Poly[oxy(methyl-1,2-ethanediyl)], α-hydro-ω-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), 2-propenoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012 - 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012 -2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, adopted July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (male and female animals were derived from different litters to rule out mating of siblings)
- Age at study initiation: 10-11 wks
- Weight at study initiation: on average: Males: 335g; Females: 197g
- Fasting period before study: no
- Housing: single (except during mating and during lactation) in Markrolon type M III cages
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse-rat “GLP”, meal ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: app. 1 week

Environmental conditions
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: drinking water

Details on route of administration:

Not available

Vehicle:

corn oil

Details on oral exposure:

Preparation of dosing soltuions:
- The desired amount of test substance was weighed, and corn oil was added up to the correct volume. To prepare a homogenous suspension, the mixture was stirred with a magnetic stirrer also during administration. The test substance preparations were produced at least once a week and were stored at room temperature. The administration volume was 4 mL/kg bw.
Vehicle:
- Justification for use and choice of vehicle (if other than water): The test substance is poorly soluble in water, but forms a homogenous suspension in corn oil, which is also non-toxic to rats.
- Concentration in vehicle: 0, 1.25, 3.75 and 12.5 g/100mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity, stability and concentration control analyses of the test substance preparations were performed in all concentrations at the start of the administration period. Additionally, samples from all concentrations as reverse samples for concentration control analysis were taken at the end of the study. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 90.0-110.0% of the nominal concentrations. Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the the test substance was distributed homogeneously in corn oil.

Duration of treatment / exposure:

Males: 35 days
Females: 56 days

Frequency of treatment:

Daily (except to animals being in labor)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

Dose selection rationale:
The selection of doses was based on the results of a test study in female Wistar rats (BASF project No. 10C0457/11S163) conducted at dose levels of 0, 600 and 1000 mg/kg bw/d. In this study, a NOAEL was not established due to erosions and ulcerations in the stomach of different animals at both dose levels of 600 and 1000 mg/kg bw/d as well as clinical findings like poor general condition, piloerection and body weight loss after 1 week of treatment.

Positive control:

No

Observations and examinations performed and frequency:

Cage side observations: Yes
- Time schedule: twice daily on workdays, daily on weekends and public holidays
- Cage side observations: check for moribund animals, pertinent behavioral changes, signs of overt toxicity, parturation, littering and lactation behavior of the dams

Detailed clinical observations: Yes
- Time schedule: prior to the first administration, weekly thereafter
- The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

Body weight: Yes
- Time schedule for examinations: day 0, weekly thereafter with the following exceptions for females: during the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 4.

Food consumption: Yes (once weekly), except during mating

Water consumption: Monitored by daily visual inspection

Other:
- Functional observation battery and motor activity measurement of 5 males and females per group 2 days prior to sacrifice
- Urinanalysis in 5 males and females per group one day prior to necropsy
- Clinicochemical and hematological examinations 5 males and females on the day of necropsy after fasting for 16h

Sacrifice and pathology:

Sacrifice:
- Male animals: All surviving animals on day 36
- Maternal animals: All surviving animals on day 57

Gross necropsy
- Gross necropsy consisted of external and internal examinations

Histopathology / Organ weights
The following tissues were weighed
- in all animals: epididymides, testes, and stages of spermatogenesis
- in 5 males and females of each group: adrenal glands, brain, heart, kidneys, liver, spleen, thymus
The uteri of all cohabited female parental animals were examined for the presence and number of implantation sites. The uteri of apparently non-pregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations.
The following tissues were examined histotechnically in at least 5 animals per sex of the control and high dose group (reproductive organs were examined in all high dose and control animals):
adrenal glands, gross lesions (all affected animals in all dosage groups), bone marrow (femur), brain, cecum, cervix, coagulation glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (auxillary and mesenteric), ovaries, oviducts, prostate gland, peyer's patches, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, stomach, testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina.

