[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Based on the available weight of evidence information from studies for substances representative of the main constituents, the test substance is not considered to pose reproductive or development concern. Considering that the esters make up more than 80% of the composition, the NOAEL based on the study with esters can be regarded as representative of the test substance. Therefore, a NOAEL of 233.4 mg/kg bw/day, which also corresponds to the lowest NOAEL, determined in a study with Isodecyl oleate has been considered further for hazard/risk assessment.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

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.

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

Justification for study design:

Investigation of the repeated-dose, reproductive and developmental toxicity of the test substance as well as the reversibility of the potential toxic changes.

Species:

rat

Strain:

other: Crj:CD(SD)IGS

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 d. During this period, the general condition, body weight, and estrous cycle (for 9 d 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 h 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 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.

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 d. 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.

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 d 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 h at room temperature after storage in light-protected containers in a cold dark place (refrigerator) for 7 d.
- 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 d before mating, 14 d during the mating period, and 14 d after the mating period, that is, 42 d in total, for the males of the main group and the males and females of the recovery group, and 14 d before mating and up to Day 4 of lactation throughout the mating and gestation periods, that is, 42 to 45 d in total, for the females of the main group. The recovery period for the males and females of the recovery group was 14 d 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 d 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.

Positive control:

No

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 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 gestation, 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 h 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. 4 h urine specimens were collected under fasting conditions of the animals with free access to water, followed by 20 h 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 h 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 h and 20 h 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.

Oestrous cyclicity (parental animals):

Vaginal smear was collected everyday from all females of the main group, 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 (estrous cycle) were calculated. During the mating period, vaginal smears were examined to confirm the presence/absence of sperms.

Sperm parameters (parental animals):

-

Litter observations:

- The number of live pups and the number of stillborn pups were counted on the day of birth. Live pups were observed once everyday up to Day 4 of lactation. Live pups were also 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 vol% formalin solution and stored.
- External anomaly rate (%) = (number of pups with external anomalies/total number of pups) × 100
- Sex ratio = (number of male pups)/(number of male plus female pups)

Postmortem examinations (parental animals):

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 examination 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 stored 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))

Postmortem examinations (offspring):

After the body weight was measured on Day 4 of lactation, all the pups were exsanguinated to death under ether anesthesia and necropsied to examine for the presence/absence of abnormalities in the organs and tissues, including the head, chest, and abdomen. Body weight was measured for individual pups and the mean body weight was calculated separately for each of the male and female littermates

Statistics:

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

Reproductive indices:

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

Offspring viability indices:

Stillbirth rate (%) = (number of stillborn pups/total number of pups) × 100
Live birth rate (%) = (number of live-born pups/total number of pups) × 100
Viability rate of live-born pups (%) = (number of pups alive on day 4 of lactation/number of pups alive on day 0 of lactation) × 100

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.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- 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.

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 Days 2 and 4 of lactation in the females of the 250 mg/kg bw 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:

no effects observed

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:

no effects observed

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

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 bw group. 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

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no animals that showed abnormal estrous cycles. No significant differences were observed in the mean length of the estrous cycle between the control group and any of the treatment groups.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- Mating results. One pair in the 500 mg/kg bw group did not copulate, whereas 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 rate, fertility rate, or conception rate between the control group and any of the treatment groups.
- 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 difference in the delivery rate, gestation period, number of corpora lutea, number of implantation sites, 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 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:

NOEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no significant systemic adverse effects

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed on reproductive parameters

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

During the lactation period, death occurred in only 4 pups in the control group and 2 pups in the 1000 mg/kg bw 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.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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 group and any of the treatment groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio: no effects

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Thymic cervical residue was observed in the 1, 1, 0 and 2 males and 3, 1, 0 and 3 females of the control group, 250, 500, and 1000 mg/kg bw groups, respectively, however, the occurrences were not dose-related.
- No significant differences in external anomalies rate were observed. A kinking of the tail in 1 animal of the 500 mg/kg bw group was recorded, but was considered to be a spontaneous occurrence as judged from the frequency of occurrence and the type of the anomaly.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Repeated-dose toxicity:

Administration of the test substance had no effect on any of the following: detailed observation of the general condition, results of function tests, grip strength, spontaneous motor activity, body weight, food consumption, or the results of urinalysis (including water intake), or hematological tests. In regard to the general condition, decreased spontaneous motility, wheezing, and decreased body weight were observed in 1 male of the 1000 mg/kg bw group in the recovery group. Gross examination showed rough mucosa in the anterior stomach and expansion of the digestive tract from the stomach to the colon due to gas accumulation, and histological examination showed thickening of the gastric mucosa. Such findings were observed in only 1 animal. Body weight in this animal showed a similar increase as that observed in the control group until the above symptoms manifested themselves on Day 37 of administration, but continuously decreased after the manifestation of these symptoms, from which these symptoms were judged to be due to inadvertent administration of the test substance into the upper respiratory tract. In regard to the blood chemistry tests, a significant increase of the serum ALT was observed in the males of the 1000 mg/kg bw group at the end of the administration period. Since the findings were very mild and within the range of physiological variations and not accompanied by histopathological abnormalities, it was judged that these findings were not caused by the test substance. In the pathological examination performed at the end of the administration period, gross observation showed indentation of the anterior stomach in the animals of the 250 mg/kg bw and higher dose groups, and rough mucosa and/or white foci in the anterior stomach in the animals of the 500 mg/kg bw and higher dose groups, and histological observation showed erosions/ulcers of the anterior stomach, thickening of the anterior stomach mucosa, and edema in the submucosal tissue, suggesting the irritability of the test substance.

Conclusions:

Based on the results of the read across study, the systemic and reproductive rat NOAELs (P0 and F1 generation) of the read across substance, were both determined 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 test substance was administered at 0 (control group), 250, 500 or 1000 mg/kg bw to male Sprague-Dawley SPF rats for 14 d 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. No test substance-related effects were observed regarding clinical signs, detailed clinical findings, function tests, grip strength, amount of spontaneous movement, body weights, food consumption, urinalysis (including water intake), and haematology or blood chemistry parameters. Gross pathological examination at the end of the administration period revealed recessed areas in the forestomach at 250 mg/kg bw and above and rough mucosa or white foci at 500 mg/kg bw and above, and erosion/ulceration, mucosal thickening and submucosal edema at 250 mg/kg bw and above on histopathological examination. In the light of the absence of a forestomach in humans, observed effects on this tissue were of questionable relevance with reference to the extrapolation of the toxic properties of a test substance in humans. Further, administration of the test substance did not have any effect on the oestrous cycle, days to copulation, copulation rate, fertility rate, or conception rate. Similarly, administration of the test substance did not have any effect on the delivery rate, gestation period, number of corpora lutea, number of implantation sites, implantation rate, stillbirth rate, number of live-born pups, live-birth rate in the mother animals, or on the sex ratio of the littermates. No abnormalities were observed in the lactating behaviour during the lactation period either. These results suggest that administration of the test substance even at 1000 mg/kg bw had no effect on the reproductive function, such as that shown by the copulation rate, of the males or females, or in the fertility rate, conception rate, or on the gestation maintenance, delivery, or lactating behaviour in the mother animals. Pups showed no changes caused by the administration of the test substance regarding the observation at birth, necropsy findings on Day 4 of lactation, body weight, or viability rate, which suggested that administration of the test substance even at 1000 mg/kg bw had no effect on the development. Under the study conditions, the systemic and reproductive rat NOAELs (P0 and F1 generation) of the read across substance, were both determined to be 1000 mg/kg bw/day (BRC, 2005). Based on the results of the read across study, similar effect levels can be considered for the constituent PSE.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

From November 01, 2012 to December 13, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification.

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)

Version / remarks:

(22 Mar 1996)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Behoerde fuer Soziales, Familie, Gesundheit und Verbraucherschutz; Hamburg, Germany

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services Germany GmbH, Sulzfeld, Germany
- Age at study initiation: males: 50 days; females: 60 days
- Mean weight at study initiation: males: 248.8 to 298.7 g; females: 195.2 to 228.1 g
- Fasting period before study: no
- Housing: single housing in MAKROLON cages (type III plus)
- Diet: commercial ssniff R-Z V1324 (ssniff Spezialdiäten GmbH, Soest, Germany); ad libitum
- Water: tap water; ad libitum
(Analyses of diet and water was performed.)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE
- Concentration in vehicle: 50, 150 and 500 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1 male and 1 female animal were placed in one cage during the dark period
- Length of cohabitation: The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 2 weeks of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged singly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle mixtures samples were taken at the following time points and stored at ≤ -20°C until analysis:
Start of treatment period; immediately after preparation of the test item-vehicle mixtures; 8 and 24 hours after storage of the test item preparations at room temperature; end of treatment period; during treatment with the test item always before administration to the last animal of the dose level group
The following parameters were determined: linearity, accuracy, precision, sensitivity, specificity, stability at +2°C to +8°C or -20°C (0, 24, 72 and 168 hours)

Duration of treatment / exposure:

Males: The daily administration of the test item was started two weeks before mating and lasted until test day 35, which was one day before sacrifice.
Females: The daily administration of the test item was started two weeks before mating and continued to at least day 3 of lactation.
Maximum: 56 days of treatment

Frequency of treatment:

once daily; 7 days/week

Dose / conc.:

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

Dose / conc.:

300 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:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a 14-day range-finding study (Leuschner, 2012)

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before and after dosing
- Cage side observations checked: skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns

MORTALITY AND CLINICAL SIGNS
- Time schedule: at least once daily (the frequency was increased when signs of toxicity were observed); deaths were recorded twice daily (animals which died or were sacrificed during the study were necropsied as soon as possible after exitus)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow within-subject comparisons) and once a week thereafter
- Paramters: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern); changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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: No data

For further systemic effects (water intake, haematology, clinical chemistry, neurobehaviour), see "Repeated dose toxicity: oral" (chapter 7.5.1)

OTHER
Reproduction paramters: number of pregnant females, pre-coital time, gestation length

Sperm parameters (parental animals):

Parameters examined in P male parental generation:
testis weight, epididymis weight, and qualitative sperm staging

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of all pups/litter

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: litter weight, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [males were sacrificed on day 36]
- Maternal animals: All surviving animals [females were sacrifices on day 4 post-partum or shorty thereafter]

GROSS PATHOLOGY: Yes
- Organ weights: epididymes and testicles (all males); adrenal gland, brain, heart, kidney, liver, spleen, thymus (5/sex/dose)
- Fixation: epididymis, gross lesions, mammary gland, ovary, prostate, seminal vesicle, testicle, uterus (incl. cervix and oviducts), vagina (all animals); adrenal gland, bone marrow (os femoris), brain (cerebrum, cerebellum, brain stem), heart (left and right ventricle, septum), intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method), intestine, large (colon, rectum), kidney and ureter, liver, lungs (with mainstem bronchi and bronchioles), preserved by inflation with fixative and then immersion, lymph node (1 cervical, 1 mesenteric), nerve (sciatic), oesophagus, spinal cord (3 sections), spleen, stomach, thyroid (incl. parathyroids), thymus, tissue masses or tumours (incl. regional lymph nodes), tongue (incl. base), trachea (incl. larynx), urinary bladder (5/sex/dose)
HISTOPATHOLOGY: Yes, all organs that were included for fixation (5/sex of control and high dose group)

Postmortem examinations (offspring):

SACRIFICE
- The surviving F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations]
Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
not performed

Statistics:

STUDENT's t-test (p ≤ 0.01): all numerical functional tests
Multiple t-test based on DUNNETT (p ≤ 0.05 and p ≤ 0.01): body weight, food consumption, haematology, clinical chemistry, absolute and relative organ weights
For all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group. In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance was p ≤ 0.01.

Reproductive indices:

For each group the gestation index was determined:
- Fertility Index female [%] = Number of pregnant rats/Number of females used x 100
- Gestation Index [%] = Number of litters with live pups/Number of pregnant rats x 100

Offspring viability indices:

For each litter and group the following indices were determined:
- Birth Index [%] = Total number of pups born (live + dead)/Number of implantation scars x 100
- Live Birth Index [%] = Number of pups born alive on day 0/1 Total number born (live + dead) x 100
- Viability Index [%] = Number of pups alive on day 4/Number of pups live on day 0/1 x 100
- Pre-implantation loss [%] = Corpora lutea - implantations/Corpora lutea x 100
- Post-implantation loss [%] = Implantations - number of pups born alive/Implantations x 100

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slight reduction in body weight and food consumption of the high dose females

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slight reduction in body weight and food consumption of the high dose females

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

increase in post implantation loss, decrease in birth index, increase in the number of stillbirths (all high dose group)

CLINICAL SIGNS AND MORTALITY
Piloerection was seen in 1 female of the high dose group at on day 2-4 of lactation.

BODY WEIGHT AND WEIGHT GAIN AND FOOD CONSUMPTION
Reduction in body weight (-9.7%) in high dose females during lactation period. Reduction in food intake (-21.7%) in high dose females during gestation/lactation.

ORGAN WEIGHTS
All effects observed (slight increase in absolute and relative liver weight in males) were still within the historical control data of the laboratory and thus not of toxicological relevance.

GROSS PATHOLOGY
No effects observed.

HISTOPATHOLOGY
No effects observed.

