O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No indication of adverse effects on fertility were observed in a screening study with a read-across substance (OECD 421, GLP).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 303 to 335 g; Females: 196 to 222 g
- Fasting period before study: none
- Housing: Single cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70%
- Air changes (per hr): 10 – 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14-Apr-2011 To: 02-Jun-2011

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance was weighed into a brown glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.

VEHICLE
- Amount of vehicle (if gavage): 4 mL/kg body weight

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 11 days
- Proof of pregnancy: daily vaginal smear was sperm positive or a copulation plug was observed
- Further matings after two unsuccessful attempts: not needed
- After successful mating each pregnant female was caged in single cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (2, 4 hrs and 48 hrs). During the second last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice and stored there at -20 ± 5 °C until analysis.
The samples were analyzed by analytical procedure provided by the Sponsor and adapted at at the CRO. The test item was used as the analytical standard.

Duration of treatment / exposure:

Males: Minimum 4 weeks
Females: Approximately 7 weeks

Frequency of treatment:

daily

Details on study schedule:

not applicable for screening study

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

250 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 dose levels were selected based on a 28-day oral toxicity study in which the NOAEL was established at 200 mg/kg body weight/day.

Positive control:

not required

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Viability/mortality was checked twice daily.
Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

OTHER:
For males: Food consumption: Weekly during pre-pairing and after pairing periods.
For females: Pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight, completeness of stages or cell populations of the testes

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in 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:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals (day 28)
- Maternal animals: All surviving animals (post partum day 4)

GROSS NECROPSY
- For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea were recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites

HISTOPATHOLOGY / ORGAN WEIGHTS
The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.
The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Organ weights: testes and epididymides

Histological examination of ovaries was carried out in female no. 55 that did not give birth. In addition, microscopic examination of the reproductive organs of the infertile male no. 15 was made.

Postmortem examinations (offspring):

SACRIFICE
- The offspring was sacrificed on day 4 post partum.

Statistics:

Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

fertility, conception and gestation indeces

Offspring viability indices:

viability indices (day 0-4)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs of discomfort such as salivation and pushing the head through the bedding material were observed at 50 and/or 250 mg/kg in males and females over the study. At 10 mg/kg body weight/day, no test item-related clinical signs were observed during the study. The only observation was hair loss on the dorsal side of the neck in female no. 60 starting on day 20 of the gestation until the termination. In control group, one male was noted to have a wound on the dorsal side of the neck at termination.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test item-related adverse effects were observed in mean body weight and mean body weight gain of males at all dose levels. At 50 and 250 mg/kg body weight/day, the statistically significantly higher mean body weight gain during the pre-pairing and pairing periods, were considered to be incidental and not indicative of any test item effect. In females, at 250 mg/kg body weight/day, mean body weight gain was statistically significantly lower on days 2, 6 and 14 of the pre-pairing period and between days 1 and 4 and on day 12 of the gestation period. These were effecs of the treatment with the test item, which did not affect the mean body weight, therefore not considered to be adverse. At 50 mg/kg body weight/day, mean body weight gain was statistically significantly lower on days 3 and 11 of the pre-pairing period and on days 1 and 2 of the gestation period. These were
transient fluctuations, which did not affect mean body weight and therefore considered not to be adverse.
At 10 mg/kg body weight/day, no test item-related effects were observed during the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No test item-related effects were observed in mean food consumption of males at all dose levels during the whole study. At 250 mg/kg body weight/day, during the pre-pairing period mean food consumption was statistically significantly lower during the first week of the pre-pairing period (-11.8% compared to the control) and slightly lower during the second week. This was a transient reduction and considered not to be adverse as, mean food consumption recovered and was similar to the control group during the gestation and lactation periods. At 10 and 50 mg/kg body weight/day, no test item-related effects were observed in mean food consumption.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All findings recorded were within the range of normal background lesions, which may be recorded in animals of this strain and age. And also, no test item-related histological finding was recorded in ovary of one female (no. 55) that did not give birth and in reproductive organs of one infertile male (no. 15). No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 2.6, 3.4, 2.0 and 2.9 days in order of ascending dose level. The median precoital time was 3, 3, 1 and 3 days in order of ascending dose level. One female (no. 55) at 10 mg/kg body weight/day was not pregnant. Thus, the fertility indices were: 100.0%, 90.0%, 100.0% and 100.0% in ascending of dose level and gave no indication of any test item-related effect. The mean duration of gestation was unaffected by treatment with the test item. Mean number of corpora lutea (determined at necropsy) was 14.0, 15.2, 13.5 and 13.3 in order of ascending dose level and gave no indication of a test item-related effect. The mean number of implantations per dam was unaffected by treatment with the test item. As the higher incidence of post-implantation loss which occurred at 250 mg/kg body weight/day (8.9% versus 5.1%) was within the range of the historical control data was not considered to be adverse. The mean number of implantations per dam were: 13.7, 14.6, 11.6 and 12.3 in order of ascending dose level. The mean incidence of post-implantation loss as a percentage of total implantations was: 5.1%, 1.5%, 0.9% and 8.9% in order of ascending dose level.