Other examinations:

Reproductive performance: see chapter 7.8.1

Statistics:

Not available

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Almost all male and most female animals of the high dose group showed salivation within 2 hours after the administration on several days of the study. Salivation within 2 hours after treatment was also seen in several mid dose male and female animals. From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female animal (in the 500 mg/kg bw/day group) was sacrificed in a moribund condition on study day 51 (GD 24). Vaginal discharge was observed on GDs 23-24 in this animal, which showed poor general state and was unable to deliver on GD 24. This animal was sacrificed moribund on the same day. According to the pathological results the findings were assessed as being incidental and not related to treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Neurobehaviour:
All deviations from "zero" values were equally distributed between all groups including controls; hence the finding were assessed as being incidental and not treatment related.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

All mean weight parameters (absolute and relative) did not show significant differences when compared to the control groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All findings were considered to be incidental or spontaneous in origin and without any relation to treatment.
One high dose female animal that was sacrificed in a moribund state revealed a decidual reaction with consequent inflammation of the uterus. Decidual reaction is a proliferation of decidual cells (tissue of endometrial origin lining of the uterus, which is in contact with the fetal membranes and the placental plate). They often are assiocated with inflammation as observed in this animal. The local inflammation in the uterus can lead to disturbance of the general condition or, if the inflammation is spreading, sepsis. This was regarded to be the reason for the moribund state of this animal but was not regarded to be treatment-related.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Reproductive toxicity results: see chapter 7.8.1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

Under the study conditions, the rat NOAEL for systemic effects was established at >=500 mg/kg bw/day for females and males.

Executive summary:

A study was conducted to determine the repeated dose toxicity of the test substance according to OECD guideline 422 and EPA OPPTS 870.3650, in compliance with GLP. The general systemic toxic potential of the substance to Wistar rats was assessed by daily oral administration (gavage). Males were dosed for 35 days and females for 56 days. Groups comprising 10 male and 10 female rats received the test substance at doses of 0, 50, 150 or 500 mg/kg bw/ day in corn oil. A detailed clinical observation was performed in all animals before initial test substance administration and thereafter at weekly intervals. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0-7, 7-14, 14-20 and lactation days 1-4. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, after the day of parturition (postnatal day [PND] 0) and on PND 4. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings. Clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed. One female animal of test group 500 mg/kg bw/day was sacrificed in a moribund condition on study day 51 (GD 24). Vaginal discharge was observed on GDs 23-24 in this animal, which showed poor general state and was unable to deliver on GD 24. This animal was sacrificed moribund on the same day. According to the pathological results the findings were assessed as being incidental and not related to treatment. Almost all male and most female animals of the high dose group showed salivation within 2 hours after the administration on several days of the study. Salivation within 2 hours after treatment was also seen in several mid dose male and female animals. From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. No test substance-related changes in body weight or body weight gain were observed for all test groups. For food and water consumption, no test substance-related, adverse findings were noted. No treatment-related changes among hematological, clinical chemistry and urinalysis parameters were observed. All deviations from "zero" values observed following neurobehavioural examination were equally distributed between all groups including controls; the findings were assessed as being incidental and not treatment related. All mean organ weight parameters (absolute and relative) did not show significant differences when compared to the control groups. All gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment. All histopathology (non-neoplastic) findings were considered to be incidental or spontaneous in origin and without any relation to treatment. The high dose female animal that was sacrificed in a moribund state revealed a decidual reaction with consequent inflammation of the uterus. Decidual reaction is a proliferation of decidual cells (tissue of endometrial origin lining of the uterus, which is in contact with the fetal membranes and the placental plate). They are often assiocated with inflammation as observed in this animal. The local inflammation in the uterus can lead to disturbance of the general condition or, if the inflammation is spreading, sepsis. This was regarded to be the reason for the moribund state of this animal but was not regarded to be treatment-related. Under the study conditions, the rat NOAEL for systemic effects was established at >=500 mg/kg bw/day for females and males (BASF, 2013).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, adopted July 2000

Deviations:

no

Principles of method if other than guideline:

The objective of the study was to detect possible effects of the test substance on the integrity and performance of male and female reproductive systems including gonadal function, mating behavior, conception, gestation and parturition.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Assessment of the effects of the test substance on the reproductive system, fertility and performance.

Test material

Test material information

Constituent 1

Reference substance name:

Poly[oxy(methyl-1,2-ethanediyl)], a-hydro-w-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), mono-2-propenoate

Cas Number:

1378240-17-5

Molecular formula:

(C3H6O)n(C3H6O)n(C3H6O)nC9H16O4

IUPAC Name:

Poly[oxy(methyl-1,2-ethanediyl)], a-hydro-w-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), mono-2-propenoate

Constituent 2

Reference substance name:

Poly[oxy(methyl-1,2-ethanediyl)], α-hydro-ω-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), di-2-propenoate

Cas Number:

104956-81-2

Molecular formula:

(C3H6O)n(C3H6O)n(C3H6O)nC12H18O5

IUPAC Name:

Poly[oxy(methyl-1,2-ethanediyl)], α-hydro-ω-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), di-2-propenoate