REPRODUCTION
The qualitative sperm staging revealed no test item-related specific spermatogenic changes in the male animals from the high dose group (1000 mg/kg b.w./day).
- Pre-coital time: No effects observed
- Gestation length: No effects observed
- Reproduction parameters of the dams: statistically significant increase in post implantation loss, non-statistically significant decrease in birth index, elevated number of stillbirths, leading to a statistically significant reduction in the live birth index.
No effects were found for the fertility index, the gestation index and the preimplantation loss.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: adverse effects on body weight and body weight gain and food consumption at 1000 mg/kg bw/d

Remarks on result:

other: (i.e., equivalent to 233.4 mg a.i./kg bw/day)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: (i.e., equivalent to 778 mg a.i./kg bw/day)

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: an increase in post-implantation loss, a decrease in the birth index and a decrease in the live birth index at 1000 mg/kg bw/d

Remarks on result:

other: (i.e., equivalent to 233.4 mg a.i./kg bw/day)

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no spermatogenic changes

Remarks on result:

other: (i.e., equivalent to 778 mg a.i./kg bw/day)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

reduced viability (high dose group)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced litter weight of the high dose female offspring

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
A test item-related decrease in the viability index was noted in the high dose group (1000 mg/kg bw/day) (71.3 % in the high dose group vs. 98.7% in the control group). The high number of dead pups in the high dose group was due to 2 dams with no surving pups on lactation day 4 (deaths partly due to cannibalization). The total litter loss in 2 of 7 dams (28.6%) was considered as test item related.

LITTER WEIGHT
A non-statistically significant reduction in mean litter weight on lactation day 1 by 17.2% was noted in the high dose group (high dose group), which is regarded to be test item-related. Total litter weight was also reduced by 24.4% in comparison to controls.

GROSS PATHOLOGY (OFFSPRING)
No effects observed.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects on litter weight and viability of the offspring at 1000 mg/kg bw/day.

Remarks on result:

other: (i.e., equivalent to 233.4 mg a.i./kg bw/day)

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

no

Relevant for humans:

not specified

Individual body weights (g) of females during the pre-mating and lactation period.

Control group	Day(s) relative to start
Animal No.	1	8	15
11	196	209	212
12	217	228	243
13	210	220	213
14	217	234	244
15	3211	215	232
16	195	208	197
17	205	224	239
18	212	238	233
19	224	236	259
20	200	218	214
Mean	209	223	229
SD	9	10	19

1000 mg/kg bw/d	Day(s) relative to start
	1	8	15
71	200	210	225
72	210	231	234
73	206	234	247
74	210	222	235
75	194	219	218
76	218	243	223
77	214	230	227
78	222	225	210
79	198	200	201
80	203	223	231
Mean	207	224	225
SD	9	12	13

Control group	Day(s) relative to littering
Animal No.	1	4
11	286	309
12	323	330
13	325	311
14	331	327
15	311	322
16	282	302
17	309	327
18	312	328
19	315	331
20	300	329
Mean	309	322
SD	16	10

1000 mg/kg bw/d	Day(s) relative to littering
Animal No.	1	4
71	270	281
72	339	301
73	289	309
75	289	317
77	253	247
78	280	271
79	290	296
80	293	308
Mean	288	291
SD	24	23

Relative food consumption (mg) of females between day 1 and 4 of lactation

Control group		1000 mg/kg bw/d
Animal No.
11	122	71	115
12	91	72	96
13	105	73	63
14	107	75	110
15	106	77	30
16	121	78	63
17	114	79	98
18	100	80	110
19	101
20	126
Mean	109	Mean	86
SD	11	SD	30

Viability index [%], day 1 to 4 of lactation

Control group		1000 mg/kg bw/d
Animal No.
11	93	71	93
12	100	72	100
13	100	73	0
14	93	74	not pregnant
15	100	75	100
16	100	76	not pregnant
17	100	77	0
18	93	78	no viable pubs
19	89	79	100
20	100	80	100
Mean	96	Mean	70
SD	4	SD	48

Summary of Live Birth Index, Pre-implantation loss, and Post-implantation loss in female animals

	Control group	100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
Live Birth Index
Mean	100	100	100	86
SD	0	0	0	35
Total %^1	100	100	100	91
Pre-implantation loss
Mean	2.4	18.3	0.6	8.1
SD	3.1	26.0	2.0	9.8
Total %^2	2.5	20.3**	0.7	9.1*
Post-implantation loss
Mean	7.6	9.0	7.5	20.8
SD	10.5	14.8	8.8	32.9
Total %^3	7.6	10.2	7.7	21.7**

*:p < 0.05 / **: p < 0.01, Chi²-test

#1: based on the total number of live born pups and the total number of pups at birth (alive and dead)

#2: based on the total number of corpora lutea and the total number of implantation sites

#3: based on the total number of implantation sites and the total number of live born pups

 

 

Conclusions:

Under the read across study conditions, NOAEL for toxicity to reproduction was determined to be 300 mg/kg bw/day (or 233.4 mg a.i./kg bw/day) and 1000 mg/kg bw/day (or 778 mg a.i./kg bw/day) for females and males, respectively. The NOAEL for development toxicity was determined to be 300 mg/kg bw/day (or 233.4 mg a.i./kg bw/day).

