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Diss Factsheets

Administrative data

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 from studies on substances representative of the main constituents, the test substance, 'mono- and di- C16 -18 and C16 -18 AE10 PSE' is not considered to pose reproductive or development concern. The NOAELs were found to be higher than the highest tested doses ranging from 250-1000 mg/kg bw/day.​

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
Good quality 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 the absence of a reproductive toxicity study with the test substance, the endpoint can be assessed based on studies for substances representative of the main constituents, which can be categorised as phosphate esters (PSE), ethoxylated phosphate ester (AE PSE) and/or free ethoxylated alcohol (AE). 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+, according to the OECD Guideline 422, in compliance with GLP. The read across substance was administered at 0 (control group), 250, 500 or 1000 mg/kg bw to male Sprague-Dawley SPF rats for 14 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 read across 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. Administration of the read across substance did not have any effect on the oestrous cycle, days to copulation, copulation rate, fertility rate, or conception rate. Similarly, administration of the read across 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 read across 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 read across substance regarding the observation at birth, necropsy findings on Day 4 of lactation, body weight, or viability rate, which suggested that administration of the read across substance even at 1000 mg/kg bw had no effect on the development. Under the study conditions, the read across substance NOAELs for maternal, reproductive and developmental toxicity were determined to be 1000 mg/kg bw/day (BRC, 2005).

Constituent AE PSE - read across study:

A screening study was conducted to determine the repeated dose and reproduction / developmental toxicity potential of the read across substance, 'C8-10 AE4 PSE', according to the OECD Guideline 422, in compliance with GLP. In the main test, the read across substance was administered at 0 (control group), 25, 50, 200, 800 mg/kg bw/day to animals from 14 days before mating period till the day before sacrifice [Female:Day 4 of lactation for female dams that delivered a litter, Day 24 of gestation for female rats that did not deliver a litter, studyDay 52 for the female rat with no confirmed day of mating]. The animals were observed for mortality, clinical signs, body weight, food consumption, reproductive as well as developmental toxicity parameters during the study.There were no read across substance related mortality observed in parent animals. In the 800 mg/kg bw/day group, excess salivation, chromorhinorrhea, urine-stained abdominal fur, red and/or dried perioral substance and red or clear perinasal substance occurred in significantly increased numbers of male parent rats. Significantly increased numbers of parent females in the 800 mg/kg bw/day group had excess salivation. All other clinical signs were considered unrelated to the treatment. No treatment related significant change in body weight, weight gain, organ weight, food consumption were observed. No biologically significant differences were observed in parent animals. No treatment related effects were observed on oestrous cycle and reproductive performance of the animals. Two pregnant females in the 800 mg/kg bw group were found dead or sacrificed before delivery. All other pregnant rats delivered a litter. Values for the numbers of dams delivering litters and the gestation index, the numbers of dams with stillborn pups and of dams with all pups dying, viability and lactation indices, and the sex ratio were comparable within all groups. In gross pathology, at 800 mg/kg bw/day dose, thickened walls of the cardiac region of the stomach and white areas on the mucosal surface (3/10), ulceration on the mucosal surface of the cardiac region of the stomach (1/10) were observed in males. In females, lesions of the cardiac region of the stomach (2/10) were observed (Parental animals). In histopathology, at 800 mg/kg bw/day dose, findings in the non-glandular stomach related to read across substance (8/8 males; 4/4 females) were noted. Also, at 200 mg/kg bw/day dose, findings in the non-glandular stomach possibly related to read across substance, irritating effects (1/5 males) were observed, whereas at 25, 50 mg/kg bw/day dose groups no adverse effects were noted during observation in parent animals. No clear and consistent treatment related differences in results for the qualitative and quantitative microscopic assessment of bone marrow smears were observed in parent animals. No treatment related adverse effects were observed in offspring animals viability, clinical signs and gross pathology. Gestation duration and pup weights per litter were increased however not statistically significantly at 800 mg/kg bw dose for offspring animals. Implantation sites per litter, corpora lutea, litter sizes, surviving pups per litter and live litter size at weighing were reduced, however not statistically significantly at 800 mg/kg bw dose for offspring animals. These changes were not considered to be biologically relevant. Changes in the forestomach in parental animals considered to be a result of local irritation of the (irritant) read across substance than a true effect of systemic toxicity. Under the study conditions, the read across substance NOAELs for maternal, reproductive and developmental toxicity were determined to be 800 mg/kg bw/day (Lech, 2009).

Constituent AE - read across study:

A 2 -generation study was conducted to determine the dermal reproductive and development toxicity of the read across substance, 'C9-11 AE6' (purity not specified), according to a method similar to OECD Guideline 416. In the study, groups of 30 weanling Fischer 344 rats of each sex were dermally exposed to 1 mL/kg bw read across substance at concentrations of 0, 1, 10 or 25% w/v three times a week except during the mating periods. This treatment equals exposure levels of about 0, 10, 100 and 250 mg/kg bw/day. No mortalities were observed in the parental generation, and the five deaths in the F1 adult males and females in the control and treatment groups were not considered to be compound related. In the highest dose group, body weights of both males and females in both treated generations were sporadically decreased compared to controls. There was no effect on maternal body weight during gestational and lactational periods in both generations. At necropsy organ weight differences in liver, lung, kidney and heart were observed in the F1 generation. However no pathological findings were associated with these affected organs. There were no compound-related effects on mating and fertility indices and mean gestational length in both generations. No effects on testicular weights, sperm counts and LDH-X activities in F0 and F1 male adults were observed. There was no compound related effect on pup weights of any dose group in the F1 generations when compared to the controls. F2 pups from all treatment groups had higher body weights but these were not statistically significantly different from controls, except in in the 10% dose group on day 21 postnatally. Thus, the effect on body weight changes appears to be of no toxicological significance. No compound related effects on Iitter size, sex ratio and number of live pups in the F1 and F2 generations were observed during lactational days 1, 4, 7, 14, 21 and 28. Macroscopic and microscopic examination of the reproductive organs did not reveal significant differences in the treated groups compared to the controls. Under the study conditions, the read across substance dermal NOAELs for maternal systemic, reproductive and developmental toxicity were determined to be 100, >250 and >250 mg/kg bw/day, respectively (Lu, 1985).