Constituent 3

Reference substance name:

Propylidynetrimethanol, propoxylated, esters with acrylic acid

EC Number:

500-123-4

EC Name:

Propylidynetrimethanol, propoxylated, esters with acrylic acid

Cas Number:

53879-54-2

Molecular formula:

(C3H6O)n(C3H6O)n(C3H6O)nC15H20O6

IUPAC Name:

Poly[oxy(methyl-1,2-ethanediyl)], .alpha.-hydro-.omega.-[(1-oxo-2-propenyl)oxy]-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol

Constituent 4

Reference substance name:

Poly[oxy(methyl-1,2-ethanediyl)], a-hydro-w-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), mono[3-[(1-oxo-2-propen-1-yl)oxy]propanoate] di-2-propenoate

Cas Number:

1378240-20-0

Molecular formula:

(C3H6O)n(C3H6O)n(C3H6O)nC15H20O6 + C3H4O2

IUPAC Name:

Poly[oxy(methyl-1,2-ethanediyl)], a-hydro-w-hydroxy-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1), mono[3-[(1-oxo-2-propen-1-yl)oxy]propanoate] di-2-propenoate

Constituent 5

Reference substance name:

2-{[1-(2-ethyl-3-hydroxy-2-{[2-(prop-2-enoyloxy)ethoxy]methyl}propoxy)propan-2-yl]oxy}propyl prop-2-enoate (TMP(PO)n(EO)mDA)

Molecular formula:

C20H34O8

IUPAC Name:

2-{[1-(2-ethyl-3-hydroxy-2-{[2-(prop-2-enoyloxy)ethoxy]methyl}propoxy)propan-2-yl]oxy}propyl prop-2-enoate (TMP(PO)n(EO)mDA)

Constituent 6

Reference substance name:

2-{[1-(2-{[2-(prop-2-enoyloxy)ethoxy]methyl}-2-({[1-(prop-2-enoyloxy)propan-2-yl]oxy}methyl)butoxy)propan-2-yl]oxy}propyl prop-2-enoate (TMP(PO)n(EO)mTA)

Molecular formula:

C26H42O10

IUPAC Name:

2-{[1-(2-{[2-(prop-2-enoyloxy)ethoxy]methyl}-2-({[1-(prop-2-enoyloxy)propan-2-yl]oxy}methyl)butoxy)propan-2-yl]oxy}propyl prop-2-enoate (TMP(PO)n(EO)mTA)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (male and female animals were derived from different litters to rule out mating of siblings)
- Age at study initiation: 10-11 wks
- Weight at study initiation: on average: Males: 335g; Females: 197g
- Fasting period before study: no
- Housing: single (except during mating and during lactation) in Markrolon type M III cages
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse-rat “GLP”, meal ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: app. 1 week

Environmental conditions
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Preparation of dosing soltuions:
- The desired amount of test substance was weighed, and corn oil was added up to the correct volume. To prepare a homogenous suspension, the mixture was stirred with a magnetic stirrer also during administration. The test substance preparations were produced at least once a week and were stored at room temperature. The administration volume was 4 mL/kg bw.
Vehicle:
- Justification for use and choice of vehicle (if other than water): The test substance is poorly soluble in water, but forms a homogenous suspension in corn oil, which is also non-toxic to rats.
- Concentration in vehicle: 0, 1.25, 3.75 and 12.5 g/100mL

Details on mating procedure:

- M/F ratio per cage: 1:1 over night
- Length of cohabitation: until sperm was detected in vaginal smear or for a maximum of 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity, stability and concentration control analyses of the test substance preparations were performed in all concentrations at the start of the administration period. Additionally, samples from all concentrations as reverse samples for concentration control analysis were taken at the end of the study. The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 90.0-110.0% of the nominal concentrations. Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the the test substance was distributed homogeneously in corn oil.

Duration of treatment / exposure:

Males: 35d
Females: 56d
(The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice.)

Frequency of treatment:

Daily (except to animals being in labor)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
The selection of doses was based on the results of a test study in female Wistar rats (BASF project No. 10C0457/11S163) conducted at dose levels of 0, 600 and 1000 mg/kg bw/d. In this study, a NOAEL was not established due to erosions and ulcerations in the stomach of different animals at both dose levels of 600 and 1000 mg/kg bw/d as well as clinical findings like poor general condition, piloerection and body weight loss after 1 week of treatment.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

Cage side observations: Yes
- Time schedule: twice daily on workdays, daily on weekends and public holidays
- Cage side observations: check for moribund animals, pertinent behavioral changes, signs of overt toxicity, parturation, littering and lactation behavior of the dams

Detailed clinical observations: Yes
- Time schedule: prior to the first administration, weekly thereafter
- The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

Body weight: Yes
- Time schedule for examinations: day 0, weekly thereafter with the following exceptions for females: during the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 4.