Executive summary:

A study was conducted to determine the repeated dose toxicity of the read across substance, 'Isodecyl oleate' (purity: 77.8%), according to OECD Guideline 422, in compliance with GLP. Three groups of ten male and ten female Sprague-Dawley rats received the read across substance daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 3 post-partum. The read across substance was administered as a suspension in the vehicle, corn oil, at dose-levels of 0, 100, 300 or 1000 mg/kg bw/day. Another group of ten males and ten females received the vehicle alone, under the same experimental conditions and acted as a control group. The systemic parameters for evaluating parental toxicity have been discussed in section 7.5.1 and only the reproductive and development parameters/effects have been described here. The animals were checked for macroscopic examination of the appearance and size of the gonads, adrenal glands, uterus, and accessory reproductive organs. Male animals were checked for testis weight, epididymis weight, and qualitative sperm staging. Female animals were checked for food consumption and body weight changes, number of pregnant females, pre-coital time, gestation length. Offspring (F1 generation) were observed for litter weight, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies and dead pups were gross examined for external and internal abnormalities. F1 pups which survived were sacrificed at 4 days age, and carefully examined externally for gross abnormalities. Changes in reproduction parameters of the dam (which could be attributed maternal toxicity) and in the development of pups were seen in the high dose females and their offspring, respectively. A statistically significant increase in post-implantation loss was noted in high dose group dams along with non-statistically significant decrease in birth index and elevated number of stillbirths leading to statistically significant reduction in the live birth index. No read across substance related influence was noted for the fertility index, gestation index and the pre-implantation loss. The qualitative sperm staging revealed no read across substance related spermatogenic changes. A read across substance-related decrease in the viability index was noted in the high dose group ((71.3 % in the high dose group vs. 98.7% in the control group). The high number of dead pups in the high dose group was due to 2 dams with no surving pups on lactation Day 4 (deaths partly due to cannibalization). The total litter loss in 2 of 7 dams (28.6%) was considered as read across substance related. Further, anon-statistically significant reduction in mean litter weight on lactation Day 1 by 17.2% was noted in the high dose group, which is regarded to be test item-related. Total litter weight was also reduced by 24.4% in comparison to controls. Therefore, the NOAEL for systemic or maternal toxicity was 300 mg/kg bw/day (i.e., equivalent to 233.4 mg a.i./kg bw/day) in females, because of reduced food consumption and body weight observed in the high dose group; and at ≥ 1000 mg/kg bw/day (i.e., equivalent to 778 mg a.i./kg bw/day) in males, due to absence of any adverse effects. Further, the NOAEL for reproductive toxicity was established at 300 mg/kg bw/day (or 233.4 mg a.i./kg bw/day), due to an increase in post-implantation loss, a decrease in the live birth index at 1000 mg/kg bw/day; and the NOAEL for development toxicity was also established at 300 mg/kg bw/day (or 233.4 mg a.i./kg bw/day), due to effects on litter weight and viability of the offspring in the high dose group (Hansen, 2013). Based on the results of the read across study, similar effect levels can be considered for the constituent esters.

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar 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:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rat Wistar aged 8 (males) - 9 (females) weeks at start of exposure period. 12M+12F/group

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

- Type of exposure: Dietary
- Duration of test/exposure: males 41-44 d, females approx. 54 d
- Treatment: Control group and treatment
- Vehicle: Diet
- Concentration in vehicle: 0, 1500, 7500 & 30,000 ppm

Details on mating procedure:

14 d premating exposure, then 1M+1F caged together. Inspection for vaginal plugs thrice daily. If mating did not occur after 14 d cohabitation the female was placed with another male for 8 d.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Actual dose received: 0, 100, 500 and 2000 mg/kg bw/day

Duration of treatment / exposure:

Males 41 - 44 d
Females up to 54 d

Frequency of treatment:

continuous in diet

Remarks:

0, 1500, 7500 & 30,000 ppm test substance in diet

Remarks:

Actual dose received: 0, 100, 500, 2000 mg/kg bw/day

No. of animals per sex per dose:

12 animals per sex per dose

Control animals:

yes

Parental animals: Observations and examinations:

Body weight, weight gain, food consumption, and food efficiency were recorded.

Oestrous cyclicity (parental animals):

Exposure was for 14 d premating covering at least 2 oestrous cycles. Ovaries were weighed and examined histopathologically at section (21 d after birth).

Sperm parameters (parental animals):

Exposure 14 d premating, no specific sperm analyses carried out, the testes & epididymis were weighed and examined histopathologically.

Postmortem examinations (parental animals):

- Organ weights P: liver, kidneys, thymus, testis, and epididymis.
- Histopathology P: liver, kidneys, adrenals, brain, heart, spleen, ovaries, thymus, testes, epididymides and any organs showing abnormality on macroscopic examination were fixed. The above tissues from all controls and top dose treated rats (except the thymus) plus abnormalities were examined.
- Macroscopic P: Full macroscopic examination.