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fertility index, viability index

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the lower food consumption and the occasional lower mean body weight gain which were not considered to be adverse in females at 250 and 50 mg/kg body weight/day.

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

At first litter check, the findings noted were: tail reduced in size in male pup at 10 mg/kg body weight/day, wound on snout in one male pup at 50 mg/kg body weight/day and in one female at 250 mg/kg body weight/day and tail missing in one male pup at 250 mg/kg body weight/day. Type and incidences of these findings did not give any indication of any test item-related effect.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The number of live pups at first litter check was unaffected by treatment with the test item. The mean number of live pups per litter was: 13.0, 14.3, 11.5 and 11.2 in order of ascending dose level.
At 10 mg/kg body weight/day, one female pup was found dead on day 2 post partum and at 50 mg/kg body weight/day one female pup was missing on day 4 post partum. The resulting viability indices were: 100.0%, 99.2%, 99.1% and 100.0% in order of ascending dose levels.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup weights on day 1 post partum and mean pup weight development during the lactation to day 4 post partum were unaffected by treatment with the test item. Mean pup weights on day 4 post partum were 9.0, 8.3, 9.2 and 9.0 g for combined data of male and female pups in order of ascending dose level.

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:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormal findings were noted at macroscopic examination of the pups.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item. The proportion of males on day 4 post partum was 49%, 52%, 52% and 58% in order of ascending dose level.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse findings, with sacrifice on day 4 post partum.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Under the conditions of this study, the NOEL (No Observed Effect Level) for reproduction/ developmental toxicity was considered to be 250 mg/kg body weight/day.

Executive summary:

In a GLP-compliant reproduction screening study following OECD guideline 421, four groups of 10 males and 10 females were treated by gavage with the test item once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum. The following dose levels were used: Group 1: 0 mg/kg body weight/day (control group); Group 2: 10 mg/kg body weight/day; Group 3: 50 mg/kg body weight/day; Group 4: 250 mg/kg body weight/day; A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water). All animals survived until the scheduled necropsy. Signs of discomfort such as salivation at dose level of 50 and 250 mg/kg body weight/day and pushing the head through the bedding material at dose level of 250 mg/kg body weight/day were observed during the course of the study. No test item-related effects were observed in mean food consumption of males during the entire study at all dose levels. At 250 mg/kg body weight/day, mean food consumption was statistically significantly lower in females during the first week of the pre-pairing period, thereafter it progressively recovered, and therefore it was not considered to be adverse. No test item-related effects were observed in mean body weight and mean body weight gain of males at all dose levels during the entire study. In females, at 250 mg/kg body weight/day, mean body weight gain was statistically significantly lower on three occasions during the pre-pairing period and at the beginning of the gestation. This was considered an effect of the test item but not to be adverse as mean body weight was not affected. At 50 mg/kg body weight/day, the occasional lower body weight gain observed during the study was not indicative of any test item-related effect. Mating performance, fertility, conception and gestation indeces were not affected by treatment with the test item. The mean duration of gestation was also unaffected by treatment with the test item. The mean number of corpora lutea, the mean number of implantations per dam, and the incidence of post-implantation losses were not affected by treatment with the test item. No test item-related effects were observed in mean values of absolute and relative weight of testes and epididymides. There was no histological evidence of toxicological properties in the organs and tissues examined. No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals. The number of live pups at first litter check and on day 4 post partum was unaffected by the treatment with the test item. No test item-related clinical signs were observed at first litter check and during the lactation. Mean pup weights and weight development were also not affected by the treatment with the test item. Taking into account the lower food consumption and the occasional lower mean body weight gain which were not considered to be adverse in females at 250 and 50 mg/kg body weight/day, the general NOAEL (No Observed Adverse Effect level) was established at 250 mg/kg body weight/day for both males and females. Under the conditions of this study, the NOEL (No Observed Effect Level) for reproduction/ developmental toxicity was considered to be 250 mg/kg body weight/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Quality of whole database:

GLP and guideline study with analogue substance.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No data for the registered substance are available. Therefore, reproduction toxicity was addressed by read across approach with data from a structural analogue. For the source chemical, a OECD 421 study is available. The purpose of this study was to generate preliminary information concerning the effects of the analogue substance on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition (OECD 421, GLP). Dose levels were chosen based on a subacute oral toxicity study:

In the 28 day oral toxicity study dose-related increases in the absolute liver weights were recorded in both sexes in all groups (50, 100, 200 mg/kg). After the treatment-free recovery period, only the liver weights of females of the 200 mg/kg group were statistically significantly higher when compared with the controls. The following effects were observed for the highest dose group (200 mg/kg): Slight decrease in the glucose level in females and slight increases in the triglyceride and phosphorus levels, and gamma-glutamyl-transferase activity were noted in females. A slight increase in the total cholesterol, phospholipid and potassium level was recorded in males. These findings primarily reflect metabolic adaptation in the liver and are of no toxicological relevance. Liver weights (absolute and/or relative) were higher in animals of both sexes. After the treatment-free recovery period only the liver weights of females were statistically significantly higher when compared with the controls. Centrilobular hepatocellular hypertrophy of the liver was the corresponding finding in all males and females. The severity of this hepatocellular hypertrophy was slight to moderate (except for 1 female rat with grade minimal). The following effects occurred at the mid dose group (100 mg/kg): Slight increases in the triglyceride and phosphorus level, and gamma-glutamyl-transferase activity were noted in females. Liver weights (absolute and/or relative) were higher in animals of both sexes. Centrilobular hepatocellular hypertrophy of the liver was noted in all males and 4 females. The severity of this hepatocellular hypertrophy was minimal to moderate.

In the OECD 421 screening study, four groups of 10 males and 10 females were treated by gavage once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum. The following dose levels were used:

Group 1: 0 mg/kg body weight/day (control group)

Group 2 10 mg/kg body weight/day

Group 3: 50 mg/kg body weight/day

Group 4 250 mg/kg body weight/day

A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

Signs of discomfort such as salivation at dose level of 50 and 250 mg/kg body weight/day and pushing the head through the bedding material at dose level of 250 mg/kg body weight/day were observed during the course of the study. At 250 mg/kg body weight/day, mean food consumption was statistically significantly lower in females during the first week of the pre-pairing period, thereafter it progressively recovered. No test item-related effects were observed in mean body weight and mean body weight gain of males at all dose levels during the entire study. For females, at 250 mg/kg body weight/day, mean body weight gain was statistically significantly lower on three occasions during the pre-pairing period and at the beginning of the gestation. This was considered an effect of the test item but not to be adverse as mean body weight was not affected. At 50 mg/kg body weight/day, the occasional lower body weight gain observed during the study was not indicative of any test item-related effect. Mating performance, fertility, conception and gestation indices were not affected by treatment with the test item. The mean duration of gestation was also unaffected by treatment with the test item. The mean number of corpora lutea, the mean number of implantations per dam, and the incidence of post-implantation losses were not affected by treatment with the test item. No test item-related effects were observed in mean values of absolute and relative weight of testes and epididymides. There was no histological evidence of toxicological properties in the organs and tissues examined. No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals. The number of live pups at first litter check and on day 4 post partum was unaffected by the treatment with the test item. No test item-related clinical signs were observed at first litter check and during the lactation. Mean pup weights and weight development were also not affected by the treatment with the test item.

Further details justifying the read-across are given in the read across Justification in IUCLID chapter 13.

The registered substance (target chemical) contains small amounts of 4-nonylphenol (0.15 %), which is classified for toxicity to reproduction and development. In the available EU risk assessment (European Union Risk Assessment Report on 4-nonylphenol (branched) and nonylphenol, 2002) the toxicity profile of nonylphenol was discussed in great detail. The authors conclude that “the observation of oestrogenic activity in the in vitro and in vivo assays, minor perturbations in the reproductive system of offspring in the multigeneration study, and testicular changes in gavage studies collectively raise concerns for reproductive toxicity, possibly mediated through action on the oestrogen receptor”. However, the EU risk assessment also emphasizes that “the effects on reproduction-related parameters in the multigeneration study were marginal and there was no evidence of functional changes in reproduction”. In addition, the reported testicular toxicity was observed at dose levels that also caused mortality. Therefore, it is reasonable to assume that the very low levels of 4-nonylphenol present in the registered substance will have no influence on the toxicity profile of the test article. Additional multigeneration studies performed since 2002 (ECHA dissemination view), confirmed the findings in the previously reported reproduction study (i.e. marginal effects). It should be noted that the test article is used as an additive at low concentrations, therefore the potential emission of 4-nonylphenol is estimated to be very low. As a result, the amount of nonylphenol consumers and workers could potentially get in contact with is estimated to be far below the DNEL available for 4-nonylphenol (published on ECHA dissemination view).