Further, as per a HERA 2009 review report, the investigated alcohol ethoxylates (AE) did not cause reproductive toxicity when applied orally or dermally and the NOAEL for reproductive toxicity is greater than 250 mg/kg bw/d for selected AEs. This conclusion is supported by the fact that none of the AEs investigated in seven sub-chronic toxicity studies caused any adverse effects on the reproductive system (HERA, 2009).

Overall, based on the available weight of evidence from studies on substances representative of the main constituents, the test substance, ‘mono- and di- C16 -18 and C16 -18 AE10 PSE’ is not considered to pose reproductive concern. The NOAELs were found to be higher than the highest tested doses ranging from 250-1000 mg/kg bw/day. Due to the absence of significant reproductive toxicity combined with the low systemic exposure for the major constituents, the NOAEL of 1000 mg/kg bw/day has been considered further for hazard/risk assessment.

Effects on developmental toxicity

Description of key information

Based on the available weight of evidence from studies on substances representative of the main constituents,the test substance, 'mono- and di- C16 -18 and C16 -18 AE10 PSE' is not considered to pose development concern. The NOAELs were found to range from 250-1000 mg/kg bw/day. As a conservative approach the lower NOAEL of 250 mg/kg bw/day based on the study with PSE, has been considered further for hazard/risk assessment.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2016
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 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 results of the read across study, the NOAEL for maternal toxicity was established at 50 mg/kg bw/day due to treatment-related significant reduction in maternal body weight and food consumption at 250 and 1000 mg/kg bw/day. The NAOEL for fetal/developmental toxicity was established at 250 mg/kg bw/day due to treatment-related significant reduction in fetal weights at 1000 mg/kg bw/day. The NOAEL for teratogenicity was set at the highest dose of 1000 mg/kg bw/day, due to the absence of any signs of teratogenicity at the test doses.
Executive summary:

A study was conducted to evaluate the prenatal developmental toxicity of the read across substance, mono- and di- C12 PSE, K+ in rats, according to OECD guideline 414, in compliance with GLP. In this study, each group 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 read across substance was 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 bw/day. The rats in the vehicle control 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 GD 0, 3, 5, 8, 11, 14, 17 and 20. 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. 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 bw/day as compared to vehicle control group along with significant reduction in corrected body weight gain at 1000 mg/kg bw/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 bw/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 bw/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 bw/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 of teratogenicity in any of the tested doses. Under the study conditions, the NOAEL for maternal toxicity was established at 50 mg/kg bw/day due to treatment-related significant reduction in maternal body weight and food consumption at 250 and 1000 mg/kg bw/day. The NAOEL for fetal/developmental toxicity was established at 250 mg/kg bw/day due to treatment-related significant reduction in fetal weights at 1000 mg/kg bw/day. The NOAEL for teratogenicity was set at the highest dose of 1000 mg/kg bw/day, due to the absence of any signs of teratogenicity at the test doses (Rao, 2017). Based on the results of the read across study, similar NOAELs is considered for the test substance.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crj:CD(SD)IGS
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.
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.
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.
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
Remarks:
0, 250, 500 and 1000 mg/kg bw/day
No. of animals per sex per dose:
A total of 4 dose groups were set up: 250, 500, and 1000 mg/kg bw/day 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/day, Bozo Research Center study No.: C-R016), administration of the test substance did not produce any effect even at 1000 mg/kg bw,/day the level defined as the limiting dose by the OECD Guideline for Testing of Chemicals 422. Therefore, 1000 mg/kg bw/day was set as the highest dose, and doses of 500 and 250 mg/kg bw/day were derived by dividing by a common factor of 2. A total of 3 doses were thus set up.
Maternal 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.

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 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)).
Ovaries and uterine content:
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.
Fetal examinations:
- 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 (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 litter mates.
Statistics:
- Bartlett test
- Dunnett’s test
- χ2 test with Yates’ continuity correction
- Fisher’s exact test
Indices:
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.
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 dose group in the main group, but this was not dose-related.
Water consumption and compound intake (if drinking water study):
no effects observed
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.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No dose-related changes in either direction (increase or decrease) were observed in either the absolute or the relative weight. Although significant differences in the weights of the organs were observed, they were considered to be within the range of normal variations, because they were neither dose-related nor were observed at the end of the administration period.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Findings at the end of the administration period. Stomach: Indentation of the anterior stomach was observed in 0, 5 and 7 males, and 1, 1 and 3 females of the 250, 500 and 1000 mg/kg bw groups, respectively. White foci were observed in 1 male of the 500 mg/kg bw group. Rough mucosa in the anterior stomach was observed in 5 and 9 males, and 5 and 6 females of the 500 and 1000 mg/kg bw groups, respectively. Indentation of the glandular stomach was observed in 1 female of the 500 mg/kg bw group. All the other findings observed in the organs and tissues were considered to be incidental, as judged from the frequency of their occurrence and the pathological findings (Dark red foci in the glandular stomach were observed in 0, 1, 2 and 1 males and 4, 2, 1 and 1 females of the control group, 250, 500 and 1000 mg/kg bw groups, respectively. Kidney: Pyelectasis was observed in 2 and 1 males of the 250 and 1000 mg/kg bw groups, respectively. Eyeballs: Corneal opacity (unilateral) was observed in 1 female of the 500 mg/kg bw group).
- Findings at the end of the recovery period. Stomach: Rough mucosa in the anterior stomach was observed in 1 male of the 1000 mg/kg bw group. This animal also showed expansion of the digestive tract from the stomach to the colon due to gas accumulation, and a mild decrease in the size of the testis. Other findings observed in the following organs were considered to be incidental as judged from the frequency of their occurrence and the pathological findings (Lung: Dark red foci were observed in 1 female of the 1000 mg/kg bw group).
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
Other effects:
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.
Details on maternal toxic effects:
- 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:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed on reproductive parameters
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no significant systemic adverse effects
Key result
Abnormalities:
no effects observed
Fetal body 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.
Changes in sex ratio:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No significant differences in external anomalies rate were observed. A kinking of the tail in 1 animal of the 500 mg/kg bw/day group was recorded, but was considered to be a spontaneous occurrence as judged from the frequency of occurrence and the type of the anomaly.
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Mortality: During the lactation period, death occurred in only 4 pups in the control group and 2 pups in the 1000 mg/kg bw/day 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.