Food consumption: Yes (once weekly), except during mating

Water consumption: Monitored by daily visual inspection

Other (for details see entry for this study in the repeated dose section):
- Functional observation battery and motor activity measurement of 5 males and females per group 2 days prior to sacrifice
- Urinanalysis in 5 males and females per group one day prior to necropsy
- Clinicochemical and hematological examinations 5 males and females on the day of necropsy after fasting for 16h

Oestrous cyclicity (parental animals):

-

Sperm parameters (parental animals):

Parameters examined in male parental animals: testis weight, epididymis weight, stages of spermatogenesis in the male gonads

Litter observations:

- Parameters examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, clinical symptoms, weight gain (on PND1 and 4)
- Gross examinations of dead pups: yes, externally and organs were assessed macroscopically

Postmortem examinations (parental animals):

Sacrifice:
- Male animals: All surviving animals on day 36
- Maternal animals: All surviving animals on day 57

Gross necropsy
- Gross necropsy consisted of external and internal examinations

Histopathology / Organ weights
The following tissues were weighed
- in all animals: epididymides, testes
- in 5 males and females of each group: adrenal glands, brain, heart, kidneys, liver, spleen, thymus
The uteri of all cohabited female parental animals were examined for the presence and number of implantation sites. The uteri of apparently non-pregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations.
The following tissues were examined histotechnically in at least 5 animals per sex of the control and high dose group (reproductive organs were examined in all high dose and control animals):
adrenal glands, gross lesions (all affected animals in all dosage groups), bone marrow (femur), brain, cecum, cervix, coagulation glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (auxillary and mesenteric), ovaries, oviducts, prostate gland, peyer's patches, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, stomach, testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina

Postmortem examinations (offspring):

Necropsy:
- All surviving pubs were subjected to postmortem examinations: external examination and macroscopic examination of organs. Animals with notable findings or abnormalitites were evaluated on a case-by-case basis.

Reproductive indices:

For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
Male mating index (%) = number of males with confirmed mating (vaginal sperm detected in females) / number of males placed with females x100
Male fertility index (%) = number of males proving their fertility (implants in utero) / number of males placed with females x100

For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
Female mating index (%) = number of females mated (vaginal sperm detected) / number of females placed with males x100
Female fertility index (%) = number of females pregnant (implants in utero) / number of females mated (vaginal sperm or implants in utero) x100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x100

Offspring viability indices:

Live birth index (%) = number of liveborn pups at birth / total number of pups born x100
Viability index (%) = number of live pups on day 4 after birth / number of live pups on the day of birth x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All findings were considered to be incidental or spontaneous and without any relation to treatment.
The high dose female animal that was sacrificed in a moribund state revealed a decidual reaction with consequent inflammation of the uterus. Decidual reaction is a proliferation of decidual cells (tissue of endometrial origin lining of the uterus, which is in contact with the fetal membranes and the placental plate). They are usually assiocated with inflammation as observed in this animal. The local inflammation in the uterus can lead to disturbance of the general condition or, if the inflammation is spreading, sepsis. This was regarded to be the reason for the moribund state of this animal but was not regarded to be treatment-related.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

For all male and female animals mating was confirmed (mating index of 100%). Male and female fertility index varied between 80% and 90%. All pregnant rats in all dose groups delivered pups with the exception of 1 high dose female which was sacrificed moribund on GD24.
One high dose female animal, that was not pregnant, revealed a decrease in ovary size and a diffuse atrophy of both ovaries and no corpora lutea. This was regarded to be the cause of the infertility in this pair. All other females and their male mating partners did not show gross lesions in reproduction relevant organs which could explain the lack of offspring.