Effects on developmental toxicity

Description of key information

No adverse effects on developmental toxicity were observed in a screening study (of an analogue substance performed according to OECD 421 andGLP).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 421

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 303 to 335 g; Females: 196 to 222 g
- Fasting period before study: none
- Housing: Single cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70%
- Air changes (per hr): 10 – 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14-Apr-2011 To: 02-Jun-2011

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance was weighed into a brown glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.

VEHICLE
- Amount of vehicle (if gavage): 4 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (2, 4 hrs and 48 hrs). During the second last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice and stored there at -20 ± 5 °C until analysis.
The samples were analyzed by analytical procedure provided by the Sponsor and adapted at at the CRO. The test item was used as the analytical standard.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 11 days
- Proof of pregnancy: daily vaginal smear was sperm positive or a copulation plug was observed
- Further matings after two unsuccessful attempts: not needed
- After successful mating each pregnant female was caged in single cages

Duration of treatment / exposure:

Males: Minimum 4 weeks
Females: Approximately 7 weeks

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

250 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 dose levels were selected based on a 28-day oral toxicity study in which the NOAEL was established at 200 mg/kg body weight/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Viability/mortality was checked twice daily.
Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

OTHER:
For males: Food consumption: Weekly during pre-pairing and after pairing periods.
For females: Pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.

SACRIFICE
- Male animals: All surviving animals (day 28)
- Maternal animals: All surviving animals (post partum day 4)

GROSS NECROPSY
- For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea were recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites

HISTOPATHOLOGY / ORGAN WEIGHTS
The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.
The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Organ weights: testes and epididymides
Histological examination of ovaries was carried out in female no. 55 that did not give birth. In addition, microscopic examination of the reproductive organs of the infertile male no. 15 was made.

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in 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:
yes, for external abnormalities

SACRIFICE
- The offspring was sacrificed on day 4 post partum.

Statistics:

Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Indices:

fertility, conception and gestation indeces, viability indices (day 0-4)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs of discomfort such as salivation and pushing the head through the bedding material were observed at 50 and/or 250 mg/kg in males and females over the study. At 10 mg/kg body weight/day, no test item-related clinical signs were observed during the study. The only observation was hair loss on the dorsal side of the neck in female no. 60 starting on day 20 of the gestation until the termination. In control group, one male was noted to have a wound on the dorsal side of the neck at termination.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test item-related adverse effects were observed in mean body weight and mean body weight gain of males at all dose levels. At 50 and 250 mg/kg body weight/day, the statistically significantly higher mean body weight gain during the pre-pairing and pairing periods, were considered to be incidental and not indicative of any test item effect. In females, at 250 mg/kg body weight/day, mean body weight gain was statistically significantly lower on days 2, 6 and 14 of the pre-pairing period and between days 1 and 4 and on day 12 of the gestation period. These were effecs of the treatment with the test item, which did not affect the mean body weight, therefore not considered to be adverse. At 50 mg/kg body weight/day, mean body weight gain was statistically significantly lower on days 3 and 11 of the pre-pairing period and on days 1 and 2 of the gestation period. These were
transient fluctuations, which did not affect mean body weight and therefore considered not to be adverse.
At 10 mg/kg body weight/day, no test item-related effects were observed during the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No test item-related effects were observed in mean food consumption of males at all dose levels during the whole study. At 250 mg/kg body weight/day, during the pre-pairing period mean food consumption was statistically significantly lower during the first week of the pre-pairing period (-11.8% compared to the control) and slightly lower during the second week. This was a transient reduction and considered not to be adverse as, mean food consumption recovered and was similar to the control group during the gestation and lactation periods. At 10 and 50 mg/kg body weight/day, no test item-related effects were observed in mean food consumption.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Weights (absolute and relative to body weight) of testes and epididymides were not affected by the treatment with the test item in any groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No test item-related abnormal findings were observed in males during the necropsy. The only findings noted were sores and kidney pelvic dilation in one male in the control group. In females, dark red or reddish discoloration of the ovaries was observed in one female at 10 mg/kg body weight/day, six females at 50 mg/kg body weight/day and three females at 250 mg/kg body weight/day. There were no hisopathologic correlates.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All findings recorded were within the range of normal background lesions, which may be recorded in animals of this strain and age. And also, no test item-related histological finding was recorded in ovary of one female (no. 55) that did not give birth and in reproductive organs of one infertile male (no. 15). No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