Sexual maturation: no effects observed

Gross Pathology: 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/day groups, respectively, however, the occurrences were not dose-related.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Based on the results of the read across study, the test substance NOAELs for maternal and developmental toxicity were determined to be 1000 mg/kg bw/day.
Executive summary:

A study was conducted to determine the repeated dose and reproduction / developmental toxicity potential of the read across substance, mono- C12 PSE, Na+, according to the OECD Guideline 422, in compliance with GLP. The read across substance was administered at 0 (control group), 250, 500 or 1000 mg/kg bw/day 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/day groups, a 14 d recovery period was allowed after the 42 d administration period to investigate the reversibility of the toxic changes. No read across 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/day and above and rough mucosa or white foci at 500 mg/kg bw/day and above, and erosion/ulceration, mucosal thickening and submucosal edema at 250 mg/kg bw/day and above on histopathological examination. Administration of the read across 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. For details on effects observed on reproductive parameters, please refer to section 7.8.1 of IUCLID or 5.9.1 of CSR. The absence of adverse effects on pups at birth, necropsy findings on Day 4 of lactation, body weight, or viability rate, suggested that administration of the read across substance even at 1000 mg/kg bw/day had no effect on the development. Under the study conditions, the NOAELs for maternal and developmental toxicity were determined to be 1000 mg/kg bw/day (BRC, 2005). Based on the results of the read across study, similar NOAELs can be considered for the test substance.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
From May 30, 2008 to February 06, 2009
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
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals or test system and environmental conditions:
Test animals
- Source: Charles River Laboratories, Inc., Portage, MI, USA
- Age at study initiation: 72 d (males), 66 d (females)
- Weight at study initiation: 306-344 (males), 208-239 (females)
- Housing: individual housing in stainless steel, wire-bottomed cages.
- Diet: Certified Rodent Diet 5002 (PMI Nutrition International, St.Louis, MO, USA), ad libitum
- Water: filtered local water (chlorine addition), ad libitum
- Acclimation period: 7 d

Environmental conditions
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Preparation of dosing solutions:
Formulations were prepared at least once daily. Formulations were used within four hours of preparation and were stirred for at least 30 ot 60 minutes before dosage administration.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
A weight/weight verification method was employed to verify the test-substance content of the prepared formulations. No analytical method was developped for this test substance.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 14 d
- Proof of pregnancy: sperm in vaginal smear and/or copulatory plug referred to as Day 0 of pre
gnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
Duration of treatment / exposure:
Males: 14 d before cohabitation period through the day before sacrifice (45 dosages)
Females: 14 d before cohabitation period through the day before scheduled sacrifice (Day 4 of lactation for dams that delivered a litter, Day 24 of gestation for rats that did not deliver a litter, study Day 52 for the rat with no confirmed day of mating). Surviving female rats were given a total of 38 to 52 dosages.
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations: 25, 50, 200, 800 mg/kg bw
Basis: actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a range-finding study rats were dosed with 100, 300, and 1000 mg/kg bw/day. At Day 6 the high dose of 1000 mg/kg bw was lowered to 750 mg/kg bw, due to excessive toxicity. Reductions of the body weight were observed within Days 4-5. In the gestation period body weights were only slightly reduced in the 300 and 750/1000 mg/kg bw dose groups. No effects were observed after Caesarean section on Day 21 of gestation up to the highest dose of 750/1000 mg/kg bw. Thus, doses of 25, 50, 200, and 800 mg/kg bw were chosen for the main study. The low dose was expected to be a NOAEL for maternal and embryo-fetal toxicity, and the 800 mg/kg bw dose was expected to produce minimal maternal toxicity and little or no developmental toxicity.
Maternal examinations:
- Cage side observations twice daily: clinical signs, abortions, premature deliveries, deaths
- Detailed clinical observations: before the first dosage and weekly thereafter
- Body weight examinations: daily
- Food consumption examinations: Males: weekly, except for the cohabitation period; females: weekly until the cohabitation period, and on Days 0, 7, 10, 12, 15, 18, 20, 24, and 25 of gestation, and on Day 1 and 4 of lactation. For the rat with no confirmed mating on study Days 28, 35, 38, 40, 43, 46, 48, and 52.
- Gross pathology: whole body
- Histopathology: small intestine, large intestine, brain, urinary bladder, heart, testes, liver, lung, trachea, lymphnodes, mesentery lymphnodes, spleen, epididymides, adrenal gland, peripheral nerve, kidney, ovary, prostate, vesicula seminalis, thyroid gland, oesophagus, thymus, uterus, bone marrow, mammary gland, spinal cord, sternum, stomach, vagina
Fetal examinations:
The following parameters were examined in F1 offspring:
- Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities
- Gross examination of dead pups: for external and internal abnormalities; possible cause of death was determined for pups born or found dead