Statistics:

Analysis of variance followed if significant differences were established by Dunnett’s T-test to assess possible intergroup differences. For pregnancy rate a Qui2-test was carried out to confirm lack of significance.

Reproductive indices:

Pregnancy rate, length of gestation, implantations, corpora lutea and resorptions were recorded.

Offspring viability indices:

Offspring (and dams) were sacrificed on post-natal Day 5 and the pups were weighed and examined for external malformations than sexed and examined for internal malformations.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

None considered of biological significance

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

None considered of biological significance

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment related histopathological changes including no effects in the testes and ovaries.

Reproductive function: oestrous cycle:

not specified

Description (incidence and severity):

Not reported

Reproductive function: sperm measures:

not specified

Description (incidence and severity):

Not reported

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no statistically significant difference in pregnancy rates although they were reduced in treated groups C 92%, 100 & 500 mg/kg bw/day 83%, 2000 mg/kg bw/day 75% these were within the normal historical control range according to the authors (actual historical control data not presented).
- Fertility index: Not reported
- Precoital interval: Not reported
- Duration of gestation: Comparable in treated and control dams (23 d in all groups).
- Gestation index: Not reported
- Changes in lactation: Not reported
- Number of implantations: No significant differences in the numbers of implantations between treated and control groups (mean 13 in control group, 14 in each treated group). There were no resorptions.
- Number of corpora lutea: No significant differences between treated and control groups (mean 14 in test and controls).
- Ovarian primordial follicle counts: Not reported

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed on reproductive parameters

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

None considered of biological significance

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

None considered of biological significance

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no treatment related histopathological changes including no effects in the testes and ovaries.

Other effects:

no effects observed

Description (incidence and severity):

- Litter size and weights: No effect of treatment. Litter size mean Controls 13.25, low dose 13.27, mid dose 13.2, high dose 13.33. Mean litter weights at day 1 were 75, 75, 71 and 77 gm and at day 4 106, 107, 101 and 104 gm for control, low, mid and high dose respectively. No statistical significance.
- Sex and sex ratios: No treatment related effects.
- Post natal survival until Day 5: Similar in treated and control groups.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at highest tested dose

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Based on the results of the read across study, the test substance, hexadecanol, NOAEL for reproductive and developmental effects is considered to be 2000 mg/kg/day.

Executive summary:

A screening study was conducted to determine the toxicity to reproduction of the read across substance, 1-dodecanol (Purity not specified) according to the method similar to the OECD Guideline 422, in compliance with GLP. The read across substance at 0, 1500, 7500 and 30000 ppm was administered as dietary feed to male Wistar rats up to 41 to 44 d, whereas to female Wistar rats up to 54 d (12 animals per sex per dose). Actual received doses were calculated to 0, 100, 500 and 2000 mg/kg bw/day. After 14 d premating exposure, one male and one female were caged together. Inspection for vaginal plugs were done thrice daily. If mating did not occur after 14 d cohabitation the female was placed with another male for 8 d. Body weight, weight gain, food consumption and food efficiency were examined. Ovaries, testes and epididymis were weighed and examined histopathologically. Pregnancy rate, length of gestation, implantations, corpora lutea and resorptions were recorded. Offspring (and dams) were sacrificed on post-natal Day 5 and the pups were weighed and examined for external malformations than sexed and examined for internal malformations. Organ weights were examined for liver, kidneys, thymus, testis, and epididymis. Liver, kidneys, adrenals, brain, heart, spleen, ovaries, thymus, testes, epididymides and any organs showing abnormality on macroscopic examination were fixed. The above tissues from all controls and top dose treated rats (except the thymus) plus abnormalities were examined. Haematological and biochemical parameters were measured. There were no treatment related effects observed on food/water consumption, body weight, organ weights, duration of gestation, biochemistry and haematological parameters. There was no statistically significant difference in pregnancy rates although they were reduced, 92% in control, 83% in 100 and 500 mg/kg bw/day, 75% in 2000 mg/kg bw/day, these were within the normal historical control range according to the study authors (actual historical control data was not presented). There were no changes attributable to exposure to the read across substance noted in gross pathology and histopathology. No significant differences in the numbers of implantations between treated and control groups (mean 13 in control group, 14 in each treated group) were noted. There were no resorptions, also no significant differences between treated and control groups (mean 14 in test and controls) were noted for number of corpora lutea. No statistical significant difference noted in litter size and weights, sex and sex ratios and post-natal survival until Day 5. No adverse effects were observed on reproductive parameters. Under the study conditions, the NOAEL for reproductive and developmental effects was established at 2000 mg/kg/day (Ernst, 1992). Based on the results of the read across study, similar effect levels can be considered for the constituent alcohol.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