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

Basis for effect level:

other: no effects observed

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean pup weights on day 1 post partum and mean pup weight development during the lactation to day 4 post partum were unaffected by treatment with the test item. Mean pup weights on day 4 post partum were 9.0, 8.3, 9.2 and 9.0 g for combined data of male and female pups in order of ascending dose level.

Details on embryotoxic / teratogenic effects:

The number of live pups at first litter check was unaffected by treatment with the test item. The mean number of live pups per litter was: 13.0, 14.3, 11.5 and 11.2 in order of ascending dose level.
At 10 mg/kg body weight/day, one female pup was found dead on day 2 post partum and at 50 mg/kg body weight/day one female pup was missing on day 4 post partum. The resulting viability indices were: 100.0%, 99.2%, 99.1% and 100.0% in order of ascending dose levels. No abnormal findings were noted at macroscopic examination of the pups. Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item. The proportion of males on day 4 post partum was 49%, 52%, 52% and 58% in order of ascending dose level.

Key result

Dose descriptor:

NOEL

Effect level:

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

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:

Under the conditions of this study, the NOEL (No Observed Effect Level) for reproduction/ developmental toxicity was considered to be 250 mg/kg body weight/day.

Executive summary:

In a GLP-compliant reproduction screening study following OECD guideline 421, four groups of 10 males and 10 females were treated by gavage with the test item once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum. The following dose levels were used: Group 1: 0 mg/kg body weight/day (control group); Group 2: 10 mg/kg body weight/day; Group 3: 50 mg/kg body weight/day; Group 4: 250 mg/kg body weight/day; A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water). All animals survived until the scheduled necropsy. Signs of discomfort such as salivation at dose level of 50 and 250 mg/kg body weight/day and pushing the head through the bedding material at dose level of 250 mg/kg body weight/day were observed during the course of the study. No test item-related effects were observed in mean food consumption of males during the entire study at all dose levels. At 250 mg/kg body weight/day, mean food consumption was statistically significantly lower in females during the first week of the pre-pairing period, thereafter it progressively recovered, and therefore it was not considered to be adverse. No test item-related effects were observed in mean body weight and mean body weight gain of males at all dose levels during the entire study. In females, at 250 mg/kg body weight/day, mean body weight gain was statistically significantly lower on three occasions during the pre-pairing period and at the beginning of the gestation. This was considered an effect of the test item but not to be adverse as mean body weight was not affected. At 50 mg/kg body weight/day, the occasional lower body weight gain observed during the study was not indicative of any test item-related effect. Mating performance, fertility, conception and gestation indeces were not affected by treatment with the test item. The mean duration of gestation was also unaffected by treatment with the test item. The mean number of corpora lutea, the mean number of implantations per dam, and the incidence of post-implantation losses were not affected by treatment with the test item. No test item-related effects were observed in mean values of absolute and relative weight of testes and epididymides. There was no histological evidence of toxicological properties in the organs and tissues examined. No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals. The number of live pups at first litter check and on day 4 post partum was unaffected by the treatment with the test item. No test item-related clinical signs were observed at first litter check and during the lactation. Mean pup weights and weight development were also not affected by the treatment with the test item. Taking into account the lower food consumption and the occasional lower mean body weight gain which were not considered to be adverse in females at 250 and 50 mg/kg body weight/day, the general NOAEL (No Observed Adverse Effect level) was established at 250 mg/kg body weight/day for both males and females. Under the conditions of this study, the NOEL (No Observed Effect Level) for reproduction/ developmental toxicity was considered to be 250 mg/kg body weight/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

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

Details on the screening study (OECD 421) are given above. The number of live pups at first litter check and on day 4 post partum was unaffected by the treatment with the test item. No test item-related clinical signs were observed at first litter check and during the lactation. Mean pup weights and weight development were also not affected by the treatment with the test item.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available screening study is reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for reproductive toxicity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218