Sacrifice
- The F1 offspring were sacrificed at Day of lactation 4 or 5
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) for gross lesions and an internal confirmation of sex.
- Gross necropsy included a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. Pups that died before examination of the litter for pup viability were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sank were identified as stillborn; pups with lungs that floated were identified as liveborn, and to have dies shortly after birth.
Indices:
Reproductive indices
- Fertility index = (number of pregnant animals / number of copulated pairs) x 100
- Gestation index = (number of pregnant females with pups alive / number of pregnant females) x 100

Offspring viability indices
- Viability index = (number of pups alive on Day 4 / number of pups alive on Day 0) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
800 mg/kg bw/day: excess salivation in both sexes
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
800 mg/kg bw/day: reduced mean bw gain and reduced food consumption (males: week 1-2; females: during gestation)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
800 mg/kg bw/day: reduced mean bw gain and reduced food consumption (males: week 1-2; females: during gestation)
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
800 mg/kg bw/day: damage on the lining of the stomach
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
800 mg/kg bw/day: findings in the non-glandular stomach related to test substance (8/8 males; 4/4 females)
Other effects:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
800 mg/kg bw/day: the duration of gestation was increased (not statistically significant).
Changes in number of pregnant:
not specified
Description (incidence and severity):
Pregnancy occurred in 90%, 100%, 90%, 90%, and 80% female rats in the control, 25, 50, 200, and 800 mg/kg bw/day dose groups, respectively. Two pregnant females in the 800 mg/kg bw group were found dead or sacrificed before delivery. All other pregnant rats delivered a litter.
Details on maternal toxic effects:
No clear and consistent treatment related differences in results for the qualitative and quantitative microscopic assessment of bone marrow smears were observed.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
changes in number of pregnant
clinical signs
dead fetuses
effects on pregnancy duration
gross pathology
maternal abnormalities
necropsy findings
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
800 mg/kg bw/day: Values for the pup weights per litter were increased (not statistically significant).
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Surviving pups per litter and live litter size at weighing were reduced (not statistically significant). These changes were not considered to be biologically relevant.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
800 mg/kg bw/day: Pup weights per litter were increased (not statistically significant). Litter sizes were reduced (not statistically significant). These changes were not
considered to be biologically relevant.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Based on the results of the read across study, the test substance NOAELs for maternal, reproductive and developmental toxicity were determined to be 800 mg/kg bw/day.
Executive summary:

A study was conducted to determine the repeated dose and reproduction / developmental toxicity potential of the read across substance, 'C8-10 AE4 PSE', according to the OECD Guideline 422, in compliance with GLP. In the main test, the read across substance was administered at 0 (control group), 25, 50, 200, 800 mg/kg bw/day to animals from 14 days before mating period till the day before sacrifice [Female:Day 4 of lactation for female dams that delivered a litter, Day 24 of gestation for female rats that did not deliver a litter, study Day 52 for the female rat with no confirmed day of mating]. The animals were observed for mortality, clinical signs, body weight, food consumption, reproductive as well as developmental toxicity parameters during the study. Except for local effects in forestomach, no treatment-related systemic effects were observed in the parental animals. For details on effects observed on maternal and reproductive parameters, please refer to section 7.8.1 of IUCLID or 5.9.1 of CSR. For offspring animals, natural delivery and litter observations were unaffected by dosages. Cold to touch, not nesting, moderate dehydration and pale offspring animals were observed at 800 mg/kg bw/day. However, they were not attributed to the treatment, as it was only observed in one litter and was without a dose response. No effects were observed on changes in sex ratio of litter. At 800 mg/kg bw/day dose, increase in pup weights per litter (not statistically significant), reduction in surviving pups per litter and live litter size at weighing (not statistically significant) were observed. However, these changes were not considered to be biologically relevant. No effects were observed in gross pathology for all offspring animals. Under the study conditions, the read across substance NOAELs for maternal and developmental toxicity were determined to be 800 mg/kg bw/day (Lech, 2009). Based on the results of the read across study, similar NOAELs can be considered for the test substance.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1985
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:
reference to same study
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Fischer 344
Details on test animals or test system and environmental conditions:
One hundred and fifty male and 150 female Fischer 344 rats, aged approximately 4 weeks old, were received on May 25, 1982 from Charles River Breeding
Laboratories, Portage, Michigan. Upon arrival the rats weee removed from their crates, examined as to sex and condition, and placed individually in wire mesh bottom polycarbonate cages. Purina Formulab Chow 500 and water were available ad libitum. The rats were held for a pre-trial period of 19 days, during which time pre-trial body
weights were taken on days 1, 7, and 14, and daily observations were made for clinical signs. Five days prior to the treatment, 5 male and 5 female rats were necropsied for determination of general health by the veterinarian. The rats were housed in TX-332 throughout the pre-trial and trial periods. Environmental conditions for TX-332 during the trial period were as follows: Temperature ranged 22+/- 1 deg C. Humidity ranged from 40% to 65%. 1.8% of the time on temperature and 1.9% of the time on humidity were outside these ranges during the course of the study. A light cycle of 12 hours on (6:30 AA - 6:30 PM) and 12 hours off was maintained throughout the study. .
Route of administration:
dermal
Vehicle:
water
Details on exposure:
Dose volume: 1 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
After 119 days of dosing the F0 rats and 133 days of dosing the F1 rats, females were housed with males of the same treatment group. The day on which evidence mating (presence of a copulatocy plug or sperm in the vaginal smear) was observed was designated as day 0 of gestation. Each mated female was placed in a polycarbonate cage with Absorbdri as nesting material. In the first week ot the mating period, 30 males and 30 females in each dose group were paired according to the randomization method. Only proven males with positive matings were used the following 2 weeks. In the first week of the mating period, 20 males and 20 females in a one to one ratio were cohabitated according to the randomization procedure provided by the statistician and sibling or half sibling matings were avoided. Only proven fertile males were used the subsequent matings with rest of females. Each proven fertile male could not be mated more than three times within the 3 week mating period. Females which failed to mate or conceive were kept for a period of at least 22 days and sent to necropsy. Genealogy data of males and females cohabitated duzing the mating periods, results of matings, pregnancy status, gestational length, Litter birth date and pups selected for breeding and for necropsy each litter in each group were tabulated.
Duration of treatment / exposure:
From Day 0 of gestation until weaning
Frequency of treatment:
3 times/week (except during mating)
Remarks:
Doses / Concentrations: 0, 1, 10, 25%
Basis: nominal in water
Remarks:
Doses / Concentrations: 0, 10, 100, 250 mg/kg bw/day
Basis: nominal in water
No. of animals per sex per dose:
30 (P)
20/40 (male/female; F1)
Control animals:
yes, concurrent vehicle
Details on study design:
Upon allocation to treatment groups, the F0 and F1 generation rats were individually identified with their permanent identification number by ear notch and tail band. On the day of initial treatment (day 0), the hair of the back of each rat was shaved, and the appropriate dosing solution was applied evenly on the shaved area (2" x 2") using a Pipetman' automatic micropipette. The rats were treated three times a week, Monday, Wednesday and Friday. The dosage applications were not missed because the holidays. The exact volume of the test solution administered to each rat was based on it 's weekly body weight, and the dose volume was 1ml/kg body weight. Back hair was shaved each week pc iot to initial weekly application and when necessary throughout the week to ensure adequate dermal exposure. No treatment was performed during the mating period. Rats the F0 and F1 generations were treated in accordance with their dose groups. The treatment periods were: 170 days (14 June - 1 December 1982) the F0 females from the initiation of the study until the F1 generation was weaned; 221 days (6 December 1982 - 15 July 1983) for the F1 females, from the 1st day of dosing to 30 days after the last Litter weaned: 102 days for the F0 males (14 June - 24 September 1982) and 123 days (6 December 1982—8 April 1983) for the F1 males. For the reversability test, F1 males also treated for 123 days (6 December 1982—8 April 1983). The skin of the treated site of each rat was evaluated for irritation and edema using the scoring method.
Maternal examinations:
Observations were made once daily during treatment and mating periods for morbidity, general physical appearance and clinicaI signs of toxicity. During gestation and lactation periods rats were observed in the morning and afternoon. Male and female body weights were taken weekly up to the time of mating. Females were weighed on days 0, 7, 14 and 20 of gestation and on days 1, 7, 14, 21, and 28 of lactation. F1 females were weighed weekly after day 28 of lactation. Pups were weighed as a litter on days 1 , 4, 7, 21, and 28 of lactation, and were also weighed individually on day 28 of lactation. Each male rat in the F0 and F1 generations was killed after breeding, and the left testis was homogenized and sonicated in distilled water. Enumeration of sperm heads and measurement of lactate dehydrogenase isozyme (LDH—X) activity were performed from testicular homogenates. The LDH—X activity was quantified spectrophometrically by assaying the oxidation ot NADH in the presence of sodium pyzuvate at 340 nm on the automated clinical analyzer. Gross necropsies and health assessments were performed on 5 males and 5 females during pre-trial period. Gross necropsies were also performed on all F0 and F1 parentals using a table of random numbers generated by the SAS. Histopathologicai examinations were performed on all reproductive organs, all lesions in the F0 parentals. Histopathological examinations of reproductive organs, livers, spleens, hearts, kidneys and lungs of the F1 parental males and females were performed. Selected organ weights were also measured for these rats.
Fetal examinations:
Litters were caged with their mothers for at least 21 days after birth. At parturition the following observations were made: litter size, sex of pups, and number of live and stillborn pups. Pups were observed daily for mortality and physical abnormalities. On day 4 of Iactation any litter with more than 10 pups was culled to 10 by using a table of random numbers generated by the SAS; when possible at least 2 pups of each sex were included in the litter after culling. Individual litter weights of the F1 and F2 generations were determined. Mean pup weight was calculated from litter weight divided by number of pups per litter (litter size) and this value was adjusted for the differences in litter size among dose groups for comparison. Gross necropsies and health assessments were performed on 5 males and 5 females during pre-trial period. Gross necropsies were also performed on selected F1 and F2 pups (5 males and 5 females per dose group) using a table of random numbers generated by the SAS. Histopathologicai examinations were performed on all tissues of the selected F1 pups (5 males and 5 females per dose group). Histopathological examinations of reproductive organs, livers, spleens, hearts, kidneys and lungs of the selected F2 pups in the highest dose group and the vehicle control group (5 males and 5 females per dose group were performed. Selected organ weights were also measured for these rats.
Statistics:
Organ weight, organ weight/body weight ratio, LDH—X enzyme activity and litter size, sex ratio, survival index and gestational length were analyzed by analysis of variance. Treatment groups were compared to the Control group by one tailed Dunnettts E test at P=0.05. Body weights were analyzed by analysis of covariance. Body weights in all dose groups were adjusted for body weight on the first day of dosing due to differences in initiaI weights among the treatment groups. The adjustment is intended to compare the body weights of the treatment groups so that they had the same initial weight. Gestational weights were adjusted to the common body weight on gestational day 0, and lactational weights were adjusted to that on lactational day 1. Pup weights and number of live and dead pups were adjusted for the differences in size among all dose groups. The analysis of covariance was used to adjust the treatment means for the differences in the covariates among the treatment groups. Subsequent treatment comparisons against the control were then made on the adjusted treatment means. The adjusted treatment means represent the means of the treatment groups if they were to have the same mean value in the covariate. Fertility indices were analyzed by one tailed Fisher's exact test. Mating indices were analyzed by Chi square with continuity correction. Sperm counts and survival indices were analyzed by nonparametric method, Kruskal wallis test. Treatment groups were compared to the control by one tailed Dunnett's test at P=0.05 on the ranked data.