233.4 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good quality Guideline compliant studies

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In absence of reproductive toxicity study with the test substance, the endpoint has been assessed based on studies for read across substances representative of the main constituents, which can be categorised as phosphate esters (PSE i.e., mono- and di C16 PSE, K+: 10 -40%), alkyl esters (i.e., C16-18 linear and branched fatty acid esters: 39 -87%) and alcohols (i.e., C16 -18 linear or branched alcohol: 10 -30%). The results are presented below:

Constituent: PSE - read across study:

A screening study was conducted to determine the toxicity to reproduction of the read across substance, mono- C12 PSE, Na+ (Purity no specified) according to the OECD Guideline 422, in compliance with GLP. The test substance was administered at 0 (control group), 250, 500 or 1000 mg/kg bw to male Sprague-Dawley SPF rats for 14 d 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. No test substance-related effects were observed regarding clinical signs, detailed clinical findings, function tests, grip strength, amount of spontaneous movement, body weights, food consumption, urinalysis (including water intake), and haematology or blood chemistry parameters. Gross pathological examination at the end of the administration period revealed recessed areas in the forestomach at 250 mg/kg bw and above and rough mucosa or white foci at 500 mg/kg bw and above, and erosion/ulceration, mucosal thickening and submucosal edema at 250 mg/kg bw and above on histopathological examination. In the light of the absence of a forestomach in humans, observed effects on this tissue were of questionable relevance with reference to the extrapolation of the toxic properties of a test substance in humans. Further, administration of the test substance did not have any effect on the oestrous cycle, days to copulation, copulation rate, fertility rate, or conception rate. Similarly, administration of the test substance did not have any effect on the delivery rate, gestation period, number of corpora lutea, number of implantation sites, implantation rate, stillbirth rate, number of live-born pups, live-birth rate in the mother animals, or on the sex ratio of the littermates. No abnormalities were observed in the lactating behaviour during the lactation period either. These results suggest that administration of the test substance even at 1000 mg/kg bw had no effect on the reproductive function, such as that shown by the copulation rate, of the males or females, or in the fertility rate, conception rate, or on the gestation maintenance, delivery, or lactating behaviour in the mother animals. Pups showed no changes caused by the administration of the test substance regarding the observation at birth, necropsy findings on Day 4 of lactation, body weight, or viability rate, which suggested that administration of the test substance even at 1000 mg/kg bw had no effect on the development. Under the study conditions, the systemic and reproductive/developmental toxicity rat NOAELs (P0 and F1 generation) of the read across substance, were both determined to be 1000 mg/kg bw/day (BRC, 2005). Based on the results of the read across study, similar NOAELs for systemic and reproductive/development toxicity can be considered for the phosphate ester constituent of the test substance.

Constituent: Alkyl ester (branched) - read across study:

A study was conducted to determine the repeated dose toxicity of the read across substance, 'Isodecyl oleate' (purity: 77.8%), according to OECD Guideline 422, in compliance with GLP. Three groups of ten male and ten female Sprague-Dawley rats received the read across substance daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 3 post-partum. The read across substance was administered as a suspension in the vehicle, corn oil, at dose-levels of 0, 100, 300 or 1000 mg/kg bw/day. Another group of ten males and ten females received the vehicle alone, under the same experimental conditions and acted as a control group.The systemic parameters for evaluating parental toxicity have been discussed in section 7.5.1 and only the reproductive and development parameters/effects have been described here. The animals were checked for macroscopic examination of the appearance and size of the gonads, adrenal glands, uterus, and accessory reproductive organs. Male animals were checked for testis weight, epididymis weight, and qualitative sperm staging. Female animals were checked for food consumption and body weight changes, number of pregnant females, pre-coital time, gestation length. Offspring (F1 generation) were observed for litter weight, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies and dead pups were gross examined for external and internal abnormalities. F1 pups which survived were sacrificed at 4 days age, and carefully examined externally for gross abnormalities.Changes in reproduction parameters of the dam (which could be attributed maternal toxicity) and in the development of pups were seen in the high dose females and their offspring, respectively. A statistically significant increase in post-implantation loss was noted in high dose group dams along with non-statistically significant decrease in birth index and elevated number of stillbirths leading to statistically significant reduction in the live birth index. No read across substance related influence was noted for the fertility index, gestation index and the pre-implantation loss. The qualitative sperm staging revealed no read across substance related spermatogenic changes. A read across substance-related decrease in the viability index was noted in the high dose group ((71.3 % in the high dose group vs. 98.7% in the control group). The high number of dead pups in the high dose group was due to 2 dams with no surviving pups on lactation Day 4 (deaths partly due to cannibalization). The total litter loss in 2 of 7 dams (28.6%) was considered as read across substance related. Further, anon-statistically significant reduction in mean litter weight on lactation Day 1 by 17.2% was noted in the high dose group, which is regarded to be test item-related. Total litter weight was also reduced by 24.4% in comparison to controls. Therefore, the NOAEL for systemic or maternal toxicity was 300 mg/kg bw/day (i.e., equivalent to 233.4 mg a.i./kg bw/day) in females, because of reduced food consumption and body weight observed in the high dose group; and at ≥ 1000 mg/kg bw/day (i.e., equivalent to 778 mg a.i./kg bw/day) in males, due to absence of any adverse effects. Further, the NOAEL for reproductive toxicity was established at 300 mg/kg bw/day (or 233.4 mg a.i./kg bw/day), due to an increase in post-implantation loss, a decrease in the live birth index at 1000 mg/kg bw/day; and the NOAEL for development toxicity was also established at 300 mg/kg bw/day (or 233.4 mg a.i./kg bw/day), due to effects on litter weight and viability of the offspring in the high dose group (Hansen, 2013). Based on the results of the read across study, similar NOAELs for systemic and reproductive/development toxicity can be considered for the ester constituent of the test substance.