Details on results (P0)

For remaining details on general toxicity refer to the study in rats under repeated dose section.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity (performance and fertility)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Ten of 14 pups in the litter of one low dose female animal showed reduced nutritional condition on PND 0. All pups in this litter died between PND 0-1. Each one pup of two mid dose female animals died on PND 0. These findings were assessed as being incidental as no dose-reponse relationship occurred. The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Single stillborn pubs were seen in the low dose and mid dose group only. The mean number of delivered pups per dam and the rate of liveborn and stillborn pups reflect the normal range of biological variation inherent in the strain used in this study.
The viability index as indicator for pup mortality between PND 0 and 4 was 89% in the low dose group (all pups of one female died), 99% in the mid dose group (1 pup of one female died) and 100% for high dose and control females. As the decreased viability index in the low dose group was still within the historical control data, statistically not significant, and related to mentioned litter loss of only one female animal, the finding was assessed to be incidental and not related to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the concurrent control values.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related changes were observed.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

no developmental toxicity was observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

For any results related to systemic toxicity or others, refer to the study listed in "cross-reference".

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the rat NOAEL for reproductive and developmental effects were established at >=500 mg/kg bw/day for females and males (BASF, 2013).

Executive summary:

A study was conducted to determine the toxicity to reproduction of the test substance according to OECD guideline 422 and EPA OPPTS 870.3650, in compliance with GLP. The toxic potential of the substance to Wistar rats was assessed by daily oral administration (gavage). Males were dosed for 35 days and females for 56 days (the duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice). Groups comprising 10 male and 10 female rats received the test substance at doses of 0, 50, 150 or 500 mg/kg bw/ day in corn oil. Apart from the systemic parameters which has been discussed under repeated dose section, the integrity and performance of male and female reproductive systems including gonadal function, mating behavior, conception, gestation and parturition were evaluated in this study. The uteri of all cohabited female parental animals were examined for the presence and number of implantation sites. The reproductive organs (including ovaries, oviducts, uterus, vagina, cervix, coagulation glands, prostate gland, seminal vesicles, testes, epididymides) were evaluated in at least 5 animals per sex of the control and high dose group. Mating and fertility indices in males and females were calculated for the F1 litters. The F1 offsprings were monitored for number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, clinical symptoms, weight gain (on PND1 and 4). Also, gross examinations externally and internally) were performed on dead pups. One female animal of test group 500 mg/kg bw/day was sacrificed in a moribund condition on study day 51 (GD 24). Vaginal discharge was observed on GDs 23-24 in this animal, which showed poor general state and was unable to deliver on GD 24. This animal was sacrificed moribund on the same day. According to the pathological results the findings were assessed as being incidental and not related to treatment. All mean organ weight parameters (absolute and relative) did not show significant differences when compared to the control groups. All gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment. All histopathology (non-neoplastic) findings were considered to be incidental or spontaneous in origin and without any relation to treatment. The high dose female animal that was sacrificed in a moribund state revealed a decidual reaction with consequent inflammation of the uterus. Decidual reaction is a proliferation of decidual cells (tissue of endometrial origin lining of the uterus, which is in contact with the fetal membranes and the placental plate). They are often assiocated with inflammation as observed in this animal. The local inflammation in the uterus can lead to disturbance of the general condition or, if the inflammation is spreading, sepsis. This was regarded to be the reason for the moribund state of this animal but was not regarded to be treatment-related. For all male and female animals, mating was confirmed (mating index of 100%). Male and female fertility index varied between 80% and 90%. All pregnant rats in all dose groups delivered pups with the exception of 1 high dose female which was sacrificed moribund on GD24. One high dose female animal, that was not pregnant, revealed a decrease in ovary size and a diffuse atrophy of both ovaries and no corpora lutea. This was regarded to be the cause of the infertility in this pair. All other females and their male mating partners did not show gross lesions in reproduction relevant organs which could explain the lack of offspring. No effects were seen on sperm measures. Single stillborn pubs were seen in the low dose and mid dose group only. The mean number of delivered pups per dam and the rate of liveborn and stillborn pups reflected the normal range of biological variation inherent in the strain used in this study. The viability index as indicator for pup mortality between PND 0 and 4 was 89% in the low dose group (all pups of one female died), 99% in the mid dose group (1 pup of one female died) and 100% for high dose and control females. As the decreased viability index in the low dose group was still within the historical control data, statistically not significant, and related to mentioned litter loss of only one female animal, the finding was assessed to be incidental and not related to treatment. 10 of 14 pups in the litter of one low dose female animal showed reduced nutritional condition on PND 0. All pups in this litter died between PND 0-1. Each one pup of two mid dose female animals died on PND 0. These findings were assessed as being incidental as no dose-reponse relationship occurred. The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4. Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the concurrent control values. The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups. No lesions were seen following gross examinations. Under the study conditions, in absence of toxicologically signficant adverse effects, the rat NOAEL for reproductive and developmental effects were established at >=500 mg/kg bw/day for females and males (BASF, 2013).