Indices:
Mating indices, fertility index and mean gestation length were calculated. Pup viability data, including number of pups born alive and dead, and pre weaning deaths for each litter per sex per dose group were calculated.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Lacrimation, chromodacryorrhea, periocular swelling, ptosis, red nasal discharge, rhinorrhea, polyuria, soft stool, alopecia, odd shaped pupil, and unkemptness were noted in the F0 and F1 parental rats in all dosage groups and controls. In addition, the two highest dose groups had a higher incidence of hunched posture. However, there were no pathological findings associated with these clinical signs.
Dermal irritation (if dermal study):
no effects observed
Description (incidence and severity):
Skin reaction scores for all rats in all dose groups was zero throughout the F0 and F1 generations. Test substance did not cause erythema or edema. However, both male and female rats in the 10% and 25% groups had dry and flaking skin. The discoloration of the application sites was noted in all treated rats including the controls. Possibly it was caused by the repeated shaving of the hair coat and dermal applications of the vehicle (water) or test solutions and was considered not to be compound related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality were noted in F0 generation animals. Five deaths were noted in the F1 generation: control male, 10% dosed female, 25% dosed male and two 25% dosed female and they were sent to necropsy. At necropsy, the posterior pharynx of each rat was filled with moist, finely ground and compacted food material that occl uded the laryngeal opening of the rats. Similar material was also found in the esophagus of two rats and the trachea of one of these two rats. Death was apparently caused by asphyxiation and was not compound related because a similar finding was noted in the same strain of rats at another (personal comunicaticn) and a control rat was also affected. This finding occurred during the similar period of 1982 - 1983, while rats were fed Purina Chow at both laboratories.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the F0 and F1 adults, body weights of the 1% and 10% dose groups were not affected by the treatment. However, significantly lower body weights of 25% dose group were observed during certain intervals when compared to the control group. For F0 generation, there were statistically significantly lower body weights in the 25% dose group males between days 35 - 105 during the dosing period; in females in body weights were only significantly lower at day 49 but were slightly lower than the control during the rest of the dosing period. For F1 generations, body weights of the 25% dose group males were lower but only statistically significant at days 21, 28, and 35 during dosing period; body weights of 25% dose group females were lower from days 42 - 119 during the dosing period when compared to the controls. In the 25% dose group, the effect on body weight changes of the F0 parental males was gradually reduced to almost a complete recovery in the F1 generation, while in the F0 parental females, the effect became more apparent in the F1 generation. It appears that male and female rats have different sensitivities to the compound toxicity at different times. There were no compound related effects on maternal body weights of all dose groups during the gestational and lactational periods in the F0 and F1 generations when compared to the controls. On lactation day 14, maternal body weights of the treatment groups were statistically significant higher than the control. These increases were of no toxicological significance. The 10% dose was a no effect level while 25% dose caused body weight over the test periods.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At necropsy organ weight differences in liver, lung, kidney and heart were observed in the F1 generation. However no pathological findings were associated with these affected organs.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At necropsy, in the F0 generation, a significantly lower carcass weight was observed only in the 25% dose group adult females, yet their absolute and relative organ weight were comparable to the controls. In the F1 generation, the carcass weights the adult males and females were not affected. However, organ weight differences were observed. In the F1 males significantly higher absolute and relative weights of liver, lung and heart in the 25% dose group, higher absolute and relative weight of lung in 10% dose group and higher relative lung weight in the 1% dose group were observed. The higher absolute and/or relative lung weight was not considered to be toxicologically significant as there was no dose related linear relationship in all dose groups among the changes 1% and 10% dose levels. Also no morphological changes were noted in the lungs. In the F1 adult females, significantly higher absolute and relative weights of liver, heart and kidney and the relative lung weight of the 25% dose group, higher absolute and relative heart weights and relative liver weights of the 10% group were noted. In the F1 pups, there were no differences in carcass weights and organ weights in any treatment groups when compared to the controls.
Description (incidence and severity):
Macroscopic and microscopic examination of the reproductive organs did not reveal significant differences in the treated groups compared to the controls.
Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Mating indices (number of females mated/ number of females housed males) were 73.3, 83.3, 83.3, and 93.3% in the F0 generation and 65, 70, 82.1 and 83.8% in the F1 generation of the 0, 1, 10 and 25% dose groups, respectively. The incidences appears to be higher in treatment groups than the control group but not significantly different from the control group by Chi square test with continuity correction. The lower mating performance data from the control rats are commonly observed in this strain of rats, Fischer 344. There were no apparent morphological differences in all rats to explain the apparent infertility. There were no compound related effects on fertility index and mean gestational length of the F0 females. The fertility indices (number of females pregnant/number of females mated) were 77.3, 64, 76, 53.6 in F0 generation and 88.5, 67.9, 87.5 and 77.4% in F1 generation of the 0, 1, 10 and 25% dose groups, respectively. Mated females were those having a copulatory plug or sperm in the vaginal smear or giving birth without the presence of copulatory plug or sperm. Mean gestational length in all dose groups in the F0 and F1 generations was approximately 22 days. Few females without the presence of copulatory plug or sperm during mating periods in both generations gave birth; their gestational lengths were therefore indeterminable.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
> 250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
changes in number of pregnant
changes in pregnancy duration
effects on pregnancy duration
maternal abnormalities
necropsy findings
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no compound related effect on pup weights of any dose group in the F1 generations when compared to the controls. F2 pups from all treatment groups had higher body weights but these were not statistically significantly different from controls, excpet in in the 10% dose group on day 21 postnatally. Thus, the effect on body weight changes appears to be of no toxicological significance.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No compound related effects on number of live pups in the F1 and F2 generations were observed during lactational days 1, 4, 7, 14, 21 and 28.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No compound related effects on sex ratio of pups in the F1 and F2 generations were observed during lactational days 1, 4, 7, 14, 21 and 28.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No compound related effects on Iitter size of pups in the F1 and F2 generations were observed during lactational days 1, 4, 7, 14, 21 and 28. Litter sizes were 8.2, 9.6, 7.9, and 6.4 in the F1 generation and 6.9, 8.3, 8 and 8.4 in the F2 generation of the 0, 1, 10 and 25% dose groups, respectively. The mean litter size the 25% dose group in the F1 generation was smaller than the control and the mean litter size of the control was the smallest in the F2 generation. However, there were no significant differences among dose groups when compared to the controls.
Changes in postnatal survival:
no effects observed
External malformations:
not specified
Skeletal malformations:
not specified
Visceral malformations:
not specified
Details on embryotoxic / teratogenic effects:
All pups dying during the pre weaning period, including the control group, were examined, recorded and discarded. Two pups, one male from litter (Dam, F1, 10% dose group) and one female from another litter (Dam, F2, 25% dose group) were found dead, and they appeared to have been dehydrated. No pathological finding was noted in these pups. One female pup in a litter (Dam, F2, 1% dose group) was born with a short right forelimb and the left hindlimb and was missing on day 4 postnataly. Presumably it was canabalized by it's mother. Another female pup in a litter (Dam, F2, 25% dose group) was dead with a short left forelimb and small eyes. The left ocular zone contained a small piece of ocular tissue, the optic cup without a lens (microphthalmia); the right eye was than usual. The low incidence of these findings in rats is apparent due to spontaneous occurance of developmental anomalies. There was no compound related effects on the development of the pups. Eye opening was observed at aprroximately 16.1-16.9 days for the F1 pups and 17.2-17.6 days for F2 pups in all dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
> 250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Based on the results of the read across study, the test substance dermal NOAELs for maternal systemic, reproductive and developmental toxicity were determined to be 100, >250 and >250 mg/kg bw/day, respectively.
Executive summary:

A study was conducted to determine the dermal reproductive and development toxicity of the read across substance, 'C9-11 AE6' (purity not specified), according to a method similar to OECD Guideline 416 (two-generation reproduction study). In the study, groups of 30 weanling Fischer 344 rats of each sex were dermally exposed to 1 mL/kg bw read across substance at concentrations of 0, 1, 10 or 25% w/v three times a week except during the mating periods. This treatment equals exposure levels of about 0, 10, 100 and 250 mg/kg bw/day. No mortalities were observed in the parental generation, and the five deaths in the F1 adult males and females in the control and treatment groups were not considered to be compound related. In the highest dose group, body weights of both males and females in both treated generations were sporadically decreased compared to controls. There was no effect on maternal body weight during gestational and lactational periods in both generations. For details on effects observed on reproductive parameters, please refer to section 7.8.1 of IUCLID or 5.9.1 of CSR. There was no compound related effect on pup weights of any dose group in the F1 generations when compared to the controls. F2 pups from all treatment groups had higher body weights but these were not statistically significantly different from controls, except in in the 10% dose group on day 21 postnatally. Thus, the effect on body weight changes appears to be of no toxicological significance. No compound related effects on Iitter size, sex ratio and number of live pups in the F1 and F2 generations were observed during lactational days 1, 4, 7, 14, 21 and 28. Macroscopic and microscopic examination of the reproductive organs did not reveal significant differences in the treated groups compared to the controls. Under the study conditions, the read across substance dermal NOAELs for maternal systemic, reproductive and developmental toxicity were determined to be 100, >250 and >250 mg/kg bw/day, respectively (Lu, 1985). Based on the results of the read across study, similar NOAELs can be considered for the test substance.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Good quality studies; Adverse effect limited to only reduced foetal weight in the presence of reduced maternal body weight and food consumption.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In absence of development toxicity study with the test substance, the endpoint can be assessed based on studies for substances representative of the main constituents, which can be categorised as phosphate esters (PSE), ethoxylated phosphate ester (AE PSE) and/or free ethoxylated alcohol (AE). The results are presented below:

Constituent: PSE - read across study:

Study 1: A study was conducted to determine the repeated dose and reproduction / developmental toxicity potential of the read across substance, mono- C12 PSE, Na+, according to the OECD Guideline 422, in compliance with GLP. The read across substance was administered at 0 (control group), 250, 500 or 1000 mg/kg bw/day 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/day groups, a 14 d recovery period was allowed after the 42 d administration period to investigate the reversibility of the toxic changes. No read across 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/day and above and rough mucosa or white foci at 500 mg/kg bw/day and above, and erosion/ulceration, mucosal thickening and submucosal edema at 250 mg/kg bw/day and above on histopathological examination. Administration of the read across 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. For details on effects observed on reproductive parameters, please refer to section 7.8.1 of IUCLID or 5.9.1 of CSR. The absence of adverse effects on pups at birth, necropsy findings on Day 4 of lactation, body weight, or viability rate, suggested that administration of the read across substance even at 1000 mg/kg bw/day had no effect on the development. Under the study conditions, the NOAELs for maternal and developmental toxicity were determined to be 1000 mg/kg bw/day (BRC, 2005).

Study 2: A study was conducted to evaluate the prenatal developmental toxicity of the read across substance, C12 PSE, K+ in rats, according to OECD guideline 414, in compliance with GLP. In this study, each group 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 read across substance was 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 bw/day. The rats in the vehicle control 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 GD 0, 3, 5, 8, 11, 14, 17 and 20. 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. 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 bw/day as compared to vehicle control group along with significant reduction in corrected body weight gain at 1000 mg/kg bw/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 bw/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 bw/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 bw/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 of teratogenicity in any of the tested doses. Under the study conditions, the NOAEL for maternal toxicity was established at 50 mg/kg bw/day due to treatment-related significant reduction in maternal body weight and food consumption at 250 and 1000 mg/kg bw/day. The NAOEL for fetal/developmental toxicity was established at 250 mg/kg bw/day due to treatment-related significant reduction in fetal weights at 1000 mg/kg bw/day. The NOAEL for teratogenicity was set at the highest dose of 1000 mg/kg bw/day, due to the absence of any signs of teratogenicity at the test doses (Rao, 2017).

Constituent AE PSE - read across study:

A study was conducted to determine the repeated dose and reproduction / developmental toxicity potential of the read across substance, 'C8-10 AE4 PSE', according to the OECD Guideline 422, in compliance with GLP. In the main test, the read across substance was administered at 0 (control group), 25, 50, 200, 800 mg/kg bw/day to animals from 14 days before mating period till the day before sacrifice [Female:Day 4 of lactation for female dams that delivered a litter, Day 24 of gestation for female rats that did not deliver a litter, studyDay 52 for the female rat with no confirmed day of mating]. The animals were observed for mortality, clinical signs, body weight, food consumption, reproductive as well as developmental toxicity parameters during the study. Except for local effects in forestomach, no treatment-related systemic effects were observed in the parental animals. For details on effects observed on maternal and reproductive parameters, please refer to section 7.8.1 of IUCLID or 5.9.1 of CSR. For offspring animals, natural delivery and litter observations were unaffected by dosages. Cold to touch, not nesting, moderate dehydration and pale offspring animals were observed at 800 mg/kg bw/day. However, they were not attributed to the treatment, as it was only observed in one litter and was without a dose response. No effects were observed on changes in sex ratio of litter. At 800 mg/kg bw/day dose, increase in pup weights per litter (not statistically significant), reduction in surviving pups per litter and live litter size at weighing (not statistically significant) were observed. However, these changes were not considered to be biologically relevant. No effects were observed in gross pathology for all offspring animals. Under the study conditions, the read across substance NOAELs for maternal and developmental toxicity were determined to be 800 mg/kg bw/day (Lech, 2009). Based on the results of the read across study, similar NOAELs can be considered for the test substance.

Constituent AE - read across study:

A study was conducted to determine the dermal reproductive and development toxicity of the read across substance, 'C9-11 AE6' (purity not specified), according to a method similar to OECD Guideline 416 (two-generation reproduction study). In the study, groups of 30 weanling Fischer 344 rats of each sex were dermally exposed to 1 mL/kg bw read across substance at concentrations of 0, 1, 10 or 25% w/v three times a week except during the mating periods. This treatment equals exposure levels of about 0, 10, 100 and 250 mg/kg bw/day. No mortalities were observed in the parental generation, and the five deaths in the F1 adult males and females in the control and treatment groups were not considered to be compound related. In the highest dose group, body weights of both males and females in both treated generations were sporadically decreased compared to controls. There was no effect on maternal body weight during gestational and lactational periods in both generations. For details on effects observed on reproductive parameters, please refer to section 7.8.1 of IUCLID or 5.9.1 of CSR. There was no compound related effect on pup weights of any dose group in the F1 generations when compared to the controls. F2 pups from all treatment groups had higher body weights but these were not statistically significantly different from controls, except in in the 10% dose group on day 21 postnatally. Thus, the effect on body weight changes appears to be of no toxicological significance. No compound related effects on Iitter size, sex ratio and number of live pups in the F1 and F2 generations were observed during lactational days 1, 4, 7, 14, 21 and 28. Macroscopic and microscopic examination of the reproductive organs did not reveal significant differences in the treated groups compared to the controls. Under the study conditions, the read across substance dermal NOAELs for maternal systemic, reproductive and developmental toxicity were determined to be 100, >250 and >250 mg/kg bw/day, respectively (Lu, 1985). Based on the results of the read across study, similar NOAELs can be considered for the test substance.

Further, as per a HERA 2009 review report, the investigated alcohol ethoxylates (AE) which were assessed based on 2 -generation toxicity studies showed only reduced pup body weights at maternally toxic doses/second generation leading to NOAELs >50 mg/kg bw/day in a study with C12 AE6. However, no further details were reported for this study. When applied dermally, no adverse effects on the growth and development of the offspring was observed during 2 -generations. Following dermal exposure, the NOAEL was assumed to be higher than the highest tested dose of 250 mg/kg bw/day (HERA, 2009; please refer to the attachment under section 13).

Overall, the NOAELs were found to range from 250-1000 mg/kg bw/day in the studies on the representative substances for the main constituents. As a conservative approach the lower NOAEL of 250 mg/kg bw/day based on the study with mono- and di- C12 PSE, K+, has been considered further for hazard/risk assessment. This NOAEL was based on reduced foetal weight observed in the presence of reduced maternal body weight and food consumption at ≥250 mg/kg bw/day. Therefore, based on the available weight of evidence from studies on substances representative of the main constituents, the test substance, ‘mono- and di- C16 -18 and C16 -18 AE10 PSE’ is not considered to pose development concern at maternally non-toxic doses.

Justification for classification or non-classification

Based on the available weight of evidence from studies on substances representative of the main constituents, the test substance, ‘mono- and di- C18-unsatd PSE and C18-unsatd. AE10 PSE’ is concluded not to warrant classification for reproductive toxicity, according to the EU CLP criteria (Regulation 1272/2008/EC).​​​​​

Additional information