant reduction in corrected body weight gain at 1000 mg/kg/day indicating maternal toxicity.
•        Gross necropsy findings of small thymus and spleen along with scanty mucoid ingesta in stomach and intestines were observed at 1000 mg/kg/day which were considered as treatment-related findings.
•    There was treatment-related significant reduction in fetal weights of males, females and total fetuses at 1000 mg/kg/day indicating developmental toxicity which is likely to be associated with secondary effects of maternal toxicity at this dose.
•        The other maternal and litter data parameters were comparable to vehicle control group up to the high dose of 1000 mg/kg/day.
•        Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs ofteratogenicity in any of the tested doses.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

•   Maternal toxicity is 50 mg/kg/day due to treatment-related significant reduction in maternal body weight and food consumption at 250 and 1000 mg/kg/day along with significant reduction in corrected body weight gain at 1000 mg/kg/day,
•  Fetal developmental toxicity is 250 mg/kg/day due to treatment –related significant reduction in fetal weights at 1000 mg/kg/day,
•  Teratogenicity is 1000 mg/kg/day as there were no signs of teratogenicity in any of the tested dose levels up to the high dose of 1000 mg/kg/day in Wistar rats whenLeomin PN pawas administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restriction

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Description of key information

no further data mandatory

Additional information

no further data mandatory

Justification for classification or non-classification

There is no evidence to suggest that a classification for reproductive toxicity is appropriate.

With reference to the OECD 422 studies performed with the structural surrogates, 1-Dodecanol and Phosphoric acid, dodecyl ester, sodium salt and the lack of teratogenic effects in an OECD 414 study performed with phosphoric acid, dodecyl ester, potassium salt , it is concluded that the registration substance is not subject to classification and labelling according Regulation 1272/2008/EC regarding reproductive toxicity.