Constituent: Alcohol - read across study:

A screening study was conducted to determine the toxicity to reproduction of the read across substance, 1-dodecanol (Purity not specified) according to the method similar to the OECD Guideline 422, in compliance with GLP. The read across substance at 0, 1500, 7500 and 30000 ppm was administered as dietary feed to male Wistar rats up to 41 to 44 d, whereas to female Wistar rats up to 54 d (12 animals per sex per dose). Actual received doses were calculated to 0, 100, 500 and 2000 mg/kg bw/day. After 14 d premating exposure, one male and one female were caged together. Inspection for vaginal plugs were done thrice daily. If mating did not occur after 14 d cohabitation the female was placed with another male for 8 d. Body weight, weight gain, food consumption and food efficiency were examined. Ovaries, testes and epididymis were weighed and examined histopathologically. Pregnancy rate, length of gestation, implantations, corpora lutea and resorptions were recorded. Offspring (and dams) were sacrificed on post-natal Day 5 and the pups were weighed and examined for external malformations than sexed and examined for internal malformations. Organ weights were examined for liver, kidneys, thymus, testis, and epididymis. Liver, kidneys, adrenals, brain, heart, spleen, ovaries, thymus, testes, epididymides and any organs showing abnormality on macroscopic examination were fixed. The above tissues from all controls and top dose treated rats (except the thymus) plus abnormalities were examined. Haematological and biochemical parameters were measured. There were no treatment related effects observed on food/water consumption, body weight, organ weights, duration of gestation, biochemistry and haematological parameters. There was no statistically significant difference in pregnancy rates although they were reduced, 92% in control, 83% in 100 and 500 mg/kg bw/day, 75% in 2000 mg/kg bw/day, these were within the normal historical control range according to the study authors (actual historical control data was not presented). There were no changes attributable to exposure to the read across substance noted in gross pathology and histopathology. No significant differences in the numbers of implantations between treated and control groups (mean 13 in control group, 14 in each treated group) were noted. There were no resorptions, also no significant differences between treated and control groups (mean 14 in test and controls) were noted for number of corpora lutea. No statistical significant difference noted in litter size and weights, sex and sex ratios and post-natal survival until Day 5. No adverse effects were observed on reproductive parameters. Under the study conditions, the NOAEL for reproductive and developmental effects was established at 2000 mg/kg/day (Ernst, 1992). Based on the results of the read across study, similar effect levels can be considered for the alcohol constituent of the test substance.

Overall, based on the available weight of evidence information from studies for substances representative of the main constituents, the test substance, ‘Reaction products of hexadecyl dihydrogen phosphate, dihexadecyl hydrogen phosphate, hexadecan-1-ol, stearic acid, esters of C18 (branched and linear) fatty acids with C18 (branched and linear) alcohols, and potassium hydroxide’ is not considered to pose reproductive or development concern. Considering that the esters make up more than 80% of the composition, the NOAEL based on the study with esters can be regarded as representative of the test substance. Therefore, a NOAEL of 300 mg/kg bw/day (i.e., 233.4 mg a.i./kg bw/day), which also corresponds to the lowest NOAEL, determined in a study with Isodecyl oleate has been considered further for hazard/risk assessment.

Effects on developmental toxicity

Description of key information

See above discussion.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available weight of evidence from read across studies on the main constituents, the test substance does not warrant classification for reproductive toxicity, according to EU CLP criteria (Regulation 1272/2008/EC).​​